The science of tumor-infiltrating lymphocytes (TIL): perspectives from the SITC Surgery Committee.

Introduction

The first approval for tumor-infiltrating lymphocyte (TIL) therapy by the Food and Drug Administration (FDA) in February of 2024 for patients with melanoma in the USA, along with regulatory approval for hospital-based TIL made available in the Netherlands and Denmark, was a remarkable landmark for the cell therapy field. In the randomized study of TIL versus immune checkpoint blockade (ICB) with ipilimumab, improved quality of life as well as response to TIL therapy was clearly shown. 1 Almost 20 biotechnology companies emerged to similarly develop TIL trials over this period, with remarkable advances in applying these strategies to other non-melanoma solid tumors. The Surgery Committee of the Society for Immunotherapy of Cancer (SITC) has recently provided a detailed review of best practice for tumor procurement for TIL purposes 2 as well as current clinical approaches for administering TIL. 3 In this study, we focus on the ‘Science of TIL’ to review in detail a more forward-looking review of advances in the field based on murine models and evolving clinical experience.

Current non-melanoma TIL therapy and expansion from the NCI to other centers

Summary of status of ‘unselected’ TIL in melanoma-comparison with selected or gene-modified TIL

TIL therapy was first successfully introduced in patients with advanced melanoma. Historical National Cancer Institute (NCI) trials reported objective response rates (ORRs) of up to 72% and complete responses (CRs) of around 22% in patients with cutaneous melanoma receiving bulk, unselected TIL (ie, autologous lymphocytes harvested from resected tumor tissue without prior selection for tumor reactivity or antigen specificity). International studies have confirmed TIL efficacy in such patients, with a meta-analysis of 410 patients across 13 trials reporting an ORR of 41% and a CR rate (CRR) of 12%, in the pre-ICB era. 4 A second meta-analysis found no difference in median overall survival between patients with (17.5 months) and without (16.3 months) prior programmed death ligand 1 (PD-(L)1) therapy. ORR and CRR were 34% and 10%, respectively, across both groups. 5 In the Phase II C-144–01 study, unselected TIL (lifileucel, Amtagvi) demonstrated ORRs of 31% and CRRs of 5%. 6 In a recent update with 5 years of follow-up, 7 the ORR was 31.4% (CR, 5.9%; partial response, 25.5%), while the median duration of response was 36.5 months (about 3 years) and the 5-year overall survival was 19.7%. These results led to FDA approval of lifileucel for patients with advanced, ICB-refractory melanoma. On the other hand, the randomized study comparing unselected TIL versus ipilimumab in second-line melanoma after PD-(L)1 failure reported superior ORR and progression-free survival in the TIL arm, leading to hospital approval in the Netherlands and Denmark. 8

The current canonical ‘industrial’ TIL protocol, approved by the FDA for patients with melanoma (and applied in patients with head and neck cancer, cervical cancer, and lung cancer) involves three steps. 9 The first step is the identification and resection of one or more tumors that in aggregate constitute at least 1–1.5 cm 3 of tumor. Although sufficient TIL have been generated from lesions with aspirates or biopsies, conservative approaches would recommend resecting sufficient tumors to both allow generation of TIL but also paired autologous organoids, when possible. 10 The second step typically involves trimming the material of associated fat or non-tumor material into 2–3 mm 3 fragments for up to 100 fragments for initiation within bottom-respiring flasks, culture with 6000 IU/mL of recombinant interleukin 2 (IL-2) for 11 days. This step is termed the ‘pre-REP’ as the third step involves the ‘rapid expansion protocol’ or REP. This takes washed and counted TIL and expands them with additional 6000 IU/mL recombinant IL-2, an agonistic anti-CD3 antibody, and three separate irradiated allogeneic peripheral blood mononuclear cell preparations to allow 100:1 feeder to TIL ratio with culture for another 11days. This was based on murine studies demonstrating its effectiveness by Riddell and Greenberg. 11 After this culture period, harvest, washing, and concentration prior to delivery as a white cell infusion to a suitable prepared patient who has undergone non-myeloid ablating chemotherapy with fludarabine and cyclophosphamide. Typically, patients receive the cells and at 12–24 hours up to six doses of 600,000 IU/kg of IL-2 administered every 8–12 hours. This constitutes a ‘TIL Procedure’. Alternatives to this regimen include culturing individual fragments in microwells, their testing in tumor-specific assays, and some variations in the length of culture up to 14–21 days for the pre-REP.

Gene-engineered TIL has been proposed to improve their potency and persistence through transgenic cytokine expression, inhibitory receptor or regulatory gene knockouts (eg, programmed death-1 (PD-1), cytokine-inducible SH2-containing protein (CISH), suppressor of cytokine signaling 1 (SOCS1), and ribonuclease involved in the regulation of immune responses (Regnase1), or dominant-negative transforming growth factor beta (TGF-β) receptors. The first application of this strategy employed introduction with retroviruses of neomycin resistance to track the adoptively transferred TIL. 12 13 Subsequently, efforts were undertaken in the early 1990s to express tumor necrosis factor (TNF) within TIL, to enhance their activity. 14 16 A Phase I trial using IL-12–engineered TIL, without IL-2 support, under the nuclear factor of activated T cells (NFAT) promoter showed tumor regression with PRR of 63% with doses between 0.3 to 3×10 9 cells, that is,1–2 log lower than conventional TIL. However, the TIL product, although potent, was constrained by poor in vivo persistence of cells, systemic toxicity at higher doses, and only transient clinical responses. 17 A Phase 1 trial is presently evaluating OBX-115—an engineered TIL product expressing regulated membrane-bound IL-15. Preliminary reports indicate a promising ORR in PD-(L)1-resistant melanoma, with no high-grade toxicities or cytokine release syndrome. 18 19 Ongoing studies with TIL transduced with membrane-bound IL-7 in patients with ovarian cancer ( NCT05468307 ) reported an ORR of 33% in 18 evaluable patients treated.

Phenotypic differences between melanoma and epithelial TIL

Identifying neoantigen-reactive T cells has proven more challenging in epithelial tumors than in melanoma. The primary reason for this is that, on average, epithelial cancers harbor fewer non-synonymous coding mutations than melanoma. A comparison of coding tumor mutational burden (TMB) between patients with melanoma and epithelial cancer revealed a decreasing trend in non-synonymous coding mutations, from PD-1-naïve melanomas to PD-1-treated melanomas, and further to epithelial cancers. When whole exome sequencing (WES) data solely from the NCI Surgery Branch was considered, to eliminate interinstitutional differences in sequencing depth, mutation-calling algorithms or filters, the highest TMB was found in checkpoint-naïve melanomas and the lowest in ovarian cancer ( figure 1 ; data updated from 20 21 ).

Within patients with melanoma as well as those with epithelial cancers, a higher number of T cell responses against neoantigens has been associated with better clinical response. In fact, across all groups, 0.7–1.6% of all non-synonymous mutations proved to be immunogenic, defined by detecting a T cell response against that neoantigen. One intriguing difference between melanoma and epithelial cancers was that a higher percent of T cell responses to neoantigens were mediated by CD4+cells and were likely major histocompatibility complex (MHC) Class II restricted in epithelial cancers (54% vs approximately 25% for melanoma). Furthermore, a higher number of infused CD4+cells was associated with better clinical responses in epithelial cancers. 22 Overall, response to autologous tumor antigens appeared less frequently in non-melanoma cancers. 23 Tumor antigens in non-small cell lung cancer (NSCLC) and melanoma recognized by T cells appear to be primarily from non-mutated genes. 24 Similar results have also been found in T cells recognizing pancreatic cancer. 25 Splicing alterations also provide a source of novel antigens in some low-TMB tumors as well. 26

The general paradigm that neoantigen-reactive tumor resident T cells were marked by inhibitory or exhaustion markers was also true in epithelial cancer TIL. CD39, T cell immunoglobulin and ITIM domain (TIGIT), Lymphocyte Activation Gene 3 (LAG-3), and PD-1 were the prominently expressed inhibitory markers on neoantigen-reactive TIL. 27 Furthermore, evidence that administered T cells upregulate PD-1 in vivo following adoptive transfer 22 28 provided the rationale for adding pembrolizumab to TIL therapy. This was associated with a higher clinical response rate in small non-randomized sequential studies of TIL from epithelial tumors. 22

Biomarkers of effective TIL

While there is no validated predictive biomarker for response to TIL therapy, current evidence indicates that clinical benefit may depend on both tumor-intrinsic as well as cell-infusion product attributes. Most of the current observations come from patients with melanoma treated with TIL-adoptive T cell therapy (ACT). Here, a high TMB and neoantigen load may enrich for responders and provide a diverse antigenic landscape for recognition, 20 29 although the results are not consistent across all cohorts. 29 30 Certain tumor RNA signatures, such as upregulation of immune system-associated genes 29 or expression of phosphodiesterase 1C (PDE1C), rhotekin 2 (RTKN2), and nerve growth factor receptor (NGFR), are associated with improved outcomes, whereas expression of the phosphatase inhibitor, ELFN1-marked tumors that are resistant to TIL therapy. 29

A higher number of infused TIL, 28 31 and of infused tumor-reactive TIL, 28 32 33 including neoantigen-reactive TIL 34 are associated with response. Attributes of less-differentiated TIL—longer telomeres and higher expression of co-stimulatory molecules such as CD27 and CD28, both characteristics of ‘younger’ TIL, 35 are also associated with response to therapy. 31 Multifunctionality 36 and PD-1 expression 22 37 were characteristics enriched among tumor-reactive and persisting TIL. Recently, single cell ribonucleic acid sequencing (scRNAseq) identified in a minor pool of stem-like, tumor-specific CD8 + TIL (CD39⁻ CD69⁻ transcription factor 7 (TCF7) + ), a subpopulation capable of driving complete melanoma regression and long-term persistence, 38 but this population was not found to be predictive in a recently reported gastrointestinal tumors. 39

