High CTLA-4 expression on Th17 cells results in increased sensitivity to CTLA-4 coinhibition and resistance to belatacept

Abstract

The CD28/cytotoxic T-lymphocyte antigen 4 (CTLA-4)blocker belatacept selectively inhibits alloreactive T cell responses but is associated with a high incidence of acute rejection following renal transplantation,which led us to investigate the etiology of belatacept–resistant graft rejection. T cells can differentiate into functionally distinct subsets of memory T cellsthat collectively enable protection against diverse classes of pathogens and can cross-react with allogeneicantigen and mediate graft rejection. T helper 17(Th17) cells are a pro-inflammatory CD4+ lineage that provides immunity to pathogens and are pathogenic in autoimmune disease. We found that T helper 1 (Th1)and Th17 memory compartments contained a similar frequency of divided cells following allogeneic stimulation.Compared to Th1 cells, Th17 memory cells expressed significantly higher levels of the coinhibitory molecule CTLA-4. Stimulation in the presence of belatacept inhibited Th1 responses but augmented Th17 cells due to greater sensitivity to coinhibition by CTLA-4. Th17 cells from renal transplant recipients were resistant to ex vivo CD28/CTLA-4 blockade with belatacept, and an elevated frequency of Th17 memory cells was associated with acute rejection during belatacept therapy. These data highlight important differences in costimulatory and coinhibitory requirements of CD4+ memory subsets, and demonstrate that the heterogeneity of pathogen-derived memory has implications for immunomodulation strategies.

Figure 1. CD45RA⁻ Th17 Cells are Alloreactive and Resistant to Belatacept

CFSE labeled T cell responders were stimulated with allogeneic MDDCs for 4 d followed by brief restimulation with PMA/Iono or not. Data depicted are from five independent stimulator/responder pairs. (A–B) Cells were gated on CD4⁺CD45RA⁺CFSE^low or CD4+CD45RA−CFSE^low, followed by either IFN-γ⁺ or IL-17⁺ as described. (A) Representative frequencies of Th1 and Th17 cells among CD45RA⁺CCR6⁺CFSE^low cells with and without PMA/Iono restimulation. (B) Summary frequencies of Th1 and Th17 cells within the CD45RA⁻CCR6⁻ and CD45RA⁻CCR6⁺ subsets. (C–E) Cells were first gated on CD4⁺CD45RA⁺IFN-γ⁺ (CD45RA⁺ Th1), CD4⁺CD45RA⁻IFN-γ⁺ (CD45RA⁻ Th1), or CD4⁺CD45RA⁻CCR6⁺IL-17⁺ (CD45RA⁻CCR6⁺ Th17), followed by CFSE^low divided cells. (C) Concatenated histograms depicting the frequency of CFSE^low alloreactive CD45RA⁺ Th1, CD45RA⁻ Th1, and CD45RA⁻ CCR6⁺ Th17 cells in the absence (left column) or presence (right column) of belatacept following allogeneic co-culture. (D) Frequency of divided CD45RA⁺ Th1, CD45RA⁻ Th1 and CD45RA⁻CCR6⁺ Th17 cells (CD45RA⁺ Th1/Th17 p = 0.310, CD45RA⁻ Th1/Th17 p = 0.421). (E) Relative frequency of divided CD45RA⁺ Th1, CD45RA⁻ Th1, and CD45RA⁻CCR6⁺ Th17 following allogeneic stimulation in the presence of belatacept compared to no treatment (CD45RA⁺ Th1/Th17 p = 0.0079, CD45RA⁻ Th1/Th17 p = 0.0079).

Figure 2. Th17 Memory Cells Express High Levels of CTLA-4

Peripheral blood T cells were stimulated briefly with PMA/Iono and analyzed by flow cytometry. CD4⁺ T cell subsets were defined by the following gating strategy: Primary Th1, CD4⁺CD45RA⁺IFN-γ⁺; Memory Th1, CD4⁺CD45RA⁻ IFN-γ⁺; Memory Th17, CD4⁺CD45RA⁻CCR6⁺IL-17⁺. (A) Representative histogram (top) and summary data (bottom) of surface CD28⁺ CD4⁺ populations resting or restimulated with PMA/Iono (p = 0.092, n = 10). (B) Top, CD28 and total CTLA-4 expression on resting and restimulated Th1 and Th17 memory cells. Quadrant gate depicts CD28⁺ and CTLA-4⁺ population, square gate defines CTLA-4^high population. Bottom, total CTLA-4 expression on resting and restimulated Th1 and Th17 memory cells. Gate depicts CTLA-4^high expression (n = 8). (C) Frequency of CTLA-4^high cells among Th1 and Th17 memory cells (p = 0.0009, n = 8). (D) Representative frequency (left) and summary frequencies of surface CTLA-4⁺ CD4⁺ populations (p < 0.0001, n = 9). (E) Mean fluorescence intensity of CTLA-4 on restimulated CD4⁺ IFN-γ⁺ and IL-17⁺ memory cells (p < 0.0001, n = 9).

