Redesigning CAR therapy to tackle immune effector cell-associated hematotoxicity.

The importance of addressing hematotoxicity during CAR T-cell therapy

Chimeric Antigen Receptor (CAR) T cell therapy has revolutionized the treatment of aggressive hematological malignancies, providing durable remissions and, in some cases, complete cures [ 1 , 2 ]. The success of CAR T-cell therapy lies in its ability to reprogram T cells to overhaul the dysfunctional cancer immunity cycle by conferring antigen-specific and durable synthetic memory to guard against potential future relapse. As research into CAR T cell therapy expands, its applications are now being explored beyond hematological malignancies to include solid tumors and autoimmune disorders, utilizing novel tumor-targeting strategies [ 3 ].

However, this expanding therapeutic scope brings increased attention to its safety profile. Despite its efficacy, all clinically studied formulations targeting CD19, CD22, and B-cell maturation antigen (BCMA) have been indicated to cause adverse effects, manifested as off-tumor recognition, cytokine release syndrome (CRS), Immune effector cell-associated neurotoxicity syndrome (ICANS), and immune cell-associated hematotoxicity (ICAHT) [ 4 , 5 ]. Prolonged trilineage cytopenias following CAR T-cell therapy are associated with an increased risk of severe infections and haemorrhagic events, limiting the affordability and adaptability of the approach in the clinics. While CRS and ICANS are extensively investigated, ICAHT, characterized by grade 3 cytopenias with frequent infections and hematopoietic insufficiency, is relatively underexplored [ 6 , 7 ]. A related and similarly concerning entity is immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS), which has emerged across multiple trials. IEC-HS is characterized by macrophage activation, inflammation, hyperferritinemia, coagulopathy, hepatic dysfunction, and cytopenias. Both ICAHT and IEC-HS occur independently of ICANS, yet share the underlying pathophysiology of CAR T cell-associated toxicities, such as dysregulated inflammation, macrophage activation, and often NK cell dysfunction [ 8 ].

Epidemiological data highlight the clinical significance of these toxicities. Cytopenias have been observed up to 94% of patients undergoing CAR T-cell therapy, with an infection rate reaching 32% [ 9 ]. CAR T cell-associated cytopenias typically persist for 30 days after CAR therapy and are characterized by anemia, neutropenia, thrombocytopenia, and bone marrow suppression. Prolonged and severe cytopenias can increase the risk of opportunistic infection, hemorrhagic complications, and non-relapse mortality (NRM) [ 4 , 10 ]. Given these challenges, it is important to deepen our understanding of the cellular and molecular mechanisms of ICAHT and IEC-HS. Innovations in CAR design, cellular formulation, and manufacturing processes, alongside refinements in clinical management protocols, hold promise for mitigating hematotoxicity and improving patient outcomes [ 4 , 11 ]. This review aims to provide a comprehensive overview of recent findings regarding ICAHT, its mechanisms, and discuss strategies for mitigating its impact in clinical practice.

Toxicity associated with CAR T-cell therapy: Insights from an Indian clinical trial

Building upon the global understanding of CAR T-cell-associated toxicities, recent clinical trials in resource-limited settings have begun to offer critical perspectives. The clinical development and deployment of talicabtagene autoleucel , a novel humanised anti-CD19 CAR T-cell therapy, in India is a notable example. This multicentre phase 1/2 trial, the first-of-its-kind in the region, evaluated 57 patients with relapsed or refractory B-cell malignancies and demonstrated promising anti-tumour activity with an overall response rate (ORR) of 73%. Notably, the trial reported a significantly improved and manageable safety profile compared to earlier murine scFv-based CAR T products widely used in Western settings.

Unlike many global studies that reported a substantial incidence of ICANS, no patients in the Indian cohort developed ICANS of any grade. Furthermore, CRS was observed in 68% of patients; however, grade 3 or higher CRS was rare, completely absent in the B-cell lymphoma subgroup, and limited to 20% in the B-ALL subgroup. These findings align with the underlying design rationale of the talicabtagene autoleucel construct, which incorporates a humanised scFv and a 4-1BB costimulatory domain, resulting in reduced proinflammatory cytokine secretion (e.g., IFN-γ, TNF-α, IL-6) compared to murine constructs​.

Hematologic toxicities, a hallmark of ICAHT, were frequent but mostly transient. While neutropenia was reported in 96% of patients, prolonged severe neutropenia (ANC < 0.5 × 10⁹/L for ≥ 14 days) was observed in 25%, and only 12% experienced delayed cytopenias beyond day 30. Anemia and thrombocytopenia were observed in 61% and 65% of patients, respectively, but these were generally manageable and shorter-lasting compared to global real-world datasets. Febrile neutropenia occurred in 47% of patients, emphasising the need for close monitoring during early post-infusion periods.

This Indian trial demonstrated that utilising a humanised CAR construct and optimising infrastructure can deliver effective and safer CAR T-cell therapy with a reduced toxicity burden. These findings advocate for region-specific CAR design and manufacturing innovations to address local healthcare challenges while maintaining global therapeutic standards [ 12 ].

Clinical manifestations and incidence

ICAHT

The manifestation of ICAHT primarily involves cytopenias, including neutropenia, anemia, and thrombocytopenia, resulting from disrupted hematopoiesis and immune-mediated damage to hematopoietic cells [ 11 ]. The severity of ICAHT varies depending on factors such as the specific CAR T-cell product ( Table 1 ) , the underlying malignancy being treated, bone marrow transplantation, lymphodepletion chemotherapy, baseline blood counts, and pre-existing patient conditions [ 13 ].