Selection strategies for TIL in the blood and tumor

Bystander, non-reactive lymphocytes are particularly abundant in epithelial cancers with low TMB. 23 40 The selective expansion of tumor-reactive and neoantigen-reactive TIL—rather than bystander, non-reactive lymphocytes—could thus prove critical to broaden the applicability of TIL therapy to these cancers. Selected TIL cultures recognizing personalized cancer neoantigens (SEL-TIL) demonstrated antitumor activity in patients with cholangiocarcinoma, colorectal, and breast cancers, 41 43 and infusion of neoantigen-targeted SEL-TIL, alone or in combination with pembrolizumab, outperformed standard, unselected TIL in patients with gastrointestinal (GI) cancers in non-randomized sequential trials. 22 Response to SEL-TIL in the latter setting was associated with increased numbers of targeted neoantigen-reactive CD4+TIL. However, variability in the frequency and proliferative capacity of reactive TIL during the rapid expansion protocol (REP) often results in inconsistent neoantigen reactivity of SEL-TIL products. 44 Thus, alternative strategies are being explored to reliably generate neoantigen-reactive TIL products. For instance, two independent studies demonstrated that early exposure of TIL to selected neoantigens markedly increased both the frequency as well as T cell receptor (TCR) diversity of neoantigen-reactive TIL, while expanding the range of neoantigens targeted. 44 45 Expansion yielded greater numbers of reactive TIL compared with the standard REP and preserved stem-like characteristics associated with improved antitumor efficacy. 44 Alternative approaches focus on enriching tumor-reactive TIL by leveraging their unique phenotypic features. Phenotypic and transcriptomic profiling of TIL isolated directly from tumors has revealed specific protein and gene signatures, for example, expression of PD-1, CD39, CD103 and TIGIT, associated with tumor reactivity across multiple cancer types. 46 48 Expression of these and other markers enables prospective isolation by sorting and subsequent expansion of CD8+or CD4+ tumor-reactive TIL subsets in an antigen-agnostic fashion. 49 51 Some of these are being investigated in various cancer types ( NCT05902520 , NCT05902520 ).

Although tumors remain the primary source for isolating tumor-reactive lymphocytes, peripheral blood represents a promising alternative. A recent study demonstrated the safety and feasibility of treating patients with advanced, refractory melanoma using peripheral blood-derived neoantigen-reactive T cell products generated through ex vivo antigen-specific stimulation. 52 Similarly, preclinical studies have shown the potential for selection and subsequent expansion of peripheral blood T cells expressing CD39 using immunomagnetic nanoparticles and evolved equipment. 53 Additionally, rare circulating lymphocyte subsets—such as PD-1 hi T cells—can be isolated to expand tumor-reactive and neoantigen-reactive populations from the blood of melanoma, but also from GI cancer patients. 37 54 This approach could enable the treatment of patients with limited and/or unresectable tumors, providing access to a broader and potentially more diverse pool of reactive T cells capable of addressing tumor heterogeneity. Mobilizing tumor-reactive T cells from lymph nodes or other sanctuary sites using brief administration of high-dose IL-2, allowing enhanced capture in the blood and/or tumor itself is suggested as a potential pre-TIL strategy ( table 1 ).

T cell growth factors and expansion of TIL, role of bispecifics, other strategies

The successful clinical application of ACT using TIL is critically dependent on the efficient ex vivo expansion of tumor-reactive T cells. Optimal expansion methods leverage key T cell growth factors, 55 including IL-2, IL-4, 56 57 IL-7, 58 IL-9, 59 IL-15, 60 61 and IL-21, 62 63 to promote proliferation while preserving or enhancing the ‘stemness’ epigenetics of TIL products that are essential for durable antitumor immunity. Among these cytokines, IL-2 has traditionally played a central role for expansion in the clinic. Recently, protocols using regulatable cell membrane-tethered IL-15 18 have shown great promise as an alternative approach. IL-2 supports robust TIL expansion. However, its use is often accompanied by preferential expansion of terminally differentiated effector T cells and regulatory T cells, potentially compromising therapeutic efficacy. Mutation-associated neoantigen reactive-TIL express C-X-C motif ligand 13 (CXCL13), CD39, and low levels of the IL-7 receptor, 64 explaining in part the limited ability of solely using exogenous IL-7 to promote TIL expansion. Use of agonistic antibodies triggering the beta and gamma chain of the IL-2R has been developed as an alternative to the use of cytokines such as IL-2 or IL-15 (Twain Therapeutics).

To overcome these limitations, recent strategies have explored alternative or complementary cytokines including IL-7, IL-15, and IL-21, alone or in combination, to foster the preferential expansion of less differentiated T cell subsets. 65 66 IL-7 and IL-15 enhance the maintenance and proliferation of memory-like and stem-like T cells, which exhibit greater persistence, improved antitumor activity, and reduced susceptibility to exhaustion. IL-21 has demonstrated capacity for sustaining a robust cytolytic and memory phenotype, providing additional functional advantages during ex vivo TIL expansion.

In parallel, bispecific antibodies have emerged as potent tools in the field of ACT, serving multiple critical roles including redirecting and amplifying T cell activity against tumor cells, and simultaneously augmenting T cell activation and proliferation. 67 69 Bispecific antibodies targeting CD28 and tumor-associated antigens can mediate effective bridging between T cells and malignant cells, 70 providing costimulation to tumor-specific TIL, and significantly enhancing the specificity and potency of adoptively transferred TIL. Furthermore, bispecific antibodies targeting other activating receptors on T cells (such as CD137/4-1BB 67 or CD134/OX40 71 ) alongside tumor antigens have been investigated to co-stimulate and expand TIL with improved antitumor efficacy. The use of such bispecific or trispecific antibodies has not been sufficiently explored in the production of TIL, nor as a pre-TIL approach, but this represents a promising future strategy.

Novel engineering strategies have further enriched TIL expansion methodologies. Genetic modifications, such as the overexpression of cytokine receptors (eg, IL-7Rα or IL-15Rα), IL-12 membrane tethered or regulated IL-12, with other engineered cytokines tethered directly to the T cell membrane, 18 have shown promise in optimizing TIL expansion, function, and persistence. Furthermore, pharmacological interventions aimed at epigenetic reprogramming (eg, histone deacetylase (HDAC) inhibitors or BET inhibitors) or serving as a faux partial second signal by antagonizing or eliminating CBL-B 72 during ex vivo culture can stabilize stem-like phenotypes, counter T cell exhaustion, and augment long-term antitumor responses. Recently, the inhibition of PGE 2 in TIL culture enhances growth and limits downregulation of the IL-2 receptor gamma chain. 73 74

Collectively, these innovative approaches—incorporating optimized cytokine supplementation, bispecific antibodies, genetic modification, and epigenetic interventions—provide powerful avenues to enhance the expansion and therapeutic potential of TIL-based ACT. 75 Future clinical applications will likely integrate such strategies to maximize the antitumor activity, durability, and clinical efficacy of adoptively transferred TIL.

High extracellular potassium (K + ), typically present at elevated levels (~40 mM) within the tumor microenvironment (TµE), paradoxically act to preserve T cell stemness, a state characterized by enhanced persistence, multipotency, and antitumor efficacy. This preservation occurs because elevated K + restricts nutrient uptake—specifically glycolysis and amino acid transport—resulting in a ‘functional caloric restriction’. Such metabolic limitation activates autophagy, reduces nuclear acetyl-CoA levels, and consequently lowers histone acetylation at loci associated with T cell differentiation and exhaustion. These epigenetic and metabolic shifts maintain expression of critical stemness markers, including TCF7, CD62L, and CD27, thereby stabilizing a rejuvenated T cell phenotype. Studies in both human TIL and mouse models confirm that T cells conditioned by elevated extracellular K + demonstrate superior persistence and enhanced tumor-clearing capabilities on adoptive transfer. These insights highlight potential therapeutic strategies aimed at modulating extracellular potassium levels or mimicking its beneficial metabolic effects to significantly improve the durability and effectiveness of T cell-based immunotherapies. 76 Modification of TIL culture media with an optimized concentration of potassium epigenetically programmed TIL into a stem-like state, promoting persistence and tumor regression in preclinical models. 76

Clinical regimen(s) for TIL intravenous, intraperitoneal, intratumoral, and intra-arterial administration

The most common route of administration of TIL therapy, in both melanoma and non-melanoma solid tumors including ovarian cancer, 77 80 cervical cancer, 81 renal cell carcinoma, 82 and NSCLC, 83 is by intravenous administration. This route of administration was developed and optimized for patients with hematologic malignancies and melanoma. While it appears to be safe, the efficacy of intravenous-administered TIL therapy in most non-melanoma solid tumors remains limited. Barriers to the effectiveness of systemically delivered adoptive cell therapies (including TIL) likely stem from multiple factors, such as on-target/off-tumor toxicities due to shared antigen expression between solid tumors and healthy tissues, the presence of stromal barriers and poor vascular access, and the influence of an immunosuppressive microenvironment. 84 To overcome these challenges, several groups have begun to examine the safety, feasibility, and effectiveness of regionally administered adoptive cell therapy, including both intralesional and intracavitary (intraperitoneal, intrathecal, intrapleural) routes of administration. For example, intertumoral administration of mouse and human TIL engineered to express IL-12 or IL-18 messenger RNA injected directly into patient-derived xenografts led to complete rejection in both injected and distant tumors, whereas intravenous administration did not. 85 86 Ongoing studies examining intracavitary and intra-arterial delivery of chimeric antigen receptor (CAR) T have not yet been repeated with TIL and are an important area of future investigation.

