Reduced CD5 on CD8+ T Cells in Tumors but Not Lymphoid Organs Is Associated With Increased Activation and Effector Function

Abstract

CD5, a member of the scavenger receptor cysteine-rich superfamily, is a marker for T cells and a subset of B cells (B1a). CD5 associates with T-cell and B-cell receptors and increased CD5 is an indication of B cell activation. In tumor-infiltrating lymphocytes (TILs) isolated from lung cancer patients, CD5 levels were negatively correlated with anti-tumor activity and tumor-mediated activation-induced T cell death, suggesting that CD5 could impair activation of anti-tumor T cells. We determined CD5 levels in T cell subsets in different organs in mice bearing syngeneic 4T1 breast tumor homografts and assessed the relationship between CD5 and increased T cell activation and effector function by flow cytometry. We report that T cell CD5 levels were higher in CD4⁺ T cells than in CD8⁺ T cells in 4T1 tumor-bearing mice, and that high CD5 levels on CD4⁺ T cells were maintained in peripheral organs (spleen and lymph nodes). However, both CD4⁺ and CD8⁺ T cells recruited to tumors had reduced CD5 compared to CD4⁺ and CD8⁺ T cells in peripheral organs. In addition, CD5^high/CD4⁺ T cells and CD5^high/CD8⁺ T cells from peripheral organs exhibited higher levels of activation and associated effector function compared to CD5^low/CD4⁺ T cell and CD5^low/CD8⁺ T cell from the same organs. Interestingly, CD8⁺ T cells among TILs and downregulated CD5 were activated to a higher level, with concomitantly increased effector function markers, than CD8⁺/CD5^high TILs. Thus, differential CD5 levels among T cells in tumors and lymphoid organs can be associated with different levels of T cell activation and effector function, suggesting that CD5 may be a therapeutic target for immunotherapeutic activation in cancer therapy.

Keywords: CD4+ T cells; CD5; CD8+ T cells; activation; exhaustion; tumor-infiltrating lymphocytes (TILs).

Figure 1

CD5 levels in T cell subsets and lymphoid organs. T cells isolated from 4T1-harboring BALB/c mice were stained with fluorescence-conjugated anti-CD3, anti-CD4, anti-CD8, and anti-CD5 MAbs. (A) CD5 levels on CD8⁺ T cells and CD4⁺ T cells in lymph nodes and spleen. (B) The fraction of CD5⁺/CD4⁺ T cell solated from the lymph nodes, spleen, and TILs. (C) The fraction of CD5⁺/CD8⁺ T cells isolated from the lymph nodes, spleen, and TILs. Data are shown as means ± SD of 3 mice per group and from one representative experiment of three independent experiments. FMO, Fluorescence Minus One Control; TILs, tumor-infiltrating lymphocytes; MFI, mean fluorescence intensity; ns, not significant. *p < 0.05 (Student’s paired two-tailed t-test).

Figure 2

CD5 levels correlate with PD-1, CD69, and CTLA-4 in lymphoid organs. Splenocytes and lymphocytes isolated from 4T1-harboring BALB/c mice were stained with fluorescence-conjugated antibodies to assess PD-1, CD69, and CTLA-4 in correlation to CD5. (A) The fraction of PD-1⁺/CD5^high/CD4⁺, CD69⁺/CD5^high/CD4⁺, CTLA-4⁺/CD5^high/CD4⁺ T, PD-1⁺/CD5^low/CD4⁺, CD69⁺/CD5^low/CD4⁺, and CTLA-4⁺/CD5^low/CD4⁺ T cells in spleen and lymph nodes, respectively. (B) The fraction of PD-1⁺/CD5^high/CD8⁺, CD69⁺/CD5^high/CD8⁺, CTLA-4⁺/CD5^high/CD8⁺ vs PD-1⁺/CD5^low/CD8⁺, CD69⁺/CD5^low/CD8⁺, and CTLA-4⁺/CD5^low/CD8⁺ T cells in spleen and lymph nodes, respectively. Data are shown as means ± SD of 5 or 3 mice per group and from one representative experiment of three independent experiments. *p < 0.05 (Student’s paired two-tailed t-test. ns, not significant).