Neutropenia stands as a key indicator, especially when severe (ANC < 500 cells/µL), as it heightens vulnerability to bacterial and fungal infections [ 13 , 14 ]. Incidence rates for severe neutropenia in the early phase post-CAR T therapy range from 59% to 95%, depending on treatment protocols and patient profiles [ 4 , 14 ]. This variability underscores the need for personalized care to manage ICAHT. Prolonged neutropenia that lasts over seven days further amplifies the risk of infection, contributing to increased morbidity and mortality. Clinical management typically involves vigilant monitoring, prophylactic antimicrobials, and administration of granulocyte colony-stimulating factors (G-CSF) [ 15 , 16 ]. Despite these measures, neutropenia remains a significant challenge, especially during the early stages of CAR T-cell therapy when CRS co-occurs [ 4 ].

Anemia, another key feature, manifests as reduced hemoglobin levels and contributes to fatigue, weakness, and decreased quality of life [ 17 , 18 ]. Reported rates of severe anemia vary between 16% and 77%, reflecting differences in patient conditions and treatment contexts [ 15 , 16 , 19 ]. ICAHT-related anemia is usually normocytic and normochromic, indicating decreased red blood cell (RBC) production rather than hemoglobin synthesis [ 20 ]. Anemia can have significant clinical consequences, especially in patients with existing cardiovascular conditions or those who have had intensive chemotherapy. Blood transfusions are commonly required for management but come with risks such as transfusion reactions and increased hospital stay durations, especially in patients with cardiovascular comorbidities. Anemia also exacerbates other symptoms of CAR T-cell therapy, necessitating comprehensive supportive care strategies to manage its impact on the patient’s overall well-being [ 14 ].

Thrombocytopenia, defined by a low platelet count, is another prominent manifestation of ICAHT. Platelets are crucial for blood clotting, and a decrease in their levels raises the risk of bleeding and bruising. Severe thrombocytopenia is reported in 28% to 65% of patients undergoing CAR T-cell therapy, with haemorrhagic complications, particularly concerning patients with coagulopathies or those receiving anticoagulant therapy [ 19 , 21 ]. Patients with thrombocytopenia require close monitoring and platelet transfusions to prevent bleeding incidents [ 22 ]. CAR construct variability contributes to the incidence of thrombocytopenia ( Table 2 ) , highlighting the importance of rationale designing of CAR constructs informed by the clinical data on ICAHT to devise individualised treatment strategies [ 23 ].

Bone marrow suppression is a hallmark of ICAHT, often resulting in pancytopenia, where RBC, WBC, and platelets are reduced [ 13 , 14 ]. This suppression is driven by multiple mechanisms, including direct cytotoxic effects of CAR T-cells, immune-mediated destruction of hematopoietic progenitors, and systemic inflammation that disrupts normal hematopoiesis [ 15 , 24 ]. Prolonged bone marrow suppression necessitates intensive support care, including transfusions, growth factor administration, and infection prophylaxis [ 2 , 14 , 15 ]. The duration and severity of bone marrow suppression are influenced by the patient’s baseline bone marrow reserve and prior exposure to chemotherapy or radiation therapy [ 25 ]. This suppression delays recovery, increases infection and bleeding risks, and limits the ability to administer additional therapies, including chemotherapy or radiation [ 10 ]. Additionally, the need for prolonged transfusion support and growth factor administration can lead to complications such as iron overload, alloimmunisation, and increased healthcare costs [ 14 ]. However, the role of CAR architecture, formulation, and therapeutic strategy in pancytopenia has yet to be determined.

Differences in ICAHT manifestations between CAR therapies targeting hematologic malignancies and solid tumors

The incidence, severity, and underlying mechanisms of immune effector cell–associated hematotoxicity (ICAHT) differ between CAR-T therapies targeting hematologic malignancies and those developed for solid tumors. These differences largely arise from variations in baseline bone marrow involvement, prior treatment exposure, lymphodepleting regimens, and the magnitude of CAR-T–induced systemic inflammation.

In hematologic malignancies such as acute lymphoblastic leukemia, lymphoma, and multiple myeloma, ICAHT is particularly common because malignant infiltration of the bone marrow frequently compromises hematopoietic reserve before therapy. In addition, patients often receive multiple lines of myelosuppressive chemotherapy or prior hematopoietic stem cell transplantation, further impairing marrow recovery [ 6 ]. Robust CAR-T expansion in these diseases—driven by the accessibility of target antigens such as CD19 or BCMA—also promotes intense cytokine production, which can suppress hematopoietic stem and progenitor cell function and contribute to prolonged cytopenias [ 21 ].

In contrast, CAR-T therapy for solid tumors generally demonstrates a different hematotoxicity profile. Not all the solid tumors involve in bone marrow infiltration, and patients often have relatively preserved baseline hematopoiesis compared with those with hematologic cancers [ 26 – 28 ]. A retrospective analysis of immune effector cell therapies involving 112 patients across leukemia, lymphoma, and solid tumors demonstrated that prolonged cytopenias were significantly more frequent in leukemia (63%) and lymphoma (44%) than in solid tumors (22.9%). Severe thrombocytopenia incidence was substantially lower in solid tumors (14.3%) versus lymphoma (46%) and leukemia (74.1%). Similarly, the duration of severe neutropenia was longest in leukemia patients (median 14 days), compared with lymphoma (7 days) and solid tumors (11 days). These findings suggest that disease context plays a major role in determining the severity and persistence of hematologic toxicity following adoptive cellular therapy [ 28 ].

Another important factor contributing to these differences is baseline hematopoietic reserve. Patients with leukemia frequently present with severe baseline cytopenias due to marrow infiltration by malignant cells, whereas patients with solid tumors typically maintain normal bone marrow function prior to therapy. As suggested in the study, this difference in hematopoietic reserve likely explains the higher incidence of prolonged cytopenias observed in leukemia patients [ 28 , 29 ]. Furthermore, lymphodepleting chemotherapy is required to promote CAR-T expansion but also contributes significantly to bone marrow suppression across disease types [ 30 ].

