T Cell-Derived CD70 Delivers an Immune Checkpoint Function in Inflammatory T Cell Responses

Abstract

The CD27-CD70 pathway is known to provide a costimulatory signal, with CD70 expressed on APCs and CD27 functions on T cells. Although CD70 is also expressed on activated T cells, it remains unclear how T cell-derived CD70 affects T cell function. Therefore, we have assessed the role of T cell-derived CD70 using adoptive-transfer models, including autoimmune inflammatory bowel disease and allogeneic graft-versus-host disease. Surprisingly, compared with wild-type T cells, CD70^-/- T cells caused more severe inflammatory bowel disease and graft-versus-host disease and produced higher levels of inflammatory cytokines. Mechanistic analyses reveal that IFN-γ induces CD70 expression in T cells, and CD70 limits T cell expansion via a regulatory T cell-independent mechanism that involves caspase-dependent T cell apoptosis and upregulation of inhibitory immune checkpoint molecules. Notably, T cell-intrinsic CD70 signaling contributes, as least in part, to the inhibitory checkpoint function. Overall, our findings demonstrate for the first time, to our knowledge, that T cell-derived CD70 plays a novel immune checkpoint role in inhibiting inflammatory T cell responses. This study suggests that T cell-derived CD70 performs a critical negative feedback function to downregulate inflammatory T cell responses.

Figure 1. T cell-derived CD70 inhibits inflammatory bowel disease (IBD)

(A) Naïve RAG1^−/− mice in the C57BL/6 strain background were injected with 1×10⁶ CD4⁺CD25⁻ T cells purified from C57BL/6 WT or CD70^−/− mice. Body weight was monitored after adoptive transfer. Representative weight data from 1 out of 3 independent experiments were shown as mean ± SD (n=4 per group), with statistical significance determined by two-way ANNOVA. (B) Kaplan-Meier survival data were summarized by combining 3 independent experiments (n=10–11 per group), with statistical significance determined by Log-rank test. (C-D) Naïve RAG1^−/− mice were injected with 1×10⁶ CD4⁺CD25⁻ T cells purified from C57BL/6 WT mice, and then treated from day 1 with 100ug of either CD70 antibody (FR70) or IgG control twice weekly for the duration of the experiment. (C) Body weight was shown as mean ± SD, with statistical significance determined by two-way ANNOVA. (D) Kaplan-Meier survival curves were shown with statistical significance determined using Log-rank test. (E-F) RAG1^−/− host mice were sacrificed on days 17, 36 and 66. A blinded pathologist scored large (E) and small (F) intestines based on disease criteria outline in the Experimental Procedures. Summary scores combined from 3 independent experiments were shown, with statistical significance determined by unpaired student t test. (G) Representative histopathological images were shown for large intestine samples harvested from RAG1^−/− host mice at day 36 after adoptive transfer.

Figure 2. T cell-derived CD70 decreases the levels of inflammatory cytokines

Naïve RAG1^−/− mice in the C57BL/6 strain background were injected with 1×10⁶ CD4⁺CD25⁻ T cells purified from C57BL/6 WT or CD70^−/− mice. Serum samples were harvested via retro-orbital eye bleeding at days 10, 20 and 30 after adoptive transfer. Luminex assays were performed to measure the levels of the indicated cytokines. Each dot on the plots represents a single mouse at the indicated time points, with statistical significance determined by two-way ANNOVA.

Figure 3. T cell-derived CD70 suppresses T cell expansion by inducing caspase-dependent apoptosis

Naïve RAG1^−/− mice in the C57BL/6 strain background were injected with 1×10⁶ CD4⁺CD25⁻ T cells purified from C57BL/6 WT or CD70^−/− mice. (A) Host mice were sacrificed at the indicated days after adoptive transfer. Absolute numbers of CD4⁺ T cells were calculated by multiplying the total numbers of spleen cells by the percentages of TCRβ⁺CD4⁺ T cells present in spleen samples determined by flow cytometry. (B–C) WT and CD70^−/− CD4⁺CD25⁻ T cells were stained with the cell proliferation dye ef670 and then injected into RAG1^−/− host mice, which were then sacrificed at day 6 after adoptive transfer. TCRβ⁺CD4⁺ T cell proliferation was measured by ef670 dilution with data shown as mean ± SD (n=5 per group), with statistical significance determined by unpaired student t test. (D–G) Day 6 after adoptive transfer, host mice were sacrificed and spleen cells were analyzed by flow cytometry. TCRβ⁺CD4⁺ T cells were gated to examine a live/dead marker versus active caspase-8 or caspase-3. Cells positive for both live/dead and caspase, considered dead via apoptosis, and cells positive for only caspase, dying via apoptosis, were added to determine the percent of cells that activated Caspase-8 and −3 pathways for apoptosis. Shown are representative flow plot (D) and summary data (E) of caspase-8 activation. Also shown are representative flow plot (F) and summary data (G) of caspase-3 activation. Summary data are combined from 3 independent experiments. Each dot represents a single mouse, with statistical significance determined by unpaired student t test.

