Graft-versus-host disease impairs vaccine responses through decreased CD4+ and CD8+ T cell proliferation and increased perforin-mediated CD8+ T cell apoptosis

Abstract

Tumor-targeted vaccines represent a strategy to enhance the graft-versus-leukemia effect after allogeneic blood and marrow transplantation (BMT). We have previously shown that graft-versus-host disease (GVHD) can negatively impact quantitative responses to vaccines. Using a minor histocompatibility Ag-mismatched BMT (B6 → B6 × C3H.SW) followed by adoptive transfer of HY-specific T cells and HY-expressing dendritic cells, we assessed whether GVHD induced by donor lymphocyte infusion (DLI) affects the persistence, proliferation, and survival of vaccine-responding, nonalloantigen reactive T cells. Both CD8(+) and CD4(+) HY-specific T cells undergo less vaccine-driven proliferation in allogeneic recipients with GVHD. Although vaccine-responding CD8(+) T cells show decreased IFN-γ and CD107a production, CD4(+) T cells exhibit increased programmed death 1 and T cell Ig mucin-like domain 3 expression. In addition, the degree of apoptosis in vaccine-responding CD8(+) T cells was higher in the presence of GVHD, but there was no difference in CD4(+) T cell apoptosis. Using Fas ligand-deficient or TRAIL-deficient DLI had no impact on apoptosis of HY-specific T cells. However, perforin-deficient alloreactive DLI induced significantly less apoptosis of vaccine-responding CD8(+) T cells and resulted in enhanced tumor protection. Thus, diminished vaccine responses during GVHD result from impaired proliferation of CD8(+) and CD4(+) T cells responding to vaccination, with an additional contribution from perforin-mediated CD8(+) T cell apoptosis. These results provide important insights toward optimizing vaccine responses after allogeneic BMT.

Figure 1. DLI-induced, mHA-mismatched non-lethal GVHD impairs efficacy of TCRTg CD8⁺ T cells

(A) AlloBMT consisted of CD45.1⁺ T cell depleted (TCD) B6 bone marrow infused into lethally irradiated recipients (B6→B6 × C3H.SW [F1]), followed by an alloreactive CD45.1⁺ B6 DLI at day +14 and adoptive transfer of CD4⁺ CD45.2⁺ or CD8⁺ CD45.2⁺ B6 TCRTg cells +/- male B6 DC vaccine at day +28. F1 recipients were CD45.1⁺CD45.2⁺ double positive. Syngeneic BMT was F1→F1 on day +0 followed by F1 DLI on day +14 and adoptive transfer of CD4⁺ CD45.2⁺ or CD8⁺ CD45.2⁺ B6 TCRTg cells +/- male B6 DC vaccine at day +28. (B) Donor chimerism assessed by flow cytometric analysis for % donor cells in BMT recipient spleens on day +14, 5 mice/group, representative of two independent experiments. (C) Weights were recorded twice weekly per mouse, averaged for the group, and then compared as a % change from the day +0 weight for syngeneic and alloBMT recipients, 5-6 mice/group, representative of three independent experiments. (D) B6 Rag^-/- hosts were injected with CFSE-labeled T cells obtained from CD4⁺ (Marilyn) or CD8⁺ (Matahari) TCRTg splenocytes, and after 7 days, the spleens of the Rag^-/- hosts were analyzed for T cell proliferation. A representative mouse from each group is depicted, n = 3, representative of three independent experiments.

Figure 2. GVHD decreases recovery of vaccine-responding T cells

Mice were transplanted per Figure 1A. (A) A sample flow cytometry gating schema on (top) a F1 recipient transplanted with syngeneic CD45.1⁺CD45.2⁺ F1 bone marrow and DLI, followed by CD45.1^-CD45.2⁺ Matahari splenocyte infusion, as well as (bottom) a F1 recipient transplanted with allogeneic CD45.1⁺CD45.2^- B6 bone marrow and DLI, followed by CD45.1^-CD45.2⁺ Matahari splenocyte infusion. The CD45.2⁺ Vβ8.3⁺ fraction in both BMTs represents the HY-reactive T cells from the Matahari infusion in the recipient spleen. (B) Absolute numbers of CD4⁺Vβ6⁺CD45.2⁺ Marilyn T cells in the host spleen were enumerated by flow cytometry 3, 5 and 7 days (days +31, 33 and 35 post-BMT) after adoptive transfer without concurrent male DC vaccination, or (C) with male DC vaccination, 4-9 mice/group, pooled from three independent experiments. (D) Absolute numbers of CD8⁺Vβ8.3⁺CD45.2⁺ Matahari T cells in the host spleen were enumerated by flow cytometry 3, 5 and 7 days (days +31, 33 and 35 post-BMT) after adoptive transfer without concurrent male DC vaccination, or (E) with male DC vaccination, 3-9 mice/group. * = p<0.05, pooled from three independent experiments.