Identity and purity: repertoire and adaptome analysis

Tracking clonotypes using TCR repertoire assays is pivotal for understanding the mechanisms of response to TIL therapy. Studies on tumor recognition by peripheral blood lymphocytes after TIL infusion provide hints on the ability of tumor-reactive TIL to persist. 36 However, tracking clonotypes by TCR repertoire analysis provides better proof of in vivo engraftment to understand responses to TIL therapy. Detailed analyses allow for precise monitoring of clonotypes in tumors, cell products and blood. Taking advantage of such TCR tracking by sequencing, responding patients receive predominantly tumor-resident clonotypes (ie, bona fide true TIL), while cell products from patients not benefiting from TIL-ACT therapy are mostly composed of blood-borne clonotypes (ie, not bona fide true TIL). 28 Post-ACT monitoring is crucial for tracking the persistence of infused clonotypes in blood and their potential infiltration into tumors following transfer, both critical metrics linked to therapeutic efficacy. 28 87 Therefore, TCR repertoire analyses—when performed with high-resolution depth—provide invaluable insights into treatment outcomes, serving as predictive biomarkers to assess cell product purity and potency (ie, the fraction of true TIL). Such analyses should provide insights into the design of strategies to enhance the precision and effectiveness of TIL therapies in the clinical settings. The adaptome refers to the complete set of expressed T and B cell receptor genes within a given sample. It encompasses seven receptor chains: the alpha/beta and gamma/delta chains for T cells, and the heavy, lambda, and kappa chains for B cells. 88 The use of broad application of massively multiplexed PCR to coordinately measure all seven chains of the ‘adaptome’ allows broad assessment of immunity in the tumor and peripheral blood to capture dynamic changes during therapy. 88 89

Combination of TIL and checkpoint blockade, timing, progression after TIL

Furthermore, in the second line, TIL outperformed anti-cytotoxic T lymphocyte antigen 4 (CTLA4) following PD-1 based therapy resistance in a heralded, randomized clinical trial. 8 The NCI Surgery Branch recently published the negative impact of prior checkpoint blockade (CPB) on TIL therapy efficacy. 90 CPB-naïve patients achieved a very high CR rate and of those, nearly all remained disease-free 10 years later, whereas CRs were distinctly less common if patients had previously received CPB 91 ; the only patients seemingly doing better were those who had received prior IL-2 treatment in this compendium. Similar results were observed in another cohort. 92 Patients traditionally receiving TIL are generally refractory to multiple prior therapies. A recent study presented at the European Society for Medical Oncology (ESMO) showed minimal post-TIL responses to retreatment with agents that failed pre-TIL therapies. 93 This could be due on the one hand to a ‘pruning’ phenomenon, whereby patients with highly immunoreactive tumors—who would benefit from TIL therapy—respond well to CPB and are not included later in the TIL cohorts. Furthermore, resistance to CPB has been associated with selection of immune escape tumor phenotypes. Potential contributory factors of post-TIL failure include TµE alteration, immunosuppressive signaling or evolution of tumor molecular profiles over space and time. 94 This could be associated with defects in the antigen processing and presentation machinery, including HLA-I/II loss of heterozygosity and loss of functional β2-microglobulin, 95 96 or loss of interferon response and immune desertification, leading to immune escape. Such tumors would likely not benefit from conventional TIL. 97 Furthermore, stroma barriers such as extracellular matrix and immunosuppressive factors produced by cancer-associated fibroblasts could contribute to driving poor clinical responses. 98 Finally, post-infusion exhaustion of transferred TIL 28 could lead to therapeutic failures (this point is also addressed later through rationally engineered TIL).

Lack of effective post-TIL therapies remains a critical unmet need ( table 2 ). From this angle, upfront combination of TIL with CPB seems rational. TIL and CPB can be combined without added toxicity. 99 100 In a study ( NCT03645928 ) of treatment-naïve melanoma, TIL+anti-PD-1 yielded a>60% ORR, with over one-third reaching CRs and duration of responses not yet reached. 101 Currently, a phase III trial TILVANCE-301 is studying CPB-naïve patients with melanoma evaluating the combination of TIL+anti-PD-1 compared with anti-PD-1 alone. 102

Potency assays

FDA approval requires demonstration that efficacy in patients is directly attributed to the pharmaceutical agent being proposed, commonly termed ‘potency’. This requirement presents unique and complex challenges for TIL therapy. Since the product is unique to each patient, a uniform assay to assess potency is much different than a conventional manufactured chemical compound with defined mechanism of action or CD19 CAR T cells. The lack of common tumor antigens across patients makes generating a standardized assay difficult. Thus, identification of quantitative tests that measure product attributes that contribute to the intended mechanism of action (T cell activation and cytokine release/target cell cytolysis/degranulation, etc) are essential with manufacturing changes. In 2011 the FDA issued specific guidance on cell and gene therapy potency assays, given the complexity of defining the quality and activity of individual products. The major purpose of the potency assay(s) is thus to define what the product is, such as to be able to assess the impact of a manufacturing change (such as a change in culture vessels or reagents), or when doing comparability assessment while scaling up into new suites or buildings. Ultimately, the potency assay is a matrix of tests that serve to help identify the product.

Currently, the only TIL therapy that is approved (Amtagvi) has multiple potency tests, which are a combination of various phenotypic and cytotoxic assays of which only one is public (total viable cells) 103 ; this paradigm continues to provide the basis for assessing the potency of unselected TIL development. However, new generations of TIL for new tumor types are under intense study. 104 105 Potency assays will continue to adapt to the needs of evolving TIL therapies and will remain part of the many challenges to bring these technologies to the level of patient care, given the substantial heterogeneity of TIL within and among individual products.

Clinical response in TIL ACT: what dictates response?

Problems and promise of mouse models, four requirements for successful immunotherapy

Requirements for successful immunotherapy

There are many critical requirements for successful adoptive T cell immunotherapy. 106 First, the transferred T cells must recognize tumor-specific antigens with sufficient avidity to ensure robust antitumor activity while minimizing damage to healthy tissues. Second, the therapeutic T cells must effectively traffic and infiltrate into the TµE—an area of significant challenge due to stromal barriers and immunosuppressive factors within tumors. Third, once within the tumor milieu, the T cells must interact with tissue resident BatF3+CD103+ dendritic cells (DCs), 107 108 overcome local immunosuppressive mechanisms, which may involve checkpoint inhibitory receptors, suppressive myeloid cells, regulatory T cells, immunosuppressive prostaglandins, or inhibitory cytokines such as TGF-β. Lastly, to achieve lasting therapeutic outcomes, transferred T cells should possess robust proliferative and persistence capabilities, ideally through the maintenance of stem-like memory phenotypes, enabling prolonged antitumor responses and sustained immunosurveillance against tumor recurrence.

Epigenetic reprogramming for T stem cell memory

Epigenetic reprogramming has emerged as a potent approach for enhancing T cell therapies by promoting T cell stemness, longevity, and function. Specifically, T stem cell memory (TSCM) cells, characterized by self-renewal, high proliferative capacity, and multipotency, have superior therapeutic potential compared with more differentiated effector T cell subsets. 106 Epigenetic modifications—such as changes in DNA methylation patterns, histone acetylation, and chromatin accessibility—can profoundly influence T cell differentiation trajectories and maintain stem-like qualities. 109 Recent advances highlight that treatment with small molecule inhibitors targeting epigenetic regulators, such as HDAC inhibitors or DNA methyltransferase inhibitors, can maintain or induce TSCM phenotypes. 110 Furthermore, genetic modulation of transcription factors and metabolic reprogramming strategies also profoundly shape epigenetic landscapes, stabilizing T cells in less differentiated, memory-like states. 111 These epigenetic approaches hold promise not only for improving the efficacy and persistence of adoptive T cell therapies but also for establishing long-lasting antitumor immunity, thus representing an exciting frontier in cancer immunotherapy research.

Intrinsic characteristics of TIL and extrinsic factors within the tumor microenvironment that affect TIL function

Multiple phenotypic and functional characteristics of the TIL product have been associated with clinical efficacy. A higher proportion of infused CD8 + T cells, particularly those with a differentiated effector phenotype (CD8+CD45RA−CD62L−CD27−), has been correlated with objective tumor regression. The expression of BTLA (B-lymphocyte and T-lymphocyte attenuator) on CD8 + T cells within the infusion product is also positively associated with TIL persistence 31 and clinical response. More recently, high-dimensional profiling revealed that TIL products enriched for CD39⁻CD69⁻ stem-like CD8 + T cells—characterized by memory-progenitor properties and the capacity for self-renewal—were predictive of durable clinical responses and long-term persistence post-infusion. Interestingly, a terminally differentiated CD39 + CD69 + phenotype was associated with poor outcomes, although others expand these cells with suitable responses (and thus is controversial). 38 The presence of CD8 + T cells targeting patient-specific neoepitopes within the TIL infusion product correlated with improved survival outcomes. Notably, these neoantigen-reactive T cells were virtually absent in patients who did not respond to therapy. 34 While most neoantigen-specific T cells are found in the differentiated CD39 + subset, responders uniquely retained a pool of stem-like CD39 − neoantigen-reactive T cells, indicating that antitumor efficacy depends not only on specificity but also on T cell differentiation state. 38

Chiffelle et al explored the clonal and phenotypic features of tumor-reactive T cells in patients with melanoma undergoing adoptive TIL therapy. 28 Using single-cell RNA sequencing and TCR tracking, the authors demonstrated that clinical responses were strongly associated with the presence of tumor-resident, tumor-reactive clonotypes in baseline tumor samples. In responders, these reactive clonotypes were successfully expanded ex vivo, resulting in TIL infusion products enriched in CD8 + tumor-specific cells. Post-infusion, these cells preferentially homed to tumors, contributing to durable clinical responses. In contrast, non-responders had infusion products dominated by peripheral blood-derived clonotypes that showed poor tumor homing and persistence, correlating with limited clinical benefit. Importantly, a substantial fraction of inferred tumor-specific clonotypes was lost during in vitro expansion. Moreover, IL-2 expansion led to partial loss of transcriptional programs that tumor-specific cells exhibited in situ, including exhaustion markers. However, following engraftment in tumors, effective TIL re-expressed a partially exhausted effector phenotype, indicative of functional reactivation. 28 Profiling tumor-reactive TCR clonotypes and their phenotypic states in baseline tumors and infusion products may help optimize patient selection and product manufacturing strategies.