Systemic inflammatory responses also vary across disease contexts. CAR-T therapy in hematologic malignancies frequently induces intense cytokine release and immune activation, which can exacerbate bone marrow suppression through inflammatory signaling pathways. In clinical cohorts, inflammatory complications have been associated with higher rates of prolonged cytopenias, supporting the concept that systemic immune activation contributes to impaired hematopoietic recovery [ 28 ].

In solid tumor CAR-T therapy, CAR-T expansion is often limited due to antigen heterogeneity, physical barriers within the tumor microenvironment, and immunosuppressive signals [ 31 – 33 ]. Consequently, systemic cytokine release and immune activation may be relatively limited, which might partly explain the lower incidence of severe or prolonged cytopenias.

However, the evidence base for solid tumors remains sparse. Most published CAR-T cytopenia literature focuses on CD19-targeted therapies in B-cell malignancies. The solid tumor cohort in the available data represents a small subset of the broader CAR-T literature, limiting generalizability. Additional prospective studies specifically examining solid tumor CAR-T trials are needed to establish robust risk profiles and management strategies.

Phases of hematological complications

Hematologic complications following CAR T-cell therapy can be categorized into three clinical phases, each presenting distinct challenges [ 34 ].

Pretherapeutic phase

This phase is influenced by the patient’s treatment history and the medications given before CAR T-cell infusion. Many patients undergoing CAR T-cell therapy are subjected to extensive prior treatments, such as chemotherapy and radiation, that result in decreased blood cell counts and bone marrow reserves, impairing the ability to produce new blood cells. It is crucial during this phase to identify high-risk patients for developing severe complications and to put in place strategies to prevent these risks [ 13 , 35 ].

Early ICAHT (Days 0–30)

The early phase of ICAHT typically involves the onset of low blood cell counts directly caused by CAR T-cell therapy within the first 30 days. During this time, the combination of reduced lymphocytes, CAR T-cell activity, and damage caused by the immune system can significantly reduce blood cell counts [ 36 ]. Pre-existing conditions and the severity of other toxicities, such as CRS, often worsen low blood cell counts during this phase. Effectively managing low blood cell counts during the early phase is crucial to prevent complications such as infections and bleeding, which contribute to illness and death [ 13 ]. Effective management during this phase revolves around vigilant monitoring, early interventions (G-CSF, transfusion), and addressing concurrent complications like CRS to prevent life-threatening infections or haemorrhagic events.

Late ICAHT (Post day 30)

Late ICAHT refers to prolonged or delayed hematological complications that persist or arise several weeks to months after CAR T-cell therapy. Prolonged cytopenias during this phase occur not only because of the initial treatment but also because of ongoing immune system dysregulation, bone marrow failure, or additional treatments [ 4 , 13 ]. Managing the late phase requires long-term support strategies, close monitoring for delayed infections, and the potential risk of secondary malignancies due to continued immune suppression [ 13 ].

ICAHT grading and CAR-HEMATOTOX score

To better assess and manage hematologic toxicity in CAR T-cell recipients, standardized frameworks have been developed by the European Hematology Association (EHA) and the European Society for Blood and Marrow Transplantation (EBMT) [ 6 , 18 ]. According to this classification, ICAHT is divided into early (within 30 days) and late (after 30 days) phases, with cytopenia graded on a scale of 1 to 4 based on the depth and duration of neutropenia ( Table 3 ) [ 7 , 37 ]. Lower grades (1–2) are associated with better outcomes and quicker neutrophil recovery, while higher grades (3–4) correlate with an increased risk of severe infections due to persistent neutropenia and immune dysregulation, requiring intensive management and prolonged supportive care [ 7 ]. Studies have reported that severe CRS and ICANS are more common in patients with grade 3 or higher ICAHT [ 13 ]. Aplastic neutrophil recovery was predominant in patients with higher ICAHT, which was associated with a more significant non-relapse mortality (NRM) due to an increased risk of infection. Further treatment plans for patients can be calculated based on ICAHT grades.

Complementing this, the CAR-HEMATOTOX score provides a pre-infusion risk stratification tool for predicting severe hematotoxicity [ 25 ]. This score is calculated before lymphodepletion chemotherapy and considers five key factors: Platelet count, ANC, Hemoglobin, C-reactive protein, and Ferritin ( Table 4 ) [ 38 ]. A score of ≥ 2 indicates a high risk of prolonged neutropenia and thrombocytopenia, often necessitating early growth factor support. However, this score does not correlate with CRS or ICANS severity and offers limited prognostic value for overall treatment outcomes [ 25 , 38 , 39 ]. Recognizing the inefficiencies of manual grading, computational methods and AI-driven algorithms are being explored to enhance predictive accuracy and automate grading based on receptor configuration, manufacturing strategy, patient characteristics, and clinical history [ 40 ].

Integrating N-ICAHT and T-ICAHT into hematotoxicity assessment frameworks

Initially, ICAHT classification focused on neutropenia-based grading, now termed N-ICAHT, which evaluates cytopenias based on the depth and duration of ANC suppression. This system stratifies toxicity into early and late phases post-CAR T-cell infusion, as discussed earlier, and remains a robust indicator of infection risk, NRM, and survival outcomes.

However, recent data underscore the distinct clinical relevance of thrombocytopenia. A multicentre study introduced T-ICAHT, a parallel grading system that categorizes thrombocytopenia severity using platelet count thresholds and timing analogous to N-ICAHT. In a cohort of 744 patients with B-cell non-Hodgkin lymphoma, 43% developed early T-ICAHT and 42% developed late T-ICAHT. Importantly, grade 3 + T-ICAHT was associated with increased transfusion requirements, higher bleeding risk, and significantly reduced overall survival (OS) (from 67% to 35%).