Figure 3. Vaccine-induced proliferation is impaired for TCRTg CD4⁺ and CD8⁺ T cells during GVHD

Mice were transplanted per Figure 1A and received CFSE-labeled CD4⁺ Marilyn or CD8⁺ Matahari splenocytes with or without a male B6 DC vaccine. Proliferation was measured by flow cytometry at 3, 5 and 7 days (days +31, 33 and 35 post-BMT) after transfer. (A) Results from a representative recipient of CD4⁺ Marilyn T cells after allogeneic and syngeneic BMT analyzed at each timepoint are depicted, with unvaccinated recipient (top), and male DC vaccinated recipient (bottom). (B) Pooled data from three independent experiments depicted for CD4⁺ Marilyn T cells in unvaccinated, and (C) male DC vaccinated recipients, as well as from three independent experiments for (D) CD8⁺ Matahari T cells in unvaccinated, and (E) male DC vaccinated recipients, 3-9 mice/group. CD8⁺ Matahari T cells from syngeneic and allogeneic recipients were analyzed 3 days following vaccination with male DC vaccine for intracellular IFNγ (F) or CD107a (G) (n=3 mice/group). (H) Representative FACS plots of PD-1 and TIM3 on CD4⁺ Marilyn T cells from syngeneic and allogeneic recipients 3 days after male DC vaccination (total of n=3 mice per group analyzed). * p<0.05, ** p<0.01, *** p<0.001.

Figure 4. GVHD increases apoptosis 7 days after adoptive transfer of TCRTg CD8⁺, but not CD4⁺, T cells

Mice were transplanted per Figure 1A and given either an infusion of Marilyn or Matahari splenocytes on day +28 with or without a male B6 DC vaccine. Apoptosis of nonproliferated, CFSE⁺, TCRTg T cells was enumerated by flow cytometric analysis of Annexin V at 3, 5 and 7 days (days +31, 33 and 35 post-BMT) after transfer of CD4⁺ Marilyn T cells in (A) unvaccinated and (B) male DC vaccinated recipient lymph nodes, and after transfer of CD8⁺ Matahari T cells in (C) unvaccinated and (D) male DC vaccinated recipient lymph nodes, 3-7 mice/group, pooled from three independent experiments. (E) Matahari T cells were cultured with male or female DCs from either allogeneic (C3H.SW) or syngeneic (B6 CD45.1⁺) donors at a 1:1 ratio for 72 hours. Cells were then counted and analyzed for expression of Annexin V on CD45.2⁺CD8⁺Vβ 8.3⁺7AAD^- cells. Triplicate co-culture wells were set up for each condition. * p<0.05.

Figure 5. Administration of perforin-deficient DLI attenuates GVHD-induced apoptosis of TCRTg CD8⁺ T cells in spleens and lymph nodes of alloBMT recipients

(A) C3H.SW recipients received TCD B6 bone marrow on day +0 followed by 30 × 10⁶ DLI from B6 (wild type, WT), B6 FasL^-/- (GLD), B6 TRAIL^-/-, or B6 prf1^-/- (perforin) donors on day +14. On day +28, 10 × 10⁶ CFSE-labeled Matahari T cells were adoptively transferred and all recipients concurrently received B6 male DC vaccine. After 7 days (day +35), nonproliferated CFSE⁺ Annexin V⁺ cells were enumerated in (B) spleens and (C) pooled axillary, inguinal and cervical lymph nodes from alloBMT recipients, 5-10 mice/group, * = p < 0.05, data pooled from two independent experiments.

Figure 6. Infusion of perforin-deficient DLI enhances tumor protection by TCRTg CD8⁺ T cells expanded in vivo with a tumor-specific vaccine

C3H.SW recipients received TCD B6 bone marrow on day +0 followed by 30 × 10⁶ DLI from B6 (wild type, WT or B6 prf1^-/-(perforin) donors on day +14. On day +28, 10 × 10⁶ Matahari T cells were adoptively transferred and all recipients concurrently received B6 male DC vaccine. (A) alloBMT recipients were weighed twice weekly, and on day +35, (B) all recipients were challenged with 1 × 10⁶ MB49 tumor cells in the flank and measured twice weekly, 5 mice/group, p < 0.01 on day 35, data representative from two independent experiments.