Importantly, responding patients generate superior products because their tumors harbor at baseline a richer repertoire of TIL, which exhibit elevated cytotoxicity, co-stimulation, and exhaustion markers, while myeloid cells in the same tumors show enhanced type I interferon responses. Indeed, spatial and transcriptomic analyses revealed that responders had pre-existing tumor-reactive T cells and activated myeloid cell networks within tumor and stromal compartments. 112 This was associated, and likely due to higher tumor-intrinsic immunogenicity and TMB at baseline. Remarkably, TIL ACT further amplified these T cell–myeloid cell interactions and reprogrammed the myeloid compartment toward a more immunostimulatory state in responders.

Microbiome and TIL

Recent advances have highlighted the critical role of the microbiome in shaping cancer immunotherapy responses, demonstrating that bacteria within both the gut and the tumor can influence the composition and function of intratumoral immune cells. Foundational insights came from preclinical murine models, where alterations in gut bacterial communities were shown to modulate the tumor immune microenvironment and affect responses to immune checkpoint inhibitors. 113 114 These findings were subsequently supported by clinical studies comparing profiles of gut microbiota between responders and non-responders to immunotherapy, which revealed that patients with a favorable gut microbiome exhibited enhanced systemic and antitumor immunity. 115 Ongoing studies continue to dissect the molecular mechanisms by which the microbiota shape immune responses, and several clinical trials are underway to investigate the therapeutic potential of modulating the gut microbiome in patients with cancer, including fecal microbial transfer and diet modification. By harnessing the immune-modulatory effects of the gut microbiome, it may be possible to shift both systemic and intratumoral immunity toward a more favorable state, thereby enhancing the efficacy of TIL and other immunotherapies.

In addition to gut bacteria, intratumoral bacteria have emerged as critical components of the TµE, capable of directly modulating local immune responses. 116 However, this area remains relatively underexplored, and the impact of intratumoral bacteria is likely to be highly dependent on the tumor site and its natural relationship to microbial exposure (eg, mucosal vs non-mucosal tumors). In certain contexts, intratumoral bacteria may exert immunostimulatory effects, whereas in others, they may promote immunosuppressive responses. The specific mechanisms underlying these associations, as well as the potential to modulate intratumoral bacteria to enhance immunotherapy, remain to be elucidated.

Specific solid tumors and TIL: experience and special considerations

Lung cancer

TIL therapy is also emerging as a promising approach for patients with advanced NSCLC. A pioneering phase 1 trial led by Creelan et al combined TIL therapy with nivolumab in patients with metastatic NSCLC who were treated with only nivolumab (but no chemotherapy) in first line, reporting an ORR of 25%, including two CRs. 117 Moreover, in the phase 2 IOV-COM-202 trial, lifileucel (LN-145) monotherapy demonstrated an ORR of 21.4%, including one CR, in heavily pretreated patients with NSCLC, many of whom had PD-L1-negative or STK11-mutant tumors. 118 Furthermore, the IOV-COM-202 trial explores the combination of lifileucel with pembrolizumab in ICI-naïve patients with NSCLC. Early findings from this cohort show an ORR of 54.5% in epidermal growth factor receptor (EGFR) wild-type, programmed death ligand 1 (PDL-1)-negative patients, highlighting the potential of TIL therapy in the frontline setting. 119 Both ‘immune evading’ and ‘non-evading’ organoids derived from three patients with NSCLC could be identified from the same patient, creating concerns about immune escape from TIL therapies. 120

Head and neck cancer

Head and neck cancer is the seventh most common cancer worldwide. 121 Most head and neck cancers arise from the squamous cells that line the mucosal surface of the upper aerodigestive tract. The two main etiologies of head and neck squamous cell carcinomas (HNSCCs) are tobacco abuse and human papillomavirus (HPV) infection. HPV type 16 is the causative agent in up to 80% of HPV-mediated HNSCCs. 122 Based on the limited number of foreign, viral antigens the cancer cells express, HPV-mediated HNSCCs are highly amenable to adoptive cell therapy. A first-in-human, phase 1 clinical trial tested T cells engineered with a TCR that recognizes the HPV16 E7 11-19 peptide in the context of HLA-A*0201 expression for the treatment of metastatic HPV-mediated cancers ( NCT02858310 ). 67 patients with recurrent and/or metastatic HPV-mediated cancers were screened for enrollment, and 12 patients were enrolled and treated in the trial. The administered cell dosing ranged between 1×10 9 to 1.2×10 11 engineered T cells. Robust tumor regression was observed with objective partial clinical responses, as determined by response evaluation criteria in solid tumors (RECIST), in 6 of 12 patients, including 4 of 8 patients with anti-PD1 refractory disease, and the duration of response ranged between 3 and 9 months. 123 In addition, significant efforts are being made to identify neoantigen-specific T cells for adoptive cell therapy in non-viral mediated HNSCCs. Some of the strategies to enhance the potency of TIL therapy in HNSCCs have been to employ single-cell sorting of patient-specific neoepitope-reactive TIL to enrich the percentage of tumor-reactive CD8+and CD4+ T cells for autologous TIL infusion ( NCT06236425 ).

Cervical cancer

Cervical cancer remains a major therapeutic challenge in the recurrent or metastatic setting, particularly as it affects a relatively young patient population, is associated with HPV infection in most patients and offers limited treatment options beyond first-line chemotherapy. Adoptive cell therapy with TIL has emerged as a promising approach. In a pioneering Phase II NCI study, Stevanović et al demonstrated an ORR of 28% in patients with metastatic cervical cancer treated with autologous TIL, including two durable CRs lasting over 4 years. Clinical benefit was associated with HPV-specific T cell reactivity in the infused product. 81 These findings were corroborated by the multicenter Phase II C-145–04 trial evaluating LN-145 (Iovance Biotherapeutics), which reported an ORR of 44% and acceptable safety in patients with heavily pretreated disease. 124 Infusion of autologous T cells engineered with TCR recognizing the HPV E7 antigen 123 has been associated with durable responses as well. Interestingly, the major clonotypes in TIL did not recognize the HPV E6 or E7 but rather neoantigens unique to the individual tumor. 81

Breast cancer

The presence of TIL in breast cancer specimens has been shown to correlate with prognosis, particularly in triple-negative breast cancer. Groups with experience in melanoma TIL began clinical development of breast cancer TIL. Harao et al successfully grew CD8+TIL from primary breast cancers resected after neoadjuvant chemotherapy by adding urelumab (anti-CD137 mAb) to standard culture conditions. 125 At the Surgery Branch, NCI, TIL were derived from breast cancer metastases and assessed and selected for recognition of non-synonymous somatic mutations. In fact, in 67% (28 of 42) of tumor-derived TIL recognized at least one neoantigen, and robust reactivity was documented in 13 patients who were deemed appropriate for adoptive transfer. Six women were finally treated with TIL products in an ongoing study. 42 Of the three responding patients, one developed a CR to therapy and was alive without disease 9 years following treatment. 126

Ovarian cancer

Conventional TIL use was first reported in advanced ovarian cancer by a Japanese team in 1991. Out of seven patients who received TIL following a single dose of intravenous cyclophosphamide—either as adjuvant first-line therapy or after relapse—experienced objective responses, including one CR. A different cohort of ten patients was treated with TIL in combination with cisplatin-based chemotherapy: nine showed objective responses, with seven achieving CR. In an intriguing follow-up study, 78 patients who achieved remission following primary debulking surgery and standard platinum-based chemotherapy were treated with TIL without lymphodepletion or IL-2 support, while a control group received no further therapy. After three years, the TIL-treated group demonstrated disease-free survival of 82% compared with 55% in the observation group, and overall survival of 100% versus 68%. However, due to the non-randomized design, these results should be interpreted with caution. More recent studies in the platinum-refractory setting were less encouraging. 80 In an interesting Phase I/II trial ( NCT04072263 ), TIL therapy was administered to patients with recurrent platinum-sensitive epithelial ovarian cancer at low doses with or without interferon (IFN)-α during platinum-based chemotherapy, without high-dose lymphodepleting chemotherapy or IL-2 support. Although IFN-α added toxicity, among 14 evaluable patients, the ORR was 86%, including three CRs. The contribution of TIL could not be discerned in this study. 127 Moreover, in a Phase I trial ( NCT05468307 ), 18 patients with recurrent ovarian cancer received engineered TIL expressing membrane-bound IL-7 following lymphodepletion, alongside anti-PD1 antibody sintilimab. The interim study results reported an ORR of 33.3%, including two CRs. Median progression-free survival was 5.5 months. Adverse events were mostly mild; only one grade 3 event was reported. 128