While T-ICAHT and N-ICAHT correlate in many patients, notable discordance was observed, emphasizing the need to evaluate thrombocytopenia independently. Approximately 40% of patients with severe thrombocytopenia (grade ≥ 3 T-ICAHT) had only mild neutropenia (grade ≤ 2 N-ICAHT), indicating that platelet-based grading captures an additional at-risk population. Furthermore, higher T-ICAHT grades were linked with elevated inflammatory toxicity (e.g., CRS, ICANS), underscoring the interrelationship between hematopoietic suppression and systemic immune dysregulation [ 41 ].

Incorporating both N-ICAHT and T-ICAHT as standard components of the ICAHT framework allows for a more nuanced understanding of hematotoxicity in CAR T recipients. This dual-grading approach improves risk stratification, informs supportive care decisions (e.g., transfusion needs, use of thrombopoietin mimetics), and may guide future CAR design modifications to reduce myelosuppressive effects without compromising antitumor efficacy.

Mechanisms of ICAHT in CAR T-cell therapy

The pathogenesis of ICAHT is multifactorial, encompassing immune activation, systemic inflammation, niche disruption, and off-target effects (Fig. 1 ). Understanding these mechanisms is essential for optimising CAR T-cell therapy while minimising its hematotoxic consequences [ 13 , 20 , 25 ].

Activation of immune cells and CRS

CRS is one of the key contributors to hematotoxicity in CAR T-cell therapy. During CRS, massive release of pro-inflammatory cytokines such as IL-6, IFN-γ, and TNF-α due to CAR T-cell activation leads to widespread tissue inflammation and disruption of bone marrow homeostasis [ 20 , 25 ]. Elevated CRP and ferritin levels during CRS correlate with hematotoxic severity, while apoptosis of hematopoietic progenitors and altered marrow microenvironment contribute to cytopenia (Fig. 1) [ 13 , 20 , 42 ].

CAR T cells may also directly or indirectly target hematopoietic cells [ 6 , 43 ]. Immune-mediated destruction of progenitors and sustained IFN-γ signaling, driven by CX3CR1 hi CD8+IFNγ + expansions, compromise HSC function. Therapeutics such as IFN-γ-neutralizing antibodies (e.g., emapalumab) and thrombopoietin receptor agonists may alleviate this dysfunction [ 44 , 45 ]. Importantly, managing CRS early with agents like tocilizumab or corticosteroids can prevent downstream hematopoietic damage [ 42 , 46 ]. Although IFNγ is implicated in the severity of cytopenia, the detailed cellular mechanism and soluble factors involved are yet to be deciphered.

Inflammatory disruption of HSC homeostasis

CAR T-cell therapy-induced inflammation also disrupts the bone marrow niche ( Fig. 2 ) [ 47 ]. Pro-inflammatory cytokines such as IFN-γ, TNF-α, and IL-6 skew hematopoiesis toward granulopoiesis, reducing erythropoiesis and megakaryocyte function [ 48 , 49 ]. Inflammation-induced differentiation of mesenchymal stem cells (MSCs) into adipocytes diminishes soluble factors like SDF-1 (CXCL12) and SCF, impairing HSC retention and quiescence. Additional mechanisms, such as Fas-mediated apoptosis, elevated MMP activity, and sympathetic nervous system hyperactivation, further compromise HSC quiescence and bone marrow function [ 47 , 50 ]. Reduced platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) further compromise osteoblast function, exacerbating bone marrow dysfunction [ 51 ]. The combined effects of inflammatory cytokines and niche dysfunction underscore the critical role of inflammation in ICAHT and highlight the need for therapeutic interventions to maintain HSC homeostasis.

Off-target effects and antigen escape

CAR T-cell therapy often targets antigens like CD19, expressed on malignant and normal B cells, leading to B-cell aplasia [ 52 ]. This off-target effect can extend to other hematopoietic cells if the target antigen is shared by non-malignant hematopoietic progenitors, leading to cytopenias [ 53 ]. Cross-reactivity and persistent CAR T-cell activity, especially post-antigen escape, extend immune activation and marrow suppression [ 54 , 55 ]. Innovations like dual-targeting CARs, modular universal immune receptors, inhibitory CARs, and gene-editing platforms (e.g., CRISPR/Cas9) offer promising avenues to enhance specificity and reduce toxicity [ 52 , 56 – 58 ].

Inflammatory cytokines in ICAHT: Role of IL-18, IL-15, and IL-10

Emerging evidence suggests that inflammatory cytokines contribute significantly to the development of ICAHT, with IL-18 emerging as a key mediator. A first-in-human clinical trial evaluating CD37-targeted CAR T cells revealed that three out of five treated patients experienced prolonged and severe pancytopenia, which could only be reversed by allogeneic hematopoietic stem cell transplantation in two cases. Notably, these patients exhibited sustained elevations of IL-18 and a concurrent deficiency of its natural inhibitor, IL-18 binding protein (IL-18BP), suggesting a mechanistic link between IL-18 dysregulation and hematotoxicity [ 59 ]. IL-18, secreted by activated macrophages and dendritic cells, activates MyD88-dependent signaling cascades, including NF-κB and MAPK pathways, ultimately leading to enhanced secretion of IFN-γ and perpetuating systemic inflammation [ 60 , 61 ]. This cytokine exacerbates HSPC dysfunction by inducing inflammatory stress, skewing differentiation, and expanding myeloid-derived suppressor cells (MDSCs), thereby suppressing hematopoietic recovery [ 62 , 63 ]. Synergistically, IL-18 enhances the effects of IFN-γ and TNF-α, further amplifies suppression of erythroid and megakaryocytic lineages [ 64 ]. IFN-γ is recently reported to cause cytopenia by way of inhibiting IL-17 A and GCSF production. These observations suggest that IL-18 may serve as a potential biomarker and therapeutic target, although validation in larger clinical cohorts is required.

