Deletion of Cbl-b inhibits CD8⁺ T-cell exhaustion and promotes CAR T-cell function

Abstract

BackgroundChimeric antigen receptor (CAR) T-cell therapy is an emerging option for cancer treatment, but its efficacy is limited, especially in solid tumors. This is partly because the CAR T cells become dysfunctional and exhausted in the tumor microenvironment. However, the key pathways responsible for impaired function of exhausted cells remain unclear, which is essential to overcome CAR T-cell exhaustion.MethodsAnalysis of RNA-sequencing data from CD8+ tumor-infiltrating lymphocytes (TILs) led to identification of Cbl-b as a potential target. The sequencing data were validated using a syngeneic MC38 colon cancer model. To analyze the in vivo role of Cbl-b in T-cell exhaustion, tumor growth, % PD1+Tim3+ cells, and expression of effector cytokines were analyzed in cbl-b+/+ and cbl-b–/– mice. To evaluate the therapeutic potential of Cbl-b depletion, we generated a new CAR construct, hCEAscFv-CD28-CD3ζ.GFP, that recognizes human carcinoembryonic antigen (CEA). cbl-b+/+ and cbl-b–/– CEA-CAR T cells were generated by retroviral transduction. Rag–/– mice bearing MC38-CEA cells were injected with cbl-b+/+ and cbl-b–/–; CEA-CAR T cells, tumor growth, % PD1+Tim3+ cells and expression of effector cytokines were analyzed.ResultsOur results show that the E3 ubiquitin ligase Cbl-b is upregulated in exhausted (PD1+Tim3+) CD8+ TILs. CRISPR-Cas9-mediated inhibition of Cbl-b restores the effector function of exhausted CD8+ TILs. Importantly, the reduced growth of syngeneic MC38 tumors in cbl-b–/– mice was associated with a marked reduction of PD1+Tim3+ CD8+ TILs. Depletion of Cbl-b inhibited CAR T-cell exhaustion, resulting in reduced MC38-CEA tumor growth, reduced PD1+Tim3+ cells and increased expression of interferon gamma, tumor necrosis factor alpha, and increased tumor cell killing.ConclusionOur studies demonstrate that deficiency of Cbl-b overcomes endogenous CD8+ T-cell exhaustion, and deletion of Cbl-b in CAR T cells renders them resistant to exhaustion. Our results could facilitate the development of efficient CAR T-cell therapy for solid tumors by targeting Cbl-b.

MAPK10 Expression as a Prognostic Marker of the Immunosuppressive Tumor Microenvironment in Human Hepatocellular Carcinoma

“…Consistently, signals such as MAPK10, which promote the continuous infiltration of immune cells into tumor microenvironment, may improve the prognosis of HCC patients in clinical immunotherapeutic treatment. In a recent clinical trial, cancer patients could not obtain desirable benefit and favorable therapeutic efficacy from immunotherapy using PD-1 antibody and PD-L1 antibody treatment if tumor infiltration lymphocytes were exhausted in the TME (81)(82)(83)(84) or if the cancer cells did not express programmed death-ligand 1 (PD-L1) (85)(86)(87)(88)(89). Under these or comparable circumstances, boosting of MAPK10-ICAM1 axis in HCC could facilitate immune activity in the TME and support the efficacy of existing immune therapies.…”

Ubiquitination in T-Cell Activation and Checkpoint Inhibition: New Avenues for Targeted Cancer Immunotherapy

“…Cbl-b −/− CD8 + T cells can also confer anti-tumor activity and reject tumors if adoptively transferred to tumor-bearing mice [ 125 , 126 , 130 ]. Cbl-b −/− mice have less exhausted T lymphocytes present in the tumor environment, and targeted depletion of Cbl-b, via CRISPR/Cas9, can restore the expression of inflammatory cytokines and cytotoxic molecules in wild-type exhausted PD1 + Tim3 + T cells [ 131 ]. Importantly, absence of Cbl-b could also render T cells resistant to the tolerogenic tumor microenvironment, since T cells from Cbl-b −/− mice cannot be anergized, neither in vitro upon incubation with ionomycin nor in vivo, in P14 or OTII TCR transgenic mouse models of anergy [ 132 , 133 ], and are resistant to Treg and TGF-β suppression [ 125 , 126 , 134 ] well as to PD-1 or CTLA-4 inhibition [ 135 , 136 ].…”

“…In mice, Cbl-b was found to be up-regulated in the PD-1 + Tim3 + exhausted CD8 + T cells infiltrating MC38 colon tumor, and Cbl-b −/− mice had reduced numbers of exhausted T cells in the tumor microenvironment. Importantly, CRISPR/Cas9-mediated depletion of Cbl-b in sort-isolated PD-1 + Tim3 + T cells could largely restore CD8 + effector functions and absence of Cbl-b ( Cbl-b −/− ) in CAR-T cells specific for the human carcinoembryonic antigen (hCEA) increased overall mice survival, enhanced anti-tumor activity, and resulted in less PD1 + Tim3 + -exhausted tumor infiltrating CAR-T cells when transferred to mice carrying hCEA-expressing MC38 tumors [ 131 ]. The other Cbl E3 ligase, c-Cbl, also affects PD-1, but contrary to Cbl-b, as a negative regulator.…”

“…Small peptides and small molecule inhibitors have been able to efficiently block in vivo Cbl-b interactions with a specific substrate [ 253 ] or inhibit directly the function of the Cbl-b relevant substrate [ 129 ]; yet, these approaches depend on substrate identification, which is often challenging. CRISPR/Cas9 technology has been successfully used in mice for targeted Cbl-b depletion in order to efficiently reconstitute the effector functions of exhausted tumor-infiltrating T cells [ 131 ]. Given that ubiquitin variants have been successfully developed to specifically inhibit the activated from of c-Cbl by blocking the E2-ubiquitin-binding site [ 254 ], it is likely that a similar approach could be utilized to inhibit Cbl-b.…”

Strengthening the CAR‐T cell therapeutic application using CRISPR/Cas9 technology

“…Cbl‐b is believed to be associated with exhaustion of T cells; therefore, Cbl‐b was knocked‐out and its deficiency in T cells was studied. Interestingly, Cbl‐b deletion suppressed CAR‐T cell exhaustion and restored their effector function (Kumar et al, 2021). Despite the remarkable results obtained from Cbl‐b deletion in T cells, more investigations are required to shed light on the mechanism of how Cbl‐b deficiency leads to reduced levels of T cell exhaustion.…”