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

Abstract

Background: Chimeric 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.

Methods: Analysis 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.

Results: Our 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.

Conclusion: Our 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.

Keywords: CD8-positive T-lymphocytes; adoptive; cell engineering; immunotherapy.

Figure 1

Cbl-b is upregulated in exhausted CD8⁺PD1⁺Tim3⁺ TILs. (A) Geo dataset (GSE85947) was analyzed and normalized read counts (TPM) for Cbl-b are shown. (B) Volcano plot of differentially expressed genes (−log₁₀ p value vs log2 fold change) in PD1⁺Tim3⁺ versus PD1^–Tim3^– TILs. (C) MC38 tumors were implanted subcutaneously into C57BL6 mice; PD1^–Tim3^– and PD1⁺Tim3⁺ TILs were sorted; and Cbl-b messenger RNA levels were analyzed by real-time PCR. (D) Immunoblot of Cbl-b protein using Fluorescence-activated cell sorted (FACS) PD1^–Tim3^– versus PD1⁺Tim3⁺ TILs. The data are representative of three independent experiments. Statistics are mean±SD, calculated by Student’s t-test (two-tailed). TIL, tumor-infiltrating lymphocyte. TPM, transcript per million; IB, Immunoblotting, FACS, Fluorescence-activated cell sorting.

Figure 2

Inhibition of Cbl-b restores effector function of CD8⁺PD1⁺Tim3⁺ TILs. Cbl-b was knocked out using Cbl-b-guide RNA-pLenti-CRISPR-v2 in sorted CD8⁺PD1⁺Tim3⁺cells from MC38 tumor-bearing C57BL6 mice. (A) Expression of IFN-γ, (B) TNF-α, (C) granzyme B and (D) IL-2 was measured by real-time PCR. The data are representative of three independent experiments. Statistics are mean±SD, calculated by Student’s t-test (two tailed). IFN-γ, intergeron gamma; IL, interleukin; TIL, tumor-infiltrating lymphocyte; TNF-α, tumor necrosis factor alpha, gRNA. guide RNAs.

Figure 3

Reduced MC38 tumor growth in cbl-b^–/– mice. cbl-b^+/+ and cbl-b^–/– mice (n=5) were subcutaneously injected with MC38-CEA cells. (A) Tumor volume. (B) Representative images of tumors. (C) Percentage of CD8⁺PD1⁺Tim3⁺ TILs. (D) Representative image showing percentage of CD8⁺PD1⁺Tim3⁺ TILs. (E) CD8⁺PD1⁺Tim3⁺ TILs were sorted and expression of IFN-γ, TNF-α and granzyme B was analyzed by real-time PCR. The data are representative of three independent experiments. Statistics are mean±SD, calculated by Student’s t-test (two-tailed). CEA, carcinoembryonic antigen; IFN-γ, intergeron gamma; TIL, tumor-infiltrating lymphocyte; TNF-α, tumor necrosis factor alpha.

Figure 4

Depletion of Cbl-b inhibits CAR T-cell exhaustion and promotes tumor regression. (A) Schematic representation of the CEA-reactive CAR construct. (B) 6×10⁶ cbl-b^+/+ and cbl-b^–/– CAR T cells were adoptively transferred into Rag^–/– mice (n=6) on days 3 and 8 after tumor inoculation. Percentage survival of Rag^–/– mice that received cbl-b^+/+ and cbl-b^–/– CAR T cells. The mice were considered dead when tumor size became 15 mm. Log-rank test was used for statistical analysis. (C) Tumor volume is shown; (D) sorted cbl-b^+/+ and cbl-b^–/– CD8⁺PD1⁺Tim3⁺ TILs were cocultured with MC38-CEA cells (target) labeled with calcein-AM. Specific lysis and expression of IFN-γ, TNF-α and granzyme B are shown. (E) Representative images of spleen and H&E-stained sections of the colon, lung and liver. The data are representative of three independent experiments. Statistics are mean±SD, calculated by Student’s t-test (two-tailed). AM, acetoxymethyl; CAR, chimeric antigen receptor; CEA, carcinoembryonic antigen; hCEA, human carcinoembryonic antigen; IFN-γ, intergeron gamma; scFv, single-chain variable fragment; TIL, tumor-infiltrating lymphocyte; TNF-α, tumor necrosis factor alpha.