In addition to IL-18, other cytokines implicated in CAR-T–associated inflammatory toxicity may also contribute to ICAHT. Among these, IL-15 has received increasing attention due to its role in promoting T-cell survival, proliferation, and persistence [ 65 ]. IL-15 is frequently incorporated into next-generation CAR-T platforms to enhance antitumor efficacy; however, sustained IL-15 signaling can also amplify immune activation and systemic inflammation [ 66 , 67 ]. Preclinical and clinical studies have shown that excessive IL-15 activity may intensify cytokine-driven toxicities, including cytokine release syndrome (CRS), by promoting prolonged expansion and activation of CAR-T cells and endogenous immune populations [ 68 – 70 ]. Notably, a recent clinical study evaluating IL-15–enhanced GPC3-targeting CAR-T cells in hepatocellular carcinoma reported significant safety concerns related to heightened inflammatory responses [ 71 ]. Persistent immune activation driven by IL-15 may indirectly contribute to hematopoietic suppression through mechanisms similar to those described for other inflammatory cytokines, including induction of IFN-γ and downstream inflammatory pathways that impair hematopoietic stem and progenitor cell function [ 72 ]. Although direct evidence linking IL-15 to ICAHT remains limited, these findings suggest that IL-15–driven immune amplification may represent a potential contributor to CAR-associated inflammatory toxicities.

Recent clinical studies have also suggested a potential role for IL-10 as a biomarker associated with late immune effector cell–associated hematotoxicity following CAR-T therapy. In a cohort of patients with B-cell malignancies treated with CAR-T cells, elevated serum IL-10 levels were associated with an increased risk of developing severe late cytopenia [ 73 ]. Incorporation of IL-10 into a clinical prediction model further improved the ability to identify patients at risk for late ICAHT, highlighting its potential value in risk stratification and early monitoring [ 73 ]. Although IL-10 is classically considered an anti-inflammatory cytokine, elevated levels after CAR-T infusion may reflect persistent immune activation and compensatory regulatory signaling within the cytokine milieu. Nevertheless, the mechanistic contribution of IL-10 to ICAHT remains incompletely understood, and current evidence is largely derived from a single clinical study, underscoring the need for further validation in larger and independent cohorts before its routine clinical implementation.

Role of immune cells in mediating hematotoxicity

Various immune cells play a role in the severity of ICAHT. While CAR T-cells targettumor cells, their interactions with hematopoietic lineage cells contribute to ICAHT [ 4 , 18 , 74 ].

Macrophages and myeloid cells

Macrophages are heavily involved in the pathogenesis of CRS and subsequent hematotoxicity. Upon activation, they release additional pro-inflammatory cytokines, such as IL-6, IL-1, and TNF-α, which amplify the inflammation and suppress bone marrow function [ 4 , 75 ]. In extreme cases, macrophages can phagocytise hematopoietic cells, contributing to conditions like hemophagocytic lymphohistiocytosis (HLH), characterized by profound cytopenias and marrow failure [ 74 , 76 ]. Patients with underlying autoimmune conditions or genetic predispositions to immune dysregulation face heightened HLH risk [ 77 ].

Bystander T-cells

Non-engineered bystander T-cells may become hyperactivated during CRS. Their contribution to the inflammatory microenvironment compounds marrow damage through non-specific immune attacks and amplifies the risk of hematologic complications [ 4 , 10 , 18 , 74 ].

Dendritic cells and other antigen-presenting cells

Dendritic cells and other antigen-presenting cells (APCs) also become hyperactivated during CRS, releasing additional pro-inflammatory cytokines, potentially presenting self or non-target antigens that provoke off-target CAR T-cell activity [ 75 ]. This “epitope spreading” can further compromise hematopoietic integrity and contribute to unintended toxicities [ 55 ].

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS)

HLH and MAS represent severe manifestations of immune dysregulation that can occur during CAR T-cell therapy. Both conditions are characterised by excessive immune activation, particularly of macrophages and T-cells, leading to the uncontrolled production of cytokines and the phagocytosis of hematopoietic cells, resulting in pancytopenia and severe bone marrow suppression [ 4 , 78 , 79 ]. Common clinical features include fever, splenomegaly, hyperferritinemia, and pancytopenia. Though often triggered by CRS, infections, or underlying immune disorders can also precipitate HLH/MAS [ 74 , 75 , 78 , 79 ]. Management typically necessitates aggressive immunosuppression, which may blunt the therapeutic effect of CAR T-cells, highlighting the delicate balance required to maintain antitumor efficacy while mitigating immune overactivation [ 2 , 78 , 79 ].

Gut microbiome-CAR T-hematopoiesis axis

Emerging evidence positions the gut microbiota as an important modulator of hematopoietic and immune homeostasis. Preclinical models show that germ-free and antibiotic-treated mice show impaired HSC function and reduced hematopoietic progenitor populations, particularly in the myeloid lineage. Microbiota-derived short-chain fatty acids (SCFAs), especially butyrate and acetate, play pivotal roles in maintaining bone marrow niche integrity and promoting erythropoiesis through macrophage-mediated iron recycling and cytokine production (e.g., SCF-induced stem cell factor from stromal cells) ​ [ 80 ].

Microbial components such as lipopolysaccharides (LPS) can act directly on HSCs via Toll-like receptor 4 (TLR4), activating TRIF-ROS-p38 signaling pathways that promote HSC proliferation but impair self-renewal [ 81 ]. Similarly, NOD1 ligands from gut bacteria signal through mesenchymal stromal cells to release hematopoietic cytokines such as IL-6, IL-7, and thrombopoietin (TPO), influencing lineage commitment and HSC quiescence [ 82 ]​.

In the context of CAR T-cell therapy, dysbiosis often induced by pre-treatment antibiotics or chemotherapy which can exacerbate hematotoxicity [ 83 ]. Loss of beneficial microbial taxa like Faecalibacterium prausnitzii and Akkermansia muciniphila , correlates with elevated cytokine levels and prolonged cytopenia [ 84 ]. Species within the Clostridia class have been linked to favorable CAR T responses, while Bifidobacterium enrichment correlates with severe CRS, both influencing systemic inflammation and HSC exhaustion [ 83 ].