Figure 4. T cell-derived CD70 is associated with higher levels of checkpoint signals on T cells

Naïve RAG1^−/− mice in the C57BL/6 strain background were injected with 1×10⁶ CD4⁺CD25⁻ T cells purified from C57BL/6 WT or CD70^−/− mice. Day 6 after adoptive transfer, host mice were sacrificed and spleens removed to analyze the expression of CTLA-4, PD-1, TIM-3, and LAG-3 by flow cytometry. Also analyzed are the purified CD4⁺CD25⁻ T cells as pre-transplant controls. TCRβ⁺CD4⁺ T cells were gated to examine the percentages of cells that are positive for the indicated checkpoint signals. Statistical significance was determined by unpaired student t test.

Figure 5. IFN-γ stimulates T cell-derived CD70 expression and function

CD4⁺CD25⁻ T cells were isolated from naïve C57BL/6 WT and CD70^−/− mice and added with soluble anti-CD28 to a 48-well plate coated with anti-CD3 the day prior. 0.5×10⁶ T cells were added into each well in 0.5ml medium and treated with IFN-γ at 10ng/ml. After 48 hours T cells were harvested and analyzed by flow cytometry for CD70 expression. Shown are representative flow plots (A) and summary data (B) from 3 independent experiments with similar results. Statistical significance was determined by unpaired student t test. (C) TCRβ⁺CD4⁺ T cells were gated to examine a live/dead marker versus active caspase-8. Cells positive for both live/dead and caspase-8 were added to show the percent of cells that activated Caspase-8 pathway for apoptosis. (D) TCRβ⁺CD4⁺ T were gated to examine expression of TIM-3. Statistical significance was determined by unpaired student t test.

Figure 6. T cell intrinsic CD70 signaling contributes to the inhibitory checkpoint function

CD4⁺CD25⁻ T cells were isolated from naïve C57BL/6 WT and CD70^−/− mice and added with soluble anti-CD28 to a 48-well plate coated with anti-CD3 the day prior. 0.5×10⁶ T cells were added into each well in 0.5ml medium and treated with IFN-γ at 10ng/ml. 22 hours later the Rat anti-CD70 antibody was added at 5ug/ml to “cross-linking” wells and then 2 hours later a secondary anti-Rat antibody was added to all wells and incubated for another 24 hours. Cells were harvested and analyzed by flow cytometry for active caspase-8 (A) and TIM-3 expression (B). (C) Naïve RAG1^−/− mice were injected with 0.5×10⁶ CD4⁺CD25⁻ T cells purified from CD45.1 mice mixed with 0.5×10⁶ CD4⁺CD25⁻ T cells from CD45.2 CD70^−/− mice. Day 17 after adoptive transfer, spleens were harvested to analyze the percentages of CD45.1 versus CD45.2 cells in the gated TCRβ⁺CD4⁺ T cells. Statistical significance was determined by unpaired student t test.

Figure 7. T cell-derived CD70 inhibits allogeneic CD4⁺ T cell response

Lethally irradiated BALB/c or FVB hosts were transplanted with CD4⁺CD25⁻ T cells purified from naïve C57BL/6 WT or CD70^−/− mice. (A) Kaplan-Meier survival curves of BALB/c hosts injected with 2×10⁴ CD4⁺CD25⁻ T cells and 3×10⁶ TCD BM. Data were summarized by combining 2 independent experiments (n=16 per group). (B) Kaplan-Meier survival curves of FVB hosts injected with 1×10⁶ CD4⁺CD25⁻ T cells and 3.5×10⁶ TCD BM. Data were summarized by combining 2 independent experiments (n=8 per group). (A–B) Statistical significance was determined by Log-rank test. (C–F) Lethally irradiated BALB/c hosts were injected with 5×10⁵ CD4⁺CD25⁻ T cells and 3×10⁶ TCD BM and sacrificed on Day 5. (C) Absolute numbers of CD4⁺ T cells were calculated by multiplying the total numbers of spleen cells by the percentages of TCRβ⁺CD4⁺ T cells present in spleen samples determined by flow cytometry. Summary data of caspase-8 activation (D) and caspase-3 activation (E) were combined from 3 independent experiments. (F) Summary data of CTLA-4, PD-1, TIM-3, and LAG-3 expression on TCRβ⁺CD4⁺ T cells at Day 5 after allo-HCT. Statistical significance was determined by unpaired student t test.

Figure 8. T cell-derived CD70 inhibits allogeneic CD8⁺ T cell response

Lethally irradiated BM1 or FVB hosts were transplanted with CD8⁺ T cells purified from naïve C57BL/6 WT or CD70^−/− mice. (A) Body weight was monitored after BM1 hosts injected with 2×10⁶ CD8⁺ T cells and 3.5×10⁶ TCD BM. Representative data from 1 of 2 experiments is shown as mean ± SD (n=5 per group), with statistical significance determined by two-way ANNOVA. (B) Kaplan-Meier survival data of BM1 hosts are combined from 2 independent experiments (n=8–9 per group). Statistical significance was determined by Log-rank test. (C–F) FVB hosts injected with 1×10⁶ CD8⁺ T cells and 3×10⁶ TCD BM and sacrificed on Day 9 or 12. (C) Absolute numbers of CD8⁺ T cells were calculated by multiplying the total numbers of spleen cells by the percentages of TCRβ⁺CD8⁺ T cells present in spleen samples determined by flow cytometry on day 12. Summary data of caspase-8 activation (D) and caspase-3 activation (E) are combined from 3 independent experiments. (F) Summary data of CTLA-4, PD-1, TIM-3, LAG-3 expression on TCRβ⁺CD8⁺ T cells at Day 9 after allo-HCT. Statistical significance was determined by unpaired student t test.