Liver metastases

The constant exposure of the liver to gut-derived antigens is considered central to its immune tolerance functions. This tolerance effect was evidenced in early transplantation of MHC mismatched miniature pig livers not being rejected. 129 Antigen-specific T cell apoptosis observed in normal liver, which can be mediated by Kupffer cells/hepatic macrophages, 130 131 has even led some to coin this organ as a T cell graveyard or killing field. 132 Low response rates to first-generation immune checkpoint inhibitors in patients with melanoma, lung and colorectal cancer bearing liver metastases may result from liver tolerance. 133 134 Patients with melanoma with liver metastases appear to draw a larger clinical benefit from the ipilimumab/nivolumab combination versus nivolumab alone in melanoma, perhaps because of the more suppressive TµE. 135 Alternative checkpoints other than PD-(L)1 and CTLA-4 could be more relevant to target in the liver, for instance, those related to extracellular adenosine. 136 Indeed, as for most other primary cancers, high density of TIL is more strongly associated with recurrence-free and overall survival than any other clinical, pathological or oncogenomic parameters in resected colorectal liver metastases. 137 Furthermore, tumor-reactive and neoantigen-reactive TIL can readily be found in liver metastases from melanoma and GI cancers. 21 138 In metastatic melanoma, TIL harvested from liver metastases are as likely as other harvesting sites to lead to successful manufacturing 139 and clinical response. 8 90 TIL therapy can also clear liver metastases, as demonstrated in many cancer types. 22 42 43 90 Surgical considerations/location of resection, 140 but there were only two liver mets. Interestingly, the first patient to respond to high-dose IL-2 administration was a patient with colorectal cancer metastases to the liver; microsatellite instability (MSI) status was not performed at that time (reported by MTL). Furthermore, the usual biology of uveal melanoma is to metastasize to the liver, and clearly, responses have been observed in this low TMB tumor to liver-derived TIL. 141 142

Overall, liver metastasis should be considered appropriate for TIL harvest and therapy. More research is needed to better understand how to modulate the T cell suppressive microenvironment to guide rational therapeutic combinations.

Hepatobiliary and pancreatic cancers

Hepatocellular carcinoma (HCC) arises from hepatocytes, generally in fibrotic or cirrhotic livers. The second most common primary liver malignancy is intrahepatic cholangiocarcinoma, which arises from bile duct epithelial cells, more often in normal liver, although cirrhosis is a risk factor. In contrast with liver metastases, approximately 25% of both HCC and cholangiocarcinoma are responsive to PD-L1 blockade combined with other drugs, supporting the notion that primary liver cancers can harbor a significant endogenous T cell response, and that PD-L1 is biologically relevant to the peculiar inflammatory microenvironment of the cirrhotic liver. 143

TIL therapy for HCC has been studied in a single feasibility study in the adjuvant setting, after radical resection, without lymphodepletion or aldesleukin (IL-2). 144 In later-stage HCC associated with cirrhosis, the risk of liver failure makes most patients ineligible for TIL immunotherapy, and chronic viral hepatitis infection contraindicates lymphodepletion. Of note, the liver plays a minimal role in clearing aldesleukin (IL-2) compared with the kidneys. 145

For metastatic cholangiocarcinoma, six reported patients have received TIL enriched for neoantigen reactivity (selected TIL) after lymphodepletion, with IL-2 and with or without anti-PD-1 combination. Of these, two patients presented objective responses, one still alive more than 10 years after treatment. 22 43 In this patient population, bile duct stenting to relieve jaundice leads to bile bacterial colonization and, when dysfunctional, cholangitis and liver abscess. Lymphodepletion cannot be administered with active infection, and TIL cultures may be at higher risk of bacterial contamination when initiated from liver metastases of stented bile ducts, thus limiting candidates for this approach.

In pancreaticobiliary cancers (PC), increased tertiary lymphoid structures correlate with improved overall survival, while higher TIL levels are associated with improved response to immunotherapy. However, the immunosuppressive TµE and stromal barriers of PC limit TIL presence, making it challenging to isolate sufficient cells for ACT. 146 147 While CD8+cells dominate antitumor response, extracted TIL are predominantly activated CD4+T cells. Expanded TIL often express both activation (CD69+, CD28+) and exhaustion (PD-1+, LAG-3+) markers, with neoantigen-specific clones mediating durable responses. 22 Strategies such as PD-1 blockade, 4-1BB stimulation, CD3 agonism, and CD8+enrichment can enhance TIL yield. 148 149

A recent phase 2 trial demonstrated objective responses in mismatch repair-proficient metastatic pancreatic cancer and cholangiocarcinoma using neoantigen-enriched TIL with lymphodepleting chemotherapy, IL-2, and pembrolizumab. 22 This study marked the first evidence of TIL efficacy in common GI cancers. Although overcoming the immunosuppressive microenvironment, stromal barriers, and challenges in standardizing ex vivo TIL expansion remain critical hurdles, neoantigen-specific TCR gene therapy represents a promising frontier for the treatment of pancreaticobiliary cancers.

Colorectal and other metastatic GI cancers

In GI malignancies, CD8+and CD3+ TIL play a central role in mediating antitumor responses. 150 High TIL density is associated with improved overall survival (HR=0.525, 95% CI 0.447 to 0.616), recurrence-free survival (HR=0.546, 95% CI 0.461 to 0.647), and disease-free survival 151 in GI cancers, while forkhead box P3 (FOXP3)+ T regulatory cells (Tregs) are associated with worse overall survival (HR=1.75, 95% CI 1.26 to 2.42). 152 In colorectal cancer, plasmacytoid DCs colocalize with T cells within tertiary lymphoid structures, enhancing antitumor immunity and correlating with improved overall survival. 153 A subset of colorectal cancers with defective mismatch repair (MMR), including those arising in individuals affected by hereditary Lynch syndrome, displays MSI and a highly immune-infiltrative TµE. Patients with deficient MMR or high MSI (MSI-H) show high response rates to PD-1 inhibitors, while those with proficient MMR (pMMR) or microsatellite stable tumors generally have poor responses. 154 By analogy to similar tumors with high neoantigen load, patients with MSI-H tumors failing CPB therapy would be reasonable candidates for TIL therapy, although the question has not been addressed clinically. A recent paper did report a durable complete responder with MSI-H colorectal cancer to CISH-ablated TIL following failure of CPB. 155

While unselected TIL therapy has shown limited efficacy in solid GI tumors, selected TIL has demonstrated greater promise. A phase 2 trial combining selected TIL with pre-infusion and post-infusion pembrolizumab and limited application of high-dose IL-2 in GI cancers (n=91) achieved a 23.5% ORR, compared with 7.7% with TIL alone (non-randomized), with responses lasting up to 5.8 years. 22 As previously noted, responding patients’ products exhibited recognition of a higher number of targeted neoantigens and contained a higher number of administered CD4 + neoantigen-reactive TIL. Pembrolizumab was administered prior to TIL-ACT to prevent TIL inactivation by the immunosuppressive TµE, while post-infusion IL-2 supported TIL proliferation. These results could be especially promising for pMMR or mismatch stable (MMS) patients, who have been historically resistant to immunotherapy. Standardizing TIL manufacturing, managing nonmyeloablative conditioning and IL-2 toxicity, and overcoming TµE suppression are still undergoing further optimization. These recent trials underscore the potential of TIL therapy in GI cancers, particularly when combined with ICB. 119

Sarcoma

The term ‘sarcoma’ refers to a heterogeneous group of tumors, derived from tissues of mesodermal origin and consequently found within mesenchymal tissues throughout the body. These rare tumors fall within two major diagnostic groups: bone sarcomas and soft tissue sarcomas. Despite the presence of>70 different histologic subtypes, all sarcomas have a very low TMB relative to other solid tumors. 156 157 Given the relationship between TMB and immunogenicity, this has historically excluded sarcoma from consideration of tumors where immunotherapy may have a role in advanced disease.

Recently published data suggested a reversal of this nihilism. In 2020, Petitprez et al described the Sarcoma Immune Classes (SIC) in a seminal article which clearly defined a subset of sarcomas with an enhanced immune infiltration. 158 Specifically, the SIC-E group exhibits increased immune infiltration and is associated with a positive response to immunotherapy. The therapeutic potential of this increased immune infiltration has now been shown with the successful expansion of TIL from a variety of soft tissue sarcoma subtypes in both adults 159 and children. 160 These methods to expand TIL from sarcoma have been tested in adults ( NCT04052334 , NCT03725605 ) and will be tested in the pediatric population as well ( NCT06566092 ). In fact, a total of four clinical trials ( NCT06474676 , NCT03935893 , NCT05607095 , NCT06566092 ) are now enrolling sarcoma patients to test the safety and/or efficacy of modified and unmodified TIL products. This indicates that the successful experience with TIL therapy in other solid tumors may produce meaningful opportunities for patients with advanced sarcoma. Despite the feasibility of the approach, clear evidence of efficacy has yet to be demonstrated. Future efforts are directed at optimizing the expanded product, noting that the lack of a substantially immune-edited tumor may paradoxically allow for expansion and efficacy of TIL transfer and CD4-positive T cells for autologous TIL infusion ( NCT06236425 ). Radiation therapy, a standard adjunct in the treatment of soft tissue sarcoma, has been proposed to enhance efficacy of T cell therapy. However, an elegant, recently reported trial of the impact of stereotactic body radiotherapy (SBRT) preoperatively in HPV-negative tumors demonstrated preferential eradication of tumor-reactive TIL and repopulation with Tregs and non-tumor-reactive TIL, providing caution for this approach. 161

Future of TIL therapies

Below we present emerging opportunities to improve the efficacy of TIL therapy. Interventions are broadly aimed at improving the potency of the TIL product, and at creating therapeutic combinations applied together or following the adoptive transfer, to maximize the ability of transferred TIL to eradicate tumors.