Furthermore, dysbiosis-induced gut barrier disruption during lymphodepletion can facilitate microbial translocation and endotoxemia, aggravating bone marrow suppression. Clinical observations reveal that a higher abundance of Enterobacteriaceae correlates with prolonged neutropenia and infection risk, while diverse, SCFA-producing microbiomes are associated with more rapid hematologic recovery ​ [ 83 ].

Interventions targeting the microbiota hold promise for mitigating ICAHT. Fecal microbiota transplantation (FMT) and supplementation with SCFA-producing probiotics (e.g., Lactobacillus , Clostridium butyricum ) have shown efficacy in restoring hematopoietic balance and limiting cytokine-mediated damage​. Furthermore, dietary interventions rich in fibre or polyphenols may enhance SCFA production and support HSC niches [ 85 , 86 ].

However, their deployment in immunocompromised, neutropenic patients requires cautious timing and individualized risk-benefit assessment. Ongoing studies are working to identify microbial biomarkers predictive of ICAHT severity to guide these interventions.

Management strategies and therapeutic innovations to mitigate ICAHT

Effective management of ICAHT necessitates a multipronged approach that combines proactive supportive care, targeted pharmacological interventions, and cutting-edge innovations in CAR T-cell design and manufacturing ( Fig. 3 ) . The goal is to mitigate the hematotoxicity while preserving the antitumor efficacy of CAR T-cells.

Supportive care

Supportive care forms the first line of defence against ICAHT, including interventions such as transfusions, growth factor support, infection prophylaxis, and management of iron overload [ 4 , 13 ].

Transfusions

RBC and platelet transfusions are routinely administered to manage anemia and thrombocytopenia. Careful monitoring of cytopenias and potential complications, such as bleeding or cardiovascular events, guides the use of transfusion [ 2 , 4 ]. However, transfusions are associated with risks such as alloimmunisation, iron overload, and transfusion-related complications, mandating vigilant monitoring [ 87 ].

Growth factor support

Agents like G-CSF and erythropoiesis-stimulating agents (ESAs) are used to stimulate the production of neutrophils and RBC, respectively. While G-CSF is critical in managing neutropenia, as it can reduce the duration of neutropenia and the risk of infections, its early use during CRS may exacerbate inflammation. Expert consensus often recommends initiating G-CSF after the resolution of CRS [ 4 , 79 , 88 – 90 ].

Infection prophylaxis

Infections can occur due to a reduced count of immune cells, especially neutrophils, macrophages, and T cells. A more significant number of previous antitumor treatments, prior allogeneic HCT, and bridging chemotherapies for lymphoma have all been recognized as risk factors for infection [ 91 ]. The regimen is tailored to neutrophil counts and patient-specific risk factors, with high-risk individuals (ANC < 500/µL) receiving broad-spectrum antibiotics, antivirals, and antifungal prophylaxis until hematologic recovery [ 4 , 79 , 89 ].

Management of iron overload

Patients receiving frequent RBC transfusions are at risk for iron overload, particularly in the liver, heart, and endocrine glands leading to organ damage [ 92 ]. Monitoring of iron levels and using iron chelation therapy may be necessary in patients with significant iron overload [ 6 , 93 ]. However, iron chelation therapy must be carefully managed to avoid exacerbating anemia or other complications [ 94 ].

Pharmacological interventions

Pharmacological interventions for ICAHT include cytokine inhibitors, immunosuppressive therapies, and other agents targeting specific aspects of the immune response.

Cytokine inhibitors

IL-6 inhibitors like tocilizumab are critical in managing CRS and its progression to severe ICAHT. The early administration of tocilizumab during CRS can prevent the progression to severe hematotoxicity and reduce the need for intensive supportive care [ 95 ]. Additional agents like siltuximab and anakinra may help in combination with tocilizumab for preventing severe CRS and neurotoxicity [ 96 ]. Ongoing research is focused on optimising the timing of tocilizumab administration and exploring novel pharmacologic targets to manage CAR T-cell therapy toxicities [ 97 ].

Immunosuppressive therapies

Corticosteroids are often used for managing HLH/MAS or severe CRS following CAR T-cell therapy, as they suppress immune activation and reduce cytokine production [ 4 , 97 , 98 ]. However, high doses of corticosteroids can compromise CAR T-cell function, so their use must be carefully weighed against therapeutic efficacy. JAK inhibitors are also being investigated for ICAHT, especially in refractory or severe cases [ 97 ].

Advances treatment options

Advanced treatment options may be necessary when supportive care and pharmacological interventions fail.

Hematopoietic stem cell boosts

For patients who are refractory to G-CSF treatment or continue to experience severe cytopenias, autologous or allogenic hematopoietic stem cell boosts can be considered. These boosts can help reconstitute blood cell count, although the availability of cryopreserved grafts is a limiting factor [ 13 , 18 , 99 ].

Thrombopoietin receptor agonists

Agents like eltrombopag for post-CAR leukopenia and thrombocytopenia have shown promise in improving platelet recovery and reducing the need for transfusion [ 15 , 100 ].

Allogeneic hematopoietic cell transplantation

In cases of persistent grade 4 ICAHT beyond day 30, allogenic HCT may considered as a last option. This procedure requires careful evaluation of donor availability and conditioning regimen to avoid further complications [ 13 ].

JAK inhibitors

JAK inhibitors, which block cytokine signaling pathways, are a promising candidate for reducing CRS severity. Itacitinib, a selective JAK1 inhibitor, has shown promising preclinical results in reducing CRS-associated cytokines while maintaining CAR T-cell proliferation and its antitumor activity [ 101 ].

