February 26, 2024

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Mechanisms and adjustment strategies of CAR-T cell dysfunction

Mechanisms and adjustment strategies of CAR-T cell dysfunction


Mechanisms and adjustment strategies of CAR-T cell dysfunction

Over the past decade, researchers have made remarkable progress in improving the efficacy of CAR-T cell therapy.

However, its clinical benefit remains limited, especially in solid tumors.

Even in hematological malignancies, patients who respond to CAR-T therapy are still at risk of relapse due to multiple factors.

This is due to the frequent dysfunction of T cells, especially CAR-T cells, in cancer, and many evasion mechanisms impair anti-tumor immunity.


Compared with dysfunctional T cells, functional T cells exhibit a range of activities upon antigen stimulation, including

(1) expansion;

(2) secretion of effector cytokines (such as IL-2, IFN-γ, TNF- α , perforin and granzyme ) and lyse target cells;

(3) still survive after removal of antigen stimulation;

(4) still have the above-mentioned response when the second antigen challenge.

It can be argued that T cells are considered dysfunctional if at least one of the above criteria is not met.


Currently, the most studied T cell dysfunctional state is exhaustion, which is characterized by the loss of all the above-mentioned properties of functional T cells.

Another type of dysfunction is T cell senescence, which occurs when T cells permanently cease their cell cycle and proliferation while maintaining their cytotoxic capacity.


Dysregulation of T cells is a major obstacle to the development of CAR-T therapies, which is encountered from the beginning of the cell manufacturing step.

Including the design and in vitro operation of CAR, as well as culture conditions, may play a key role in it.

Therefore, engineering CAR-T cells to increase their survival and reverse or prevent the exhausted phenotype may improve CAR-T cell therapy. Several approaches to confining and creating anti-exhausted T cells have been taken by several studies so far and tested in several pivotal clinical trials.

These methods may improve the dysfunction of CAR-T cells and bring hope to patients.



T cell exhaustion

T cell exhaustion is closely related to the persistent driving effect of antigen. In the state of acute infection, naive CD8+ T cells differentiate into short-lived effector cells ( SLEC) or memory precursor effector cells ( MPEC ).

After antigen clearance, most SLECs die, while MPECs survive to form memory CD8+ T cells, forming long-term protective immunity.


However, under chronic infection, it was observed that high-affinity antigen-specific CD8+ T cells differentiated into SLECs, rapidly cleared and died, but did not form MPEC subpopulations.

In contrast, CD8+ T cells directed against low antigen affinity expand, become exhausted, exhibit marked reductions in proliferation, cytotoxicity, and cytokine production, and die in a stalemate against persistent antigens.


Mechanisms and adjustment strategies of CAR-T cell dysfunction


A common feature of chronic viral infection and cancer is that both are long-term diseases characterized by the persistence of antigens.

Naive CD8+ T cells targeting tumor antigens are first activated in peripheral lymphoid tissues to generate stem cell-like PD-1 lo CD8+ T cells with self-renewal properties. Under the action of chemokines CCL5 and CXCL9, they migrate to TME and form Effector PD-1 lo CD8+Tex .

However, persistent antigenic load in the TME ultimately forces these cells to continue to differentiate into loss-of-function PD-1 hi CD8+Tex .

PD-1 hi status was accompanied by enhanced expression of co-inhibitory receptors (including Tim-3, LAG-3, CD160, 2B4, TIGIT, and CTLA-4) and progressive loss of effector function.

Once CD8+Tex enters the PD-1 hi state, epigenetic enforcement prevents dedifferentiation back to a functional stem cell-like and effector-like PD-1 lo state. ICB (e.g., anti-PD-1)-promoted anti-tumor responses arise only from lymphoid or intratumoral amplification of PD-1 lo CD8+Tex subsets, hypofunctional, ICB-resistant PD-1 hi CD8+Tex eventually undergo apoptosis .





Both tumor and chronic virus-specific CD8+ T cells had significant enrichment of genes related to TCR signaling ( Batf, Egr2, Ezh2, Irf4, Nfatc1, Nfatc2, Nr4a1, Nr4a2, and Nr4a3 ).

This observation further supports the persistent engagement of antigens as a major driver of exhaustion.




T cell senescence

Cellular senescence is a typically multicausal process that occurs in a variety of cell types and is characterized by cell cycle arrest.

T cell senescence and T cell exhaustion are two distinct processes, and senescent cells exhibit a specific senescence-associated secretory phenotype ( SASP ) with paracrine effects on other cells.

Senescent T cells often exhibit a CD45RA+CD27−CD28−KLRG1+CD57+ phenotype, they lose the ability to proliferate and secrete IL-2, but express cytolytic molecules, IFN-γ and TNF-α, and are cytotoxic in vitro .