Improving the potency of the product

Since the potency of the product depends on the frequency and the absolute number of tumor-specific T cells as well as the quality of the cells at the end of expansion, therapeutic interventions aimed at enhancing the tumor density of tumor-specific T cells, when administered for some time prior to TIL harvest, could in theory improve the TIL product potency. Furthermore, specific manipulations have been introduced during TIL expansion with the purpose of producing cells of better quality. These are referred to collectively as ‘pre-TIL’ strategies here.

Pre-TIL strategies using approved agents

Substantial variation exists within individual tumor metastases in both the genetic makeup of the tumor as well as the infiltrating TIL, over ‘space and time’. 94 Thus, treating all sites of disseminated tumor with T cells derived from a single site, theoretically and operationally, are issues of profound importance. Overcoming suppressive factors that limit T cell homing or proliferation in tumors could prove important as a pre-TIL conditioning strategy. Recruiting new T cells to the tumor from regional lymph nodes, other tissues, or the peripheral blood may also enable acquisition of better T cells, more capable of expansion and/or antitumor activity. In considering the literature, one must distinguish the possible effects of a short-term preconditioning therapy, given for a few weeks specifically to improve TIL harvest or bridge the patient, from the effects of prior therapies to which candidate patients have failed to respond. In review of therapies preceding TIL therapy for melanoma, patients who had received prior anti-PD-1 blockade therapy or B rapidly accelerated fibrosarcoma (BRAF) inhibitors exhibited worse clinical response and survival following TIL-ACT relative to historic patients who had not received these treatments. 90 In addition, pretreatment with CTLA-4 blockade enhanced the generation of TIL in the setting of ovarian cancer, but these were not tested in patients. 162 In other studies, the use of a short pretreatment with a BRAF inhibitor did not enhance the quality or efficacy of subsequent TIL harvest and administration, 163 nor did administration of prior blockade of CTLA-4 30 in patients with melanoma. The only prior treatment noted to be associated with improved response was prior administration of IL-2. Although recruitment of new T cells into tissues and tumors is well known following IL-2, these studies were done more than 30 years previously and would need to be tested anew. Other strategies that one could consider include the use of herpes oncolytic viruses, such as that approved in melanoma therapy 164 or anti-angiogenesis therapy to enhance T cell homing in T cell-excluded tumors. 165 Although preoperative radiation might be appealing, an elegant, recently reported trial of the impact of SBRT preoperatively in HPV-negative tumors demonstrated preferential eradication of tumor-reactive TIL and repopulation with Tregs and non-tumor-reactive TIL, providing caution for this approach 161 . However, low-dose radiation could favor the infiltration of CD8 T cells over Treg cells 166 and could be explored in this setting ( table 1 ).

Pre-TIL strategies using novel agents, panKRASi, bispecifics

There are several agents, not yet approved for clinical use, that may enhance the quality or the number of TIL within the tumor. A highly effective immune adjuvant in murine models and possibly in humans is the Toll-like receptor 9 (TLR9) agonists, specifically hypomethylated cytidine phosphate guanosine (CpG) oligodinucleotides. 167 Recently, intratumoral administration of vidutolimod, a virus-like particle containing a CpG-A TLR9 agonist along with CPB, has been shown to reverse resistance to PD-1 blockade. The agent triggered a strong IFN response and induced expression of T cell-attracting chemokines such as CXCL10, resulting in increased antitumor T cells in previously cold tumors. 168 Alternative promising strategies are systemic administration of any of several bispecific antibodies that promote antitumor effector function of resident T cells using anti-CD3 coupled with antibodies to cell surface tumor antigens. 169 Perhaps the most promising among these agents are those, rather than targeting CD3 alone, are those which target the second signal, CD28. 70 Combinations of bispecifics targeting both CD3 and CD28 appear to have promise in murine models 170 and may also be potentially useful when applied in TIL growth in vitro. Small molecule drugs targeting driver mutations associated with immunosuppression such as Wnt/beta-catenin, 171 p38 kinase, 172 and most recently pan Kirsten rat sarcoma virus inhibitor (KRAS), 173 which had previously been identified in genetic models driven by KRAS. 174 Remarkably, this notion of ‘oncogenes’ may need to be revised in the current era, appreciative of the role of the host response to cancer, to include their role in mediating immunosuppression, and thus these could be rather named ‘immuno-oncogenes’ ( table 1 ).

Role of small molecule inhibitors during TIL expansion

Novel and FDA-approved small molecule inhibitors of T cell signaling pathways limiting activation or exhaustion hold promise in their precision and simplicity in safely modulating the phenotype and function of TIL during ex vivo manufacturing. The E3 ubiquitin ligase, CBL-B, is a negative regulator of T cell activation that promotes antigen-specific tolerance in the absence of co-stimulation. 175 Small molecule inhibition of CBL-B increases central memory and CD39− CD69− ‘stem-like’ TIL expansion, decreases co-expression of inhibitory receptors PD-1, LAG-3, and T cell immunoglobulin and mucin-domain containing-3 (TIM-3) in T cells, decreases Treg and TGF-β mediated suppression, increases TCR diversity, and enhanced efficacy and persistence in preclinical models. 176 179

Several inhibitors of various kinases in the TCR signaling and T cell proliferation pathways have also been shown to improve the memory phenotype and in vivo preclinical ACT efficacy when administered during the ex vivo expansion of antigen-specific or genetically modified T cells. In CAR T cells displaying high levels of tonic signaling, inhibition of lymphocyte-specific protein tyrosine kinase (Lck) with ex vivo dasatinib has been shown to alter the differentiation trajectory of T cells while maintaining the original TCR clonotypic diversity. 180 This functional reinvigoration of a T cell memory phenotype, with reduced exhaustion, and enhanced in vitro and in vivo tumor cytotoxicity is superior to control CAR T cells. 180 Inhibition of phosphatidylinositol 3 (PI3) kinase (PI3K) with ex vivo idelalisib in gp100-specific CD8 T cells increased the number of infiltrating central memory T cells and survival in a B16F10 model of melanoma. 181 182 In a phase 1 clinical trial of B cell maturation antigen (BCMA) targeted CAR T cells in patients with multiple myeloma, addition of a PI3K inhibitor to the manufacturing process resulted in comparable response rates and improved the median duration of response (17 vs 11 months) compared with control BCMA targeted CAR T cells. 183 Additionally, ex vivo administration of the CDK4/6 inhibitor palbociclib on Ley-specific CAR T cells demonstrated a dose-dependent increase in central memory phenotype and improved in vivo tumor control in an ovarian cancer 3 (OVCAR-3) model of ovarian cancer. 184 These remain to be tested in human clinical trials of TIL.

Repealing or replacing the REP-adverse consequences of the REP

Both the application of IL-2 as well as the rapid expansion protocol 185 are known to promote terminal differentiation of T cells during culture. Means to overcome this using lower concentrations of IL-2, other T cell growth factors, and abrogation of the REP step have been goals for many pursuing improvements in TIL expansion. CD137 is a convenient marker to identify tumor-reactive T cells in TIL cultures. 186 Thus, the use of agonistic antibodies to CD137 could promote the outgrowth of tumor-specific T cells. 187

Tumor-reactive TIL stemming from the tumor are reinvigorated on expansion in culture. 28 However, they migrate back to the same suppressive TµE which restricts their function. Various strategies have been developed to overcome this difficulty, including genetic modification to either confer new functionality or ablate intrinsic suppressor functions. In fact, the first genetically modified cells administered to humans were gene-marked TIL, designed to enable distinguishing them from other endogenous T cells. 188 Retroviral delivery of a gene conferring resistance to an antibiotic, neomycin, allowed selection and enrichment in vitro. This enabled detection of transferred cells in the host several months following transfer using sensitive PCR techniques to identify the gene-marked TIL. 12 14 More recently, efforts were directed towards developing TIL engineered with cell membrane expressed IL-15, regulatable by the small molecule carbonic anhydrase inhibitor acetazolamide, to enhance TIL expansion and functionality and overcome the need to administer exogenous IL-2. This allowed demonstrable effectiveness and ability to deliver enhanced TIL for tumor eradication, with controlled expansion of cells mediated by expression of the transgene induced by acetazolamide. 189 Early consideration of TNF delivery or modifying cells to express a CAR T construct was also considered. 190 Furthermore, murine ovalbumin reactive T cells 1 (OT1) modified with LIGHT ("homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes") were tested. In the cluster of differentiation terminology it is classified as CD258 or tumor necrosis factor superfamily member 14 (TNFSF14). It normalized tumor blood vessels and drove the formation of intratumoral lymphoid structures in syngeneic tumor models, while CAR-T cells armored with LIGHT resulted in increased efficacy in preclinical models in immunodeficient mice. 191

Eliminating intrinsic suppressors of T cell functionality has been a goal of T cell engineering over the past decade. Cytokine-inducible SH2-containing protein (Cish) was first identified as a candidate for genetic deletion. A member of the suppressor of cytokine signaling (SOCS) family, Cish is upregulated in CD8+T cells following TCR stimulation and acts to suppress their functional avidity. Its deletion led to enhanced expansion and function of adoptively transferred CD8+T cells in preclinical tumor models. 192 Recently, 12 patients have been treated with Cish-ablated T cells, with a single MSI-hi but ICB-resistant colorectal cancer patient enjoying a sustained CR. 155 Similar use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens 193 in mice enabled identification of several genes, whose ablation could enhance expansion of T cells within murine tumors. These included SOCS1, Regnase 1, and CBL-B, allowing consideration of moving into clinical trials. SOCS1 and Regnase1 have been initiated in patients receiving TIL and are being considered in even more imaginative means with in vivo CRISPR screens targeting multiple genes in patients in either CAR T or TIL. 194 Application of CRISPR technologies to TIL has proven to be feasible, 195 allowing initiation of ablation of PD-1 in TIL ( NCT06783270 ).