Engineering innovations in CAR T-cell therapy to mitigate hematotoxicity

Tuning CAR construct

To mitigate ICAHT while maintaining the efficacy of CAR T-cell therapy, researchers are exploring innovative modifications to CAR constructs and alternative cell sources ( Fig. 4 ) . Inhibitory CARs (iCARs) integrate inhibitory signaling domains to suppress activation upon encountering non-target antigens, reducing off-target damage to hematopoietic progenitors and mitigating cytopenias. Inducible apoptotic genetic switches provide a safety mechanism to eliminate CAR T-cells in cases of severe toxicity or excessive inflammatory responses [ 102 , 103 ]. Dual-target CARs, which recognize two antigens simultaneously, improve specificity and reduce unintended damage to the bone marrow niche. In contrast, switchable CAR systems, regulated by small molecules, allow precise modulation of CAR T-cell activity in patients experiencing high-grade inflammatory responses [ 104 ].

Gene editing technologies like CRISPR/Cas9 can enhance CAR specificity, safety, and reduce toxicity by targeting genes that regulate inflammatory cytokine release (e.g., IL-6R) or exhaustion markers (e.g., PD-1). Also, they can create universal CAR-T cells by disrupting TCR and B2M genes. Additionally, Deletion of IFNγ from CAR T cells or secretion of IL-18 soluble receptor may be tested in the preclinical settings of CAR T cells for its impact on anaemia, neutropenia, and thrombocytopenia and infections [ 105 , 106 ].

modified CAR constructs, such as cytokine-modulating CARs that inducibly secrete anti-inflammatory agents like IL-10, TGFβ, is another mode of modification that enhances the tumor-targeting efficacy with reduced ICAHT [ 107 , 108 ]. GCSF and GM-CSF secreting CAR T cells or strategic administration of these cytokines in ICAHT patients needs to be explored. These advances mark a significant step toward safer, more effective immunotherapies.

Alternative cell sources

Alternative cell sources are emerging as promising strategies to overcome the limitations of traditional T-cell-derived CAR therapies ( Fig. 5 ) . CAR-NK cells offer a reduced risk of CRS and graft-versus-host disease (GVHD) due to their innate immune characteristics and limited reliance on HLA matching [ 109 ]. Additionally, CAR macrophages are being explored for their ability to infiltrate solid tumors and modulate the tumor microenvironment while posing a lower risk of systemic hematotoxicity [ 110 ]. Memory T-cell-derived CAR T-cells, such as those derived from central or stem-like memory T cells, show enhanced persistence, reduced exhaustion, and a lower risk of hematopoietic suppression due to their long-term proliferative capacity [ 111 , 112 ].

Another emerging platform involves CAR-engineered natural killer T (CAR-NKT) cells, which combine innate and adaptive immune properties and have demonstrated promising preclinical and early clinical activity in multiple malignancies [ 119 – 122 ]. These cells exhibit dual antitumor activity through direct CAR-mediated targeting and endogenous NKT cell functions, while also modulating the tumor microenvironment [ 122 , 123 ]. Notably, CAR-NKT cells have been reported to limit cytokine release syndrome by eliminating pro-inflammatory macrophages, suggesting a potentially distinct toxicity profile compared with conventional CAR-T cells, although their impact on ICAHT remains to be defined [ 123 ].

Engineering CARs on pathogen specific T cells to palliate post therapy infections

Pathogen-specific CAR-T cells represent another emerging alternative cellular source, leveraging antigen-experienced virus- or pathogen-specific T lymphocytes for CAR engineering. Adoptive cell therapy using ex vivo manipulated pathogen-specific T cells (PSTs) has been increasingly explored, particularly in immunocompromised patients with delayed cellular immune reconstitution following stem cell transplantation. Early clinical studies have demonstrated encouraging efficacy against viral infections such as cytomegalovirus (CMV), Epstein–Barr virus (EBV), and adenovirus, with expanding interest in targeting less common pathogens including polyomaviruses and fungal infections [ 124 ]. Importantly, pathogen-specific CAR-T cells retain dual functionality through both CAR-mediated target recognition and native T-cell receptor (TCR)–dependent activation, which may enhance persistence, promote physiological stimulation, and limit tonic signaling–associated exhaustion [ 125 – 127 ]. Owing to their predefined memory phenotype and more controlled cytokine production, this approach may also reduce the risk of severe CRS and therapy-associated hematotoxicity. However, pathogen-specific CAR-T cell therapy remains at an early clinical stage and requires further optimization to achieve broad applicability across diverse pathogens.

Off-the-shelf or universal CAR T-cells eliminate the need for patient-specific customisation as their antigen recognition mechanisms are independent of classical MHC presentation, resulting in reduced risk of off-target effects [ 113 ]. Tissue resident γ9δ2 T cell engineering, which is less accumulated in the bone marrow, and inhibition of CXCR4 may restrict CAR T cells from entering the bone marrow to target solid tumors and certain lymphomas [ 114 , 115 ]. For lymphoma and multiple myeloma, IFNγ secreting CXCR1 + T cell deletion might palliate hematotoxicity without compromising the efficacy [ 116 – 118 ].

Manufacturing modifications to reduce ICAHT

Innovations in manufacturing CAR T-cells have emerged as crucial strategies to mitigate ICAHT while preserving therapeutic efficacy. Non-viral transduction methods, such as electroporation-based transfection, allow for the introduction of CAR constructs using DNA or RNA, with RNA-based transient CAR expression being particularly effective in providing short-term activity while reducing prolonged immune activation and off-target effects [ 128 – 130 ]. Optimising viral vectors, such as self-inactivating lentiviral systems, enhances CAR specificity and minimises insertional mutagenesis [ 131 ].

The ex vivo expansion process also plays a significant role in determining the functionality of CAR T-cells. Using IL-7 and IL-15 instead of IL-2 during expansion promotes the development of memory-like T-cells with reduced exhaustion and lower inflammatory potential [ 132 – 134 ]. Defined serum-free media further reduces variability and the risk of introducing immunogenic factors, while JAK inhibitors can suppress hyper-inflammatory T-cell development during manufacturing [ 101 , 135 ]. Controlled activation protocols using anti-CD3/CD28 beads prevent terminal differentiation, ensuring the production of less inflammatory CAR T-cells and enriching specific T-cell subsets, such as central memory T-cells (Tcm) or stem-cell memory T-cells (Tscm), improves persistence and reduces the risk of hematotoxicity [ 134 ].