Figure 3. CD45RA⁻ Th17 Cells are Resistant to Inhibition by Belatacept and More Dependent on Coinhibition by CTLA-4

Peripheral blood T cells from healthy donors were stimulated with anti-CD3 in the presence of blocking molecules or relevant control for 3 d followed by brief restimulation with PMA/Iono. CD4⁺ T cell subsets were defined by the following gating strategy: CD45RA⁺ Th1, CD4⁺CD45RA⁺IFN-γ⁺; CD45RA⁻ Th1, CD4⁺CD45RA⁻ IFN-γ⁺; CD45RA⁻CCR6⁺ Th17, CD4⁺CD45RA⁻CCR6⁺IL-17⁺. (A) Representative frequency of CD45RA⁺ Th1, CD45RA⁻ Th1, and CD45RA⁻CCR6⁺ Th17 after stimulation with anti-CD3 and belatacept or IgG-Fc control. (B) Relative change in frequencies of CD45RA⁺ Th1, CD45RA⁻ Th1, and CD45RA⁻CCR6⁺ Th17 cells after anti-CD3 stimulation and treatment with belatacept compared to IgG-Fc among multiple individuals (CD45RA⁺ Th1/Th17 p = 0.0006, CD45RA⁻ Th1/Th17 p = 0.0006, n = 7). (C) Representative frequency of CD45RA⁺ Th1, CD45RA⁻ Th1, and CD45RA⁻CCR6⁺ Th17 cells after stimulation with anti-CD3/CD28 beads and anti-CTLA-4 or IgG control. (D) Relative change in frequencies of CD45RA⁺ Th1, CD45RA⁻ Th1, and CD45RA⁻CCR6⁺ Th17 cells after stimulation with anti-CD3/CD28 beads and anti-CTLA-4 or IgG control (CD45RA⁺ Th1/Th17 p = 0.0152, CD45RA⁻ Th1/Th17 p = 0.0152, n = 6).

Figure 4. Renal Transplant Recipient Lymph Node CD45RA⁻ Th17 Cells are Resistant to Belatacept

T cells from explanted renal transplant recipient lymph nodes were stimulated with anti-CD3 for 3 d in the presence of belatacept or IgG-Fc control followed by brief restimulation with PMA/Iono (n = 5/group). CD4⁺ T cell subsets were defined by the following gating strategy: CD45RA⁺ Th1, CD3⁺CD4⁺CD45RA⁺IFN-γ⁺; CD45RA⁻ Th1, CD3⁺CD4⁺CD45RA⁻IFN-γ⁺; CD45RA⁻ Th17, CD3⁺CD4⁺CD45RA⁻IL-17⁺ (A) Representative frequency and (B) summary data of CD45RA⁺ Th1, CD45RA⁻ Th1, and CD45RA⁻ Th17 cells after proliferation and brief restimulation (CD45RA⁺ Th1/Th17 p = 0.0049, CD45RA⁻ Th1/Th17 p = 0.0049).

Figure 5. Renal Transplant Patients Experiencing Acute Rejection on Belatacept-based Regimens Have Elevated Frequencies of Th17 Memory Cells

Peripheral T cells from renal transplant patients classified as Belatacept Stable, Belatacept Rejectors, or Tacrolimus Rejectors (as described in the Methods) were stimulated for 4 h with PMA/Iono (n = 4–8/group). CD4⁺ T cell subsets were defined by the following gating strategy: Th1 Memory, CD4⁺CD45RA⁻CCR6⁻ IFN-γ⁺, Th17 Memory, CD4⁺CD45RA⁻CCR6⁺IL-17⁺ (A) Representative frequencies from a single patient in each group and (B) summary frequencies of Th1 memory cells (Bela Stable/Bela Rejector p = 0.755, Bela Rejector/Tac Rejector p = 0.612) and Th17 memory cells (Bela Stable/Bela Rejector p = 0.106, Bela Rejector/Tac Rejector p = 0.400) collected at baseline. (C) Representatiave frequencies of a single patient in each group and (D) summary frequencies of Th1 memory cells (Bela Stable/Bela Rejector p = 0.200, Bela Rejector/Tac Rejector p = 0.886) and Th17 memory cells (Bela Stable/Bela Rejector p = 0.0286, Bela Rejector/Tac Rejector p = 0.0286) collected at 1 month following transplantation in Bela Stable or at the time of rejection in Belatacept and Tacrolimus Rejectors.