T cell senescence is often associated with the activation of the DNA damage response ( DDR ), which is triggered by telomere erosion, DNA damage by reactive oxygen species ( ROS ), glucose or growth factor deprivation, and activation of the cAMP pathway.

Senescent T cells exhibit p38 activation and increased accumulation of reactive oxygen species, impaired mitochondrial activity, downregulation of mTOR signaling, and shortened telomeres.

Despite undergoing cell cycle arrest, these cells were cytotoxic, similar to terminal effector T cells.


In addition, repeated activation during chronic infection or autoimmune disease has also been associated with T cell senescence.

Tumor-induced senescent T cells have been shown to suppress the activity of other T cells in the tumor microenvironment.

Accumulating evidence suggests that tumor-induced T cell senescence is an escape mechanism.



Mechanisms of CAR-T cell dysfunction

Transduced CAR-T cells exhibited similar dysfunctional mechanisms as unmodified T cells.

Indeed, CD8+ CAR-T cells and TILs isolated from the same tumor-bearing mice shared similar transcriptional and epigenetic profiles. Targeting pathways known to contribute to T cell dysfunction may improve CAR T cell function.


Mechanisms and adjustment strategies of CAR-T cell dysfunction


Design of the CAR

The type of extracellular CAR domain, the type of spacer linking the scFv to the transmembrane domain, and the costimulatory domain that constitutes the CAR have been shown to have profound effects on T cell function and persistence.

Most CARs used in clinical trials are derived from mice, which may lead to humoral and CD8+ T cell-mediated immune responses, leading to immune rejection.


The affinity of CAR affects tonic signaling, and too strong affinity for target cells may lead to T cell exhaustion.

One study developed a CAT-CAR ( CD19 single-chain antibody ) with a 40-fold lower affinity to CD19 than conventional CARs.

The construct was tested in relapsed or refractory pediatric BLL patients, and clinical studies found that 11 of 14 patients with increased CAR-T cell expansion showed persistence.


In addition, costimulatory domains also play key roles in the persistence and effectiveness of CAR-T cells, and costimulatory molecules include CD28, ICOS, CD27, 4-1BB, OX40, and CD40L.

Several studies found enhanced T cell persistence in CAR constructs equipped with 4-1BB domains but not CD28 domains.

Other studies have reported that 4-1BB-containing CAR-T cells display an enhanced memory T cell ( TEM ) phenotype, which may delay CAR-T cell exhaustion.

ICOS belongs to the family of CD28 co-stimulatory molecules. Studies have shown that the combination of ICOS and 4-1BB co-stimulatory domain in the CAR structure significantly increases the persistence of T cells.

These observations suggest that the structure of the co-stimulatory domain is an important factor affecting the persistence of CAR-T cells.


In vitro operation

The process of making T cells requires obtaining sufficient numbers of healthy T cells from the patient.

However, it is possible to obtain senescent T cells from elderly patients or patients pretreated with chemotherapy/total body irradiation .

Especially those chemotherapy containing clofarabine or doxorubicin, resulting in lymphopenia will make the quality of final CAR-T cells unsatisfactory, and the type of T cells used for infusion seriously affects the effect of treatment.


Indeed, better persistence was obtained in preclinical models by normalizing the CD4/CD8 ratio or using naive or memory cell subsets. Several studies have shown that the use of T cell populations enriched in early lineage cells enables better expansion and improves the durability and efficacy of CAR-T cell therapy.

In addition, multiple studies have shown that the addition of antioxidants, such as N-acetylcysteine, to cell culture during manufacturing also inhibits effector differentiation and promotes the expansion of TSCM cells.


Immunosuppressive microenvironment

In the TME, T cell exhaustion results from the presence of myeloid-derived suppressor cells ( MDSCs ), cancer-associated fibroblasts ( CAFs ), and immunosuppressive cytokines produced by tumor cells.

Chronic exposure to antigens can also lead to T cell exhaustion, and if antigenic stimulation persists, T cells undergo a series of epigenetic, metabolic, and transcriptional changes that are characteristic of an exhausted state.

This process occurs in a progressive manner, with IL-2 and TNF-α lost early and IFN-γ and chemokine secretion reduced later in the progression of exhaustion.

Although high proliferative capacity is also lost early, exhausted T cells are capable of limited proliferation when stimulated in vivo.

Depleted cells also showed high expression of inhibitory receptors, such as PD-1, TIM-3, LAG-3, CD160, BTLA, CTLA-4, and TIGIT.



Strategies to improve CAR-T cell dysfunction

Expression of cytokines and their receptors

Fourth-generation CAR T cells have recently been developed to counteract the immunosuppressive environment in the TME while overcoming immune exhaustion.

The TRUMKS design combines the cytotoxic function of CAR-T cells with the in situ delivery of cytokines with immunomodulatory capabilities.

Under the action of the induction system, after the CAR binds the antigen, cytokines are synthesized and act in an autocrine manner to increase the survival and expansion of T cells.