These advances, combined with rigorous post-manufacturing quality control measures, such as cytokine profiling and CAR expression testing, transform CAR T-cell production, ensuring safer and more effective patient therapies.

Conclusions and future perspectives

Current evidence indicates that ICAHT can lead to severe clinical complications, including opportunistic infections and hemorrhagic events, thereby substantially impacting patient safety and therapeutic outcomes. Optimization of supportive care measures—such as transfusion support, growth factor administration, and judicious use of immunosuppressive agents—remains a clinical priority. Although predictive tools such as the CAR-HEMATOTOX score assist in risk stratification and clinical decision-making, accurate prediction of thrombocytopenia, anemia, and susceptibility to infectious complications will likely require the integration of advanced artificial intelligence–based algorithms trained on multidimensional clinical and biological datasets. In addition, emerging biomarkers—including inflammatory cytokines such as IL-18, IL-15, and IL-10—may further refine predictive models for identifying patients at risk of severe or prolonged hematologic toxicity. Advances in CAR design offer promising avenues to reduce hematologic toxicities. These include the incorporation of humanized single-chain variable fragments (scFvs), safety switches, selective deletion of CD8 + CXCR1 + IFNγ + cells, split-CAR systems requiring dual antigen recognition, inhibitory CARs that deactivate upon engagement with healthy tissues, and cytokine-modulating approaches such as soluble IL-18 receptor CARs. Collectively, these innovations aim to uncouple antitumor efficacy from excessive immune activation that contributes to bone marrow suppression. Notably, the development of immunocompetent animal models and advanced three-dimensional human physiological systems is essential to accurately recapitulate CAR therapy–induced neutropenia, thrombocytopenia, anemia, and infection risk, thereby enabling more effective therapeutic and clinical management strategies. Beyond CAR construct engineering, alternative cellular platforms represent an important future direction for mitigating ICAHT. In this context, antigen- or pathogen-specific CAR-T cells emerge as a particularly attractive strategy. CAR T derived from antigen-experienced memory T cells, these cells retain native T-cell receptor (TCR)–mediated signaling in addition to CAR-dependent activation, enabling more physiological immune engagement. Such dual functionality may enhance persistence, reduce tonic signaling and exhaustion, and promote more controlled cytokine release, thereby potentially lowering the incidence and severity of ICAHT. Furthermore, pathogen-specific CAR-T cells may be especially beneficial in immunocompromised settings, where they could simultaneously provide antimicrobial protection while maintaining antitumor activity. Other emerging platforms, including CAR-NK and CAR-NKT cells, CAR macrophages. universal CAR T-cells, Tissue resident γ9δ2 T cell may also offer distinct safety profiles by modulating inflammatory signaling and tumor–immune interactions, although their implications for ICAHT require further investigation. The gut microbiome has also emerged as a critical modulator of CAR T-cell toxicity and hematopoiesis [ 80 , 84 ]. Dysbiosis has been linked to severe CRS and prolonged neutropenia, while microbial taxas, such as Faecalibacterium prausnitzii and Akkermansia muciniphila , appear to mitigate systemic inflammation. Regional and population-specific differences in susceptibility to ICANS and ICAHT may further reflect underlying microbiome heterogeneity, underscoring the need for geographically informed analyses. Therapeutic manipulation of the microbiome—through probiotics, fecal microbiota transplantation (FMT), or dietary strategies—represents a promising adjunct to existing ICAHT mitigation approaches, including growth factor support, stem cell boosts, and pharmacologic cytokine blockade. However, such interventions must be strategically timed and personalised, especially in immunocompromised hosts, to balance efficacy and infection risk. Future studies should prioritise refining CAR T-cell construct design to mitigate ICAHT while maintaining antitumor efficacy. Innovations such as low-affinity scFvs, costimulatory domain tuning, and modular or switchable CAR architectures offer promising routes to minimise hematoxicity. In parallel, integrating cytokine profiling, microbiome composition, pre-treatment history, and hematotoxicity risk scores such as N-ICAHT and T-ICAHT into clinical workflows may enable more precise risk stratification and personalised intervention strategies . Importantly, pre-CAR therapeutic regimens and their frequency—particularly lymphodepleting chemotherapy—can critically influence post-CAR toxicity profiles, including ICANS and ICAHT. In addition, disease context-including difference between hematologic malignancies and solid tumors-may significantly influence ICAHT incidence and severity, highlighting the need for disease specific risk models. An integrated, systems-level approach encompassing CAR design, cellular source selection, microbiome modulation, and patient-specific treatment history is therefore likely to be essential for achieving broader clinical adoption and global applicability of CAR-based immunotherapies.

Acknowledgements

We acknowledge Indian Council of Medical Research for research funding, DBT Government of India for the fellowship support, and MS. Archana Praveen and Ashik Anil Mathews for carefully going through the manuscript.

Author contributions

Muhamad Shafi Kooniyil: Writing - original draft, review, editing, validation, software, and conceptualization. Pranav Alex Thomas: Writing – review and editing. Muthuganesh Muthuvel: Writing – review and editing. Alok Srivastava: Writing – review and editing. Sunil Martin: Writing – review and editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Funding

This work was supported by grant from the Indian Council of Medical Research (IIRPIG-2023-0000559, “Affinity-tuned CAR T cells targeting refractory CD20 (+) B lineage malignancies for enhanced safety and antitumor functions.“) to Dr. Sunil Martin, and Muhamad Shafi Kooniyil was supported by fellowship from DBT, India.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

Data Availability Statement

No datasets were generated or analysed during the current study.