Post-TIL strategies

Modifications to the TIL regimen with development of new combinations, rather than changes to the TIL product per se, may also enhance clinical efficacy of TIL-ACT. At the end of REP culture, the TIL products in patients with melanoma appear to exhibit no phenotypic exhaustion based on single-cell RNA sequencing analysis. However, the same cells acquire an intermediate exhausted phenotype in the TµE following infusion, based on 30-day biopsy analyses. 28 Intermediate exhausted cells are likely to be reinvigorated by PD-1 blockade in vivo , 196 creating a solid rational basis for combining PD-1 blockade with TIL-ACT. In a recent study of patients with GI cancer, most of the TIL product upregulated PD-1 on the cell surface at the time of infusion. When a short course of pembrolizumab was added to the TIL regimen, more patients experienced an objective decrease in target tumors. 22 To a lesser extent, upregulation of PD-1 was seen in TIL derived from lung cancer and the regimen included post-treatment nivolumab. 117 Analysis of products has demonstrated upregulation of other immune checkpoints that could be explored as adjuncts to the TIL regimen ( table 2 ).

The role of IL-2 in the regimen has also been investigated, with evidence that durable responses are more likely to be achieved with high-dose IL-2 than lower dose regimens based on a meta-analysis of TIL studies. 4 The structure of the IL-2 receptor suggested that lower doses were more likely to engage the trimeric form found on Treg cells. Alternative cytokines (eg, IL-12, IL-15) had unacceptable toxicity when administered systemically. 197 However, alternative delivery systems, including genetic modification of TIL, remain in development.

Combination of vaccines and TIL

Strong preclinical evidence from mouse models 198 199 underscores the potential benefit of combining therapeutic cancer vaccines with adoptively transferred T cells. These studies have demonstrated that tumor-specific vaccines, particularly those encoding neoantigens, significantly enhance the proliferation, trafficking, and effector function of infused T cells. Vaccination not only expands antigen-specific T cells but also facilitates their infiltration into tumors and augments their antitumor activity through improved cytokine production and cytotoxicity. Furthermore, vaccines can help counteract the immunosuppressive TµE, transforming it into a more permissive milieu for TIL-mediated responses. Clinically, this combined strategy holds considerable promise for amplifying the effectiveness of TIL therapy by promoting durable immune responses, overcoming T cell exhaustion, and potentially preventing tumor recurrence. Clinical validation of the concept comes from two recent studies, one that used vaccine-primed peripheral blood T cells, and the other T cells transduced with a CAR. The first study showed that vaccination with autologous DCs pulsed with oxidized autologous tumor lysate, following adoptive transfer of ex vivo -expanded autologous peripheral blood T cells previously primed with the same vaccine, amplified neoantigen recognition in ovarian cancer patients. Interestingly, post-ACT vaccination was conducted in the context of a seemingly improved host immune milieu, with an increase in the CD8+:CD4+ and CD8+:Treg cells in peripheral blood, attributed to the lymphodepletion by cyclophosphamide and fludarabine. The second study tested CAR T cells targeting claudin 6 (CLDN6), an oncofetal antigen in solid tumors with or without a boosting CLDN6 RNA vaccine. A trend for greater expansion of CAR T cells was observed in the blood of patients receiving low-dose CAR T cells and vaccine 4 days post-ACT, compared with patients receiving CLDN6 CAR T cells alone. Furthermore, a transient increase in circulating CAR T cell counts was observed following administration of vaccine 23 or more days following transfer of the higher dose of CAR Ts 200 ( table 2 ).

Compelling evidence from preclinical and clinical studies showed that in vivo expansion of adoptively transferred CD8+T cells is a critical determinant of antitumor efficacy of ACT. 106 This can be mediated by induction and direct stimulation of defined molecules expressed on DCs (eg, CD40 and TLR) with tumor antigens 201 or vaccination with antigen-loaded DCs. A recent study showed that tumor lysate-pulsed DCs not only induced a neoantigen-reactive immune response in vivo but also generated neoantigen-reactive T cells in vitro. 202 These findings suggest that DC-based vaccination can be used to re-stimulate adoptively transferred CD8 T cells in vivo as well as to generate polyclonal CD8 T cells specific to diverse neoantigens expressed in the heterogeneous tumors in vitro.

Timing of TIL

TIL therapy has been tested mainly as a later-line therapy. There are several reasons to believe that administering TIL in earlier settings can yield higher overall and CRRs. Late-stage cancers are expected to present less tumor antigens, to bear a more immunosuppressive microenvironment and as a result, to be infiltrated by less tumor-reactive TIL. This would be the result of evolutionary cancer immunoediting and evasion, 203 characterized by the elimination of the most immunogenic cancer cells first and the persistence of those capable of immune escape over time, a phenomenon supported in findings in human cancers. 94 Progression despite immunogenic cell death-inducing chemotherapy 204 and immune checkpoints is also likely associated with poor remaining endogenous antitumor T cell response. Indeed, patients who exhibit resistance to PD-1 blockade therapy harbor tumors with increased loss of class I restricted antigen presentation, 205 206 which is essential in tumor recognition by CD8 TIL. Thus, harvesting TIL and treating patients prior to treatment resistance, after some forms of immune induction along the pre-TIL approach ( table 1 ), is an area that deserves further investigation.

The greater efficacy of TIL in treatment-naïve patients with melanoma or as an early line of treatment is supported by sequential trials. Before anti-PD-1 became a central component of front-line therapy, the objective response to TIL in anti-PD-1 naïve patients was as high as 56%, with up to 25% complete and durable responses to be expected. 90 TIL as a second line after anti-PD-(L)1 may still be significant, as supported by a randomized trial and a meta-analysis, 5 8 but the objective response after exposure to both anti-CTLA-4 and anti-PD-1 has been lower in most studies, ranging between 22% and 46%, with single-digit CRR in most studies, 30 90 207 except in one where the confirmed response rate was 15%. 112 Importantly, as first-line treatment, a pilot phase 2 study of TIL combined with anti-PD-1 in 23 patients was associated with an objective response of 65.2%, with 30.4% CR rate, most of which are ongoing at a median follow-up of 21.7 months. 101 Furthermore, in lung cancer, harvesting TIL upfront before initiating first-line anti-PD-1 monotherapy and administering TIL after progression yielded 2 out of 13 CRs and four additional objective responses. 117

TIL-ACT has also been studied in the adjuvant setting; however, without lymphodepletion and in an earlier period, 20–30 years ago. TIL generated from metastatic lymph nodes and administered with IL-2 did not show convincing benefits in patients with stage III metastatic melanoma. 208 209 In a randomized trial, adjuvant radiotherapy and TIL with low-dose IL-2 was superior to radiotherapy with vinblastine and cisplatin in patients with lung cancer, especially in Stage IIIb, where the overall survival was doubled. 210 Small phase I trials of adjuvant TIL after complete resection of colorectal liver metastases and HCC have also shown feasibility of TIL manufacturing. 144 211

Overall, the science of immunoediting and clinical trial results support that TIL could be more efficacious when harvested and administered in early lines of treatment. There are no reasons to think that skewing a patient’s T cell repertoire toward antitumor immunity could be deleterious for later lines of treatment, but rather the contrary. Innovative trial designs allowing tumor harvest as early as possible in the treatment scheme, not delaying systemic treatments to control disease progression, and aiming to administer TIL prior to resistance to multiple lines of treatments represent a more promising future approach than TIL studied as it currently is as a later line therapy.

Biobanking tumors for timely TIL therapy

One of the great challenges of TIL as a potential therapy for late-stage refractory cancers is that disease progression outpaces TIL manufacturing time and often renders patients unfit for treatment. One of the great dramas of archiving resected cancers in formaldehyde in patients at high risk of recurrence is the destruction of tumor-reactive T cells, which could otherwise have been used therapeutically at time of recurrence or progression.

Although recurrent metastases or those progressing despite systemic treatment may be clonally distinct from earlier tumors, the altered cancer genome of all daughter cells necessarily overlap, and the great hallmark of TIL is its polyclonality and capacity to target multiple tumor antigens. 21 22 34 The use of single T cell sequencing is also elucidating at a rapid pace the phenotype of neoantigen-reactive TIL. 27 46 48 212 213 This new knowledge is likely to guide the selection of tumors more likely to generate tumor-reactive TIL, to improve manufacturing of tumor-reactive TIL products rather than bystander T cells, and to better select patients more likely to respond to this therapy. Antigen-agnostic methods to select neoantigen-reactive T cells based on surface protein expression, using clinical-grade cell sorters, also expand efficient manufacturing from cryopreserved tumor cell suspensions. 3749 51 As a proof of principle, patients with metastatic melanoma have received bulk TIL manufactured from cryopreserved tumor fragments in at least one center. 214

Multiple models of cellular biobanks, such as for cord blood, already embody this paradigm in our healthcare systems. Biobanking cryopreserved tumor fragments or cell suspensions along good manufacturing process (GMP) methods to preserve TIL viability for later manufacturing and adoptive transfer appears as a promising avenue for patients at high risk of recurrence or progression with standard of care treatments. Concerns have been raised about the ethical issues associated with the treatment imperative when TIL are available but the patient’s condition is considered poor. Furthermore, coevolution of the tumor and the individual T cell response at sites raises concerns whether the appropriate TIL are available within much earlier cryopreserved tumors.

