Host-Derived CD70 Suppresses Murine Graft-versus-Host Disease by Limiting Donor T Cell Expansion and Effector Function

Abstract

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative treatment for hematologic and immunologic diseases. However, graft-versus-host disease (GVHD) may develop when donor-derived T cells recognize and damage genetically distinct normal host tissues. In addition to TCR signaling, costimulatory pathways are involved in T cell activation. CD27 is a TNFR family member expressed on T cells, and its ligand, CD70, is expressed on APCs. The CD27/CD70 costimulatory pathway was shown to be critical for T cell function and survival in viral infection models. However, the role of this pathway in allo-HCT is previously unknown. In this study, we have examined its contribution in GVHD pathogenesis. Surprisingly, Ab blockade of CD70 after allo-HCT significantly increases GVHD. Interestingly, whereas donor T cell- or bone marrow-derived CD70 plays no role in GVHD, host-derived CD70 inhibits GVHD as CD70^-/- hosts show significantly increased GVHD. This is evidenced by reduced survival, more severe weight loss, and increased histopathologic damage compared with wild-type hosts. In addition, CD70^-/- hosts have higher levels of proinflammatory cytokines TNF-α, IFN-γ, IL-2, and IL-17. Moreover, accumulation of donor CD4⁺ and CD8⁺ effector T cells is increased in CD70^-/- versus wild-type hosts. Mechanistic analyses suggest that CD70 expressed by host hematopoietic cells is involved in the control of alloreactive T cell apoptosis and expansion. Together, our findings demonstrate that host CD70 serves as a unique negative regulator of allogeneic T cell response by contributing to donor T cell apoptosis and inhibiting expansion of donor effector T cells.

Figure 1. Blockade of CD70 exacerbates GVHD

(A) BALB/c and C57BL/6 mice were given 792 and 965 cGy irradiation, respectively, on day -1 and transplanted day 0 with 2×10⁶ WT C57BL/6 BM only (n=2-4) ± 3×10⁶ C57BL/6 splenocytes (spl) (n=4-10). Recipient mice were either treated with 25μg control IgG or 25μg anti-CD70 blocking antibody 4 and 6 days post-transplant and then monitored for survival. Results are pooled from two individual experiments. (B-C) BALB/c mice were given 792 cGy irradiation on day -1 and transplanted day 0 with 2×10⁶ WT BM (n=4) ± 3.5×10