Cytokines can also act in a paracrine manner, modulating the surrounding environment and interfering with immunosuppressive cytokines present in the TME.

A series of cytokines including IL-12, IL-7, IL-15, IL-18, IL-21, and IL-23 are currently under investigation and have entered early clinical trials.


IL-12 is a proinflammatory cytokine that induces Th1CD4+ T cell responses and promotes CD8+ clonal expansion and persistence.

It is also responsible for regulating the cytotoxic activity of CTLs and natural killer ( NK ) cells, reactivating anergic tumor-infiltrating lymphocytes, recruiting NK cells, and suppressing Tregs.

Preclinical studies of CD19-CAR-T cells constitutively expressing IL-12 have shown that it enhances tumor-killing efficacy and immune memory against cancer antigens.

However, the potentially lethal toxicity associated with IL-12 necessitated the development of an induction system that limits IL-12 secretion only upon CAR activation.

Several clinical studies ( NCT02498912, NCT03932565, and NCT03542799 ) are ongoing and recruiting.


IL-15 is a cytokine that stimulates the activation, proliferation and cytotoxic activity of CD8+ T cells and NK cells.

IL-18 increases Th1 cell cytokine production while inhibiting IL-10 synthesis. Both IL-15 and IL-18 are boosters of the immune response and have been tested on CAR-T cells.

IL-18- and IL-15-secreting CAR-T cells showed enhanced expansion and persistence in tumor-bearing mice compared with conventional CAR-T cells, and enhanced tumor cell growth both in vitro and in vivo toxicity.

Multiple clinical trials are currently recruiting to test IL-15- and IL-18-secreted CARs in solid and hematologic tumors using engineered T and NK cells.


Recent studies have shown that it is possible to co-express multiple cytokines in the same CAR-T cell.

NCT04833504 is a recently completed clinical trial in which CD19+ CART cells expressing IL-7 and CCL19 were tested in patients with relapsed or refractory B-cell lymphoma, but the results have not yet been reported.

Two additional clinical trials are currently recruiting to test the secretion of IL-7-expressing CAR-T cells in combination with PD-1 blockade or other cytokines.


Combined Checkpoint Blockade Therapy

Strategies to reduce depletion by inhibiting checkpoint signaling include knockdown of co-repressor molecules via shRNA expression vectors or CRISPR/Cas9.

Many studies have reported that blocking checkpoint inhibition restores cytokine production and promotes CAR-T cell survival.

Furthermore, simultaneous blockade of multiple immune checkpoints, such as PD-1, TIM-3, and LAG-3, synergistically increases the effector function of CAR-T cells.


Combining CAR-T cells with immune checkpoint blockade therapy may be an effective strategy to enhance antitumor activity, persistence, and memory cell formation.

In glioblastoma and breast cancer cell lines, anti-PD-1 antibodies enhanced the antitumor activity of anti-HER2 CAR-T cells.

However, several clinical trials have reported negative results after treating neuroblastoma patients with a combination of PD-1 inhibitors and anti-GD2 CAR-T cells.


In addition, some studies have employed other strategies to block PD-1, such as gene editing to enable CAR-T cells to secrete PD-1 blocking antibodies or downregulate PD-1.


Use stem T cells

Effector T cells were initially considered the best product of ACT because of their ability to kill tumor cells. However, they have limited persistence, poor expansion and are prone to depletion.


TSCMs are ideal candidates for ACTs due to their long lifespan, strong self-renewal capacity, and ability to differentiate in diverse T cell populations.

Clinical studies have shown that infusion of phenotypic and functional TSCM – like CAR-T cells ( CD62L+, CD28+, and CD27+ ) yields favorable outcomes.

For example, CLL and multiple myeloma patients treated with anti-CD-19 CAR-T cells showed favorable responses associated with CD27 + CD45RO − CD8 cell populations.

Furthermore, studies in mice have shown that infusion of CD62L+-enriched T cell populations increases expansion and persistence, leading to durable tumor regression.





Despite the remarkable progress in CAR-T cell therapy in the past decade, the limited persistence of CAR-T cell dysfunction in patients remains a challenge, mainly due to T cell exhaustion and aging.

By designing the CAR structure, changing the production conditions, or introducing new therapeutic methods, we may create engineered T cells that are resistant to exhaustion, thereby further expanding the clinical application of CAR-T cells.

While results so far are still limited, the possibilities are endless and a breakthrough may be just around the corner.








1. CD8+ T Cell Exhaustion in Cancer. Front Immunol. 2021; 12: 715234.

2. Improving CAR T-Cell Persistence. Int J MolSci. 2021 Oct; 22(19): 10828.
3. Knowns and Unknowns about CAR-T Cell Dysfunction. Cancers (Basel). 2022 Feb; 14(4): 1078.

Mechanisms and adjustment strategies of CAR-T cell dysfunction

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