Present limitations and challenges

Broad application and equitable access to cellular-based therapeutic approaches for cancer patients is currently hindered by several practical challenges:

High costs associated with drug manufacturing and patient management: The time and economic cost for large-scale manufacturability of cell therapy products is a significant barrier to wide accessibility of this technology on a global scale. The per-patient cost of FDA-approved CAR T cell therapies ranges between US$700,000 to over US$1 million per patient (US$373,000–US$475,000 for the cell product and over US$500,000 in estimated costs of managing treatment-related adverse events and subsequent procedures). 215 However, even in the face of present-time economics, T cell therapy still outperforms the costs of chronic treatment alternatives and their complications. For example, the comparable cost of TIL in both hospital costs, cost of the drug, and cost of TIL generation was favorable for TIL compared with antibody to CTLA-4. 216

A large interpatient heterogeneity of starting tumor material: This, along with numerous variations in the large-scale manufacturing process, poses challenges with product consistency and phenotype of in vitro generated cellular therapies, a quality control and regulatory approval challenge. This may be somewhat reduced as TIL expansion methods become more standardized and, in the future, possibly more automated within closed systems.

Patient safety and associated toxicities: The administration of autologous TIL-ACT products has not been associated with severe off-target effects causing organ damage or fatalities. Nevertheless, cross-reactivity against melanoma self-antigens has been observed, with vitiligo and rarely uveitis, which can be managed with local steroids. In a recent meta-analysis, the total pooled estimate for the probability of severe ‘immunologic reaction’ events was 4%. 217 Luckily, no autoimmune reactions have been reported to date in patients with other solid tumors receiving TIL or neoantigen-enriched TIL products.

High-dose lymphodepleting chemotherapy with cyclophosphamide and fludarabine can produce rarely fatal sepsis or hemorrhage due to severe myelosuppression, cardiac toxicity from cyclophosphamide, or myelodysplastic syndrome. Furthermore, prolonged lymphodepletion in patients who have received prior chemotherapy may induce viral reactivation complications. For example, a case of Guillain-Barré syndrome, attributed to cytomegalovirus reactivation, has been reported in a heavily pretreated patient with melanoma three weeks after TIL-ACT. 218 In addition, one patient has been reported with treatment-refractory Epstein-Barr virus B-cell lymphoma four months after TIL-ACT. 219 Heavily pretreated patients who have received prior chemotherapy with reduced bone marrow reserve require dose adjustment of the cyclophosphamide. High-dose IL-2, if given beyond patient tolerability, can produce fatal complications from capillary leak syndrome, including respiratory failure or ventricular arrhythmia. Luckily, these fatal events remain quite rare. In the above meta-analysis examining 670 patients with available toxicity information, who received high-dose IL-2 in 12 studies, the overall fatal (grade 5) adverse event rate was 1.3%, 217 and these were either not attributed to the treatment or were attributed to lymphodepleting chemotherapy, not the IL-2. In the era of PD-1 blockade, special attention needs to be paid to those patients who approach TIL-ACT having experienced prior immune-mediated toxicities attributed to PD-1 blockade. While lymphodepleting chemotherapy may, in theory, significantly reduce the overall population of T cells involved in autoimmune responses, it may not completely eradicate autoimmune T cell clones. Patients who have residual organ dysfunction should be triaged particularly carefully. Specifically, drug-induced immune pneumonitis has been reported in patients with solid tumors associated with PD-1 inhibitors, and tends to be higher in patients with advanced NSCLC, 220 221 requiring particular attention to IL-2 dosing due to reduced lung reserve.

Mechanisms of resistance/escape

TIL therapy can achieve durable CRs for up to a quarter of patients with cutaneous melanoma, but it is still not curative for most patients. As TIL for epithelial cancers is being explored, especially in patients who received prior anti-PD-1 therapy, this has been even more of an issue. Treatments fail due to primary resistance, where tumors never show response, while others encounter acquired or secondary resistance, where relapses occur following initial response. Analysis of patient T cell and antigen repertoire and presentation analysis at a molecular level can shed light on common mechanisms of immune escape. For regressions induced by MHC class I restricted CD8+T cells, probably the most common escape mechanism is loss of the antigen or its presentation from the tumor. 222 This was underappreciated for a long time because the prototypical event of β2M loss (easily detectable by pan-class I immunohistochemistry) requires a biallelic loss and is a relatively uncommon resistance mechanism. Much more common was the allelic loss of the class I heavy chain presenting a specific antigen to clonal T cell populations. This could be detected imperfectly through the finding of loss of heterozygosity (LOH) at the Chromosome 6p MHC locus because large chromosomal deletions were the most common mediating event. 223 This has been found as the cause of primary immune resistance to T cell therapy with class I reactivity and as the cause of secondary resistance when the deletion is selected for after therapy with a CD8+population (perhaps best demonstrated using PBL transduced with a class I restricted TCR; figure 2 ). The current tools for detecting MHC allele loss when sequencing bulk fresh tumor (such as LOHHLA) have poor sensitivity when tumor cell fractions are low, so these events are under-detected. WES of patient-derived tumor organoids can demonstrate false negatives from fresh tumor WES. Although loss of the target antigen can be minimized by selecting clonal or driver mutations as the neoantigen, this does not guarantee that antigen loss cannot occur. 22

Another major demonstrated tumor-intrinsic resistance mechanism (primarily through studies of checkpoint inhibition) is loss of IFN-γ signaling, either through mutation of an IFN-γ receptor or defects in janus kinase (JAK), signal transducer and activator of transcription protein (STAT) signaling 224 and is an area in need of further investigation for TIL. A further obstacle to understanding immune resistance is our failure to understand the mechanisms of tumor rejection by class II restricted CD4+T cells (which may predominate in TIL from epithelial tumors). Multiple investigators have shown that tumors in which tumor cells are MHC deficient can still respond to adoptive therapy with class II restricted T cells, even in hosts that are T cell deficient. 225 How this happens and which immune cell subsets are involved in enhancing and driving CD4 T cell responses is still unresolved.

Pathologic considerations

TIL therapy presents opportunities for pathologists to contribute to advances in immunotherapy. During tumor harvest, pathologists have a key role to play in aliquoting clinical samples for harvest while simultaneously ensuring that appropriate clinical assessment of the harvested lesion is provided, particularly in small tumors or in cases where margin assessment is critical. Standardization and dissemination of best practices in tumor harvesting will be a vital collaborative task between surgeons and pathologists, including maintenance of sterility, cell preservation and thorough documentation. 226 Application of deep learning methods to accurately assess TIL infiltrate in tumors on H&E sections, as demonstrated in nasopharyngeal carcinoma, will allow reproducible utility as an emergent essential biomarker. 227 Similar direct multicenter direct comparators of melanoma demonstrate the superiority of artificial intelligence algorithms when compared directly with assessment by pathologists. 228 Currently, the International Immuno-Oncology Biomarker Working Group on Breast Cancer recommends routine assessment of TIL number in the setting of triple-negative and Her2/Neu+ breast cancer. 229

Pathologists also have a potential role in predicting successful outcomes in TIL therapy, an open area of investigation. Clinical and pathologic predictors of success have been described in melanoma and include female sex, young age, and specimen lymphocyte content. 139 230 In a more futuristic sense, one could imagine a time in which pathologists use intra-operative assessment with conventional (eg, frozen section) or novel biomarker-based point-of-care tests to choose among multiple candidate lesions or ideal regions from within a single tumor, or even to capture transcriptomically selected TIL 142 from within a tumor for subsequent harvest and transfer. Features of the tumor (as in gastric carcinoma) including lympho-vascular invasion and presence of TIL can predict nodal metastases. 231

Conclusion

The interaction between the host and the tumor over time and space is now well defined. Most adult tumors arise in the setting of chronic inflammation with inception and initiation (the three I’s) of a tumor 232 followed by immune editing, characterized by elimination, equilibrium, and escape (the three E’s) of a tumor. 233 Thus, TIL, both in melanoma and in other tumor types, are the highly selected and evolved progeny of an initially robust repertoire, contracted by sustained antigen, resultant apoptosis and/or anergy with clear exhaustion biomarker expression. Although there are only limited studies, the diminished repertoire found in the peripheral blood (such as in patients with head and neck cancer) is associated with worse outcome and response to immunotherapy. 234 Still, clear evidence of sustained and durable responses in patients with melanoma, leading to TIL approval in this setting, provides hope for a more robust and durable response, particularly in epithelial tumors. Up to this time, many of the responses in other tumor types are of limited quality and duration, and quite clearly anecdotal. Still, strategies to genetically modify TIL and/or select them based on neoantigen recognition also provide hope that steadfast progress will be made. The critical needs of rapid expansion of selected TIL outside of the influence of profound and highly evolved tumor-based immunosuppression will likely be necessary until well-defined principles, such as those allowing CAR T to be considered with transfection and expansion in vivo 235 . Clear, stepwise advances, as have been made over the last 40 years with TIL, once clarity emerges as to the critical factors associated with successful treatment, may allow this type of advance as well.

Acknowledgements

The authors acknowledge the SITC Surgery Committee members who did not serve as authors on this piece: Genevieve Boland, MD, PhD – Massachusetts General Hospital; William E Carson III, MD, FACS – Ohio State University; Michael Lowe, MD, MA – Emory University School of Medicine; Giao Phan, MD – UConn Health; Craig Slingluff, Jr, MD – University of Virginia. The authors also acknowledge SITC staff for their contributions, including Peter J Intile, PhD and Eleanor (Ellie) Rickman for project management and assistance. Additionally, the authors wish to thank SITC for supporting the manuscript development.

Funding: The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors except as noted below.

Patient consent for publication: Not applicable.

Ethics approval: Not applicable.

Provenance and peer review: Not commissioned; externally peer reviewed.

Collaborators: Not applicable.

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