Selective CD28 blockade impacts T cell differentiation during homeostatic reconstitution following lymphodepletion

Abstract

Introduction: Costimulation blockade targeting the CD28 pathway provides improved long-term renal allograft survival compared to calcineurin inhibitors but may be limited as CTLA-4-Ig (abatacept, belatacept) blocks both CD28 costimulation and CTLA-4 coinhibition. Directly targeting CD28 while leaving CTLA-4 intact may provide a mechanistic advantage. Fc-silent non-crosslinking CD28 antagonizing domain antibodies (dAb) are currently in clinical trials for renal transplantation. Given the current standard of care in renal transplantation at most US centers, it is likely that lymphodepletion via thymoglobulin induction therapy could be used in patients treated with CD28 antagonists. Thus, we investigated the impact of T cell depletion (TCD) on T cell phenotype following homeostatic reconstitution in a murine model of skin transplantation treated with anti-CD28dAb.

Methods: Skin from BALB/cJ donors was grafted onto C56BL/6 recipients which were treated with or without 0.2mg anti-CD4 and 10μg anti-CD8 one day prior to transplant and with or without 100μg anti-CD28dAb on days 0, 2, 4, 6, and weekly thereafter. Mice were euthanized six weeks post-transplant and lymphoid cells were analyzed by flow cytometry.

Results: Anti-CD28dAb reversed lymphopenia-induced differentiation of memory CD4+ T cells in the spleen and lymph node compared to TCD alone. Mice treated with TCD+anti-CD28dAb exhibited significantly improved skin graft survival compared to anti-CD28dAb alone, which was also improved compared to no treatment. In addition, the expression of CD69 was reduced on CD4+ and CD8+ T cells in the spleen and lymph node from mice that received TCD+anti-CD28dAb compared to TCD alone. While a reduced frequency of CD4+FoxP3+ T cells was observed in anti-CD28dAb treated mice relative to untreated controls, this was balanced by an increased frequency of CD8+Foxp3+ T cells that was observed in the blood and kidney of mice given TCD+anti-CD28dAb compared to TCD alone.

Discussion: These data demonstrate that CD28 signaling impacts the differentiation of both CD4+ and CD8+ T cells during homeostatic reconstitution following lymphodepletion, resulting in a shift towards fewer activated memory T cells and more CD8+FoxP3+ T cells, a profile that may underpin the observed prolongation in allograft survival.

Keywords: T cell depletion; alloimmunity; costimulation blockade; homeostatic reconstitution; lymphodepletion; transplantation.

Figure 1

Selective CD28 blockade reverses lymphopenia-induced differentiation of memory CD4⁺ T cells in the lymph node. (A) Fully MHC-mismatched skin from BALB/c donors were grafted onto C57BL/6 recipients. Recipients were treated with or without 0.2mg anti-CD4 and 10μg anti-CD8 one day prior to transplant and with or without 100μg anti-CD28dAb on days 0, 2, 4, 6, and weekly thereafter. Mice were euthanized six weeks post-transplant and lymphoid cells from the blood, lymph node, spleen, bone marrow, kidney, liver, and lung were analyzed by flow cytometry. Immediately prior to euthanasia fluorescently labeled anti-CD4 and anti-CD8 antibody were administered IV to label circulating T cells. (B) Representative flow cytometry plots of CD4⁺ and CD8⁺ T cells on day 21 in the blood. (C) Frequency of CD4⁺ and CD8⁺ T cells in the blood over time. (D) Left, absolute count of CD4⁺ T cells per mL of blood over time. Right, absolute count of CD4⁺ T cells at day 21 and day 42. (E) Left, absolute count of CD8⁺ T cells per mL of blood over time. Right, absolute count of CD8⁺ T cells at day 21 and Day 42. (F) Proportion of CD44 expressing CD4⁺ and CD8⁺ T cells in the blood over time. (G) Proportion of CD44 expressing CD4⁺ and CD8⁺ T cells in the blood at day 21 and in the blood, spleen, and lymph node at day 42. Experiment shown is representative of 2 independent experiments with a total of 8-10 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001 by one way ANOVA correcting for multiple comparisons.

Figure 2

Selective CD28 blockade during T cell lymphodepletion and reconstitution improves skin graft survival and reduces expression of CD4⁺ T cell activation and senescence markers. (A) Median survival time of skin grafts was 19 days with no treatment (No Rx), 26 days for CD28dAb alone, and 37 days for CD28dAb+TCD. *p<0.05, **p < 0.01 by Mantel-Cox log-rank test. (B) Representative flow cytometric data depicting the frequency of CD69⁺CD4⁺ and CD69⁺CD8⁺ T cells in the lymph node at day 42. (C) Frequency of CD69⁺ CD4⁺ and CD8⁺ T cells in the spleen and lymph node at day 42. (D) MFI of PD-1 and TIGIT on CD4⁺ T cells in the spleen, lymph node, and bone marrow at day 42. Experiment shown is representative of 2 independent experiments with a total of 8-10 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one way ANOVA correcting for multiple comparisons.

Figure 3

Selective CD28 blockade reduces the frequency of CD4⁺ and CD8⁺ T_RM in the kidney but not during T cell lymphopenia-induced reconstitution. (A) Flow cytometric gating strategy to identify tissue resident T cells in the kidney. (B) Frequency of tissue resident CD69⁺CD103⁺ CD4⁺ and CD8⁺ T cells in the kidney at day 42. (C) Frequency of tissue resident CD69⁺CD103^- CD4⁺ and CD8⁺ T cells in the kidney at day 42. (D) Frequency of PD-1⁺ resident CD4⁺ and CD8⁺ T cells in the kidney day 42. N=3-5 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, by one way ANOVA correcting for multiple comparisons.

Figure 4

Selective CD28 blockade reduces the frequency of FoxP3⁺ CD4⁺ T cells but increases the frequency of FoxP3⁺ CD8⁺ T cells following T cell homeostatic reconstitution. (A) Representative flow cytometric data depicting the frequency of FoxP3⁺CD4⁺ T cells in the blood and kidney at day 42 (B) Frequency of FoxP3⁺CD4⁺ T cells in the blood, lymph node, spleen, kidney, liver, and lung at day 42. (C) Representative flow cytometric data depicting the frequency of FoxP3⁺CD8⁺ T cells in the blood and kidney at day 42. (C) Frequency of FoxP3⁺CD8⁺ T cells in the blood, lymph node, spleen, kidney, liver, and lung at day 42. Experiment shown is representative of 2 independent experiments with a total of 8-10 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by one way ANOVA correcting for multiple comparisons.

Figure 5

FoxP3⁺CD8⁺ T cells exhibit distinct cell surface expression profiles compared to FoxP3⁺CD4⁺ T cells in the blood and kidney. (A) Representative flow cytometric data of the indicated cell surface proteins expressed on total CD4⁺ and CD8⁺ T cells in the blood stratified by FoxP3 expression. (B) Representative flow cytometric data of the indicated cell surface proteins expressed on tissue resident CD4⁺ and CD8⁺ T cells in the kidney stratified by FoxP3 expression. Experiment shown is representative of 2 independent experiment with a total of 8-10 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001 by one way ANOVA correcting for multiple comparisons.