Figure 3

CD5 levels and their correlation with PD-1, CD69 and CTLA-4 in TILs. TILs isolated from 4T1-harboring BALB/c mice were stained with fluorescence-conjugated antibodies to assess PD-1, CD69 and CTLA-4 in correlation with CD5. (A) The fraction of PD-1⁺/CD5^high/CD4⁺, CD69⁺/CD5^high/CD4⁺, CTLA-4⁺/CD5^high/CD4⁺ vs PD-1⁺/CD5^low/CD4⁺, CD69⁺/CD5^low/CD4⁺, and CTLA-4⁺/CD5^low/CD4⁺ TILs. (B) The fraction of PD-1⁺/CD5^high/CD8⁺, CD69⁺/CD5^high/CD8⁺, CTLA-4⁺/CD5^high/CD8⁺ vsPD-1⁺/CD5^low/CD8⁺, CD69⁺/CD5^low/CD8⁺, and CTLA-4⁺/CD5^low/CD8⁺ TILs. Data are shown as means ± SD of 4 or 3 mice per group and from one representative experiment of three independent experiments. *p < 0.05 (Student’s paired two-tailed t-test. ns, not significant).

Figure 4

CD5 levels and their correlation with IFNγ, CD107a and CD137 in lymphoid organs. Splenocytes and lymphocytes isolated from 4T1-harboring BALB/c mice were stained with fluorescence-conjugated antibodies to assess IFNγ, CD107a and CD137 in correlation with CD5. (A) The fraction of IFNγ⁺/CD5^high/CD4⁺, CD107a⁺/CD5^high/CD4⁺, CD137⁺/CD5^high/CD4⁺ vs IFNγ⁺/CD5^low/CD4⁺, CD107a⁺/CD5^low/CD4⁺, and CD137⁺/CD5^low/CD4⁺ T cell in spleen and lymph nodes, respectively. (B) The fraction of IFNγ⁺/CD5^high/CD8⁺, CD107a⁺/CD5^high/CD8⁺, CD137⁺/CD5^high/CD8⁺ vs IFNγ ⁺/CD5^low/CD8⁺, CD107a ⁺/CD5^low/CD8⁺, and CD137⁺/CD5^low/CD8⁺ T cells in spleen and lymph nodes, respectively. Data are shown as means ± SD of 3 mice per group and from one representative experiment from two independent experiments. *p < 0.05 (Student’s unpaired two-tailed t-test. ns, not significant).

Figure 5

CD5^-/low/CD4⁺ and CD5^-/low/CD8⁺ TILs have increased effector function (IFNγ, CD107a, and CD137 expression) compared to CD5^high TILs. TILs isolated from 4T1-harboring BALB/c mice were stained with fluorescence-conjugated antibodies to assess IFNγ, CD107a and CD137 in relationship with CD5. (A) The fraction of IFNγ⁺/CD5^high/CD4⁺, CD107a⁺/CD5^high/CD4⁺, CD137⁺/CD5^high/CD4⁺ vs IFNγ⁺/CD5^-/low/CD4⁺, CD107a⁺/CD5 ^-/low/CD4⁺, and CD137⁺/CD5 ^-/low/CD4⁺ TILs, respectively. (B) The fraction of IFNγ⁺/CD5^high/CD8⁺, CD107a⁺/CD5^high/CD8⁺, CD137⁺/CD5^high/CD8⁺ vs IFNγ ⁺/CD5^-/low/CD8⁺, CD107a ⁺/CD5^-/low/CD8⁺, and CD137⁺/CD5^-/low/CD8⁺ TILs, respectively. Data are shown as means ± SD of 4 or 3 mice per group and from one representative experiment of two independent experiments. *p < 0.05 (Student’s paired two-tailed t-test).

Figure 6

Both CD5^-/low/CD4⁺ and CD5^-/low/CD8⁺ TILs have increased effector function (co-expression of PD-1 and CD69). (A) The fraction of PD-1⁺/CD69⁺/CD5^-/low/CD4⁺ TILs and PD-1⁺/CD69⁺/CD5^high/CD4⁺ TILs. (B) The fraction of PD-1⁺/CD69⁺/CD5^-/low/CD8⁺ TILs and PD-1⁺/CD69⁺/CD5^high/CD8⁺ TILs. Data are shown as means ± SD of 3 mice per group and from one representative experiment of three independent experiments. *p < 0.05 (Student’s paired two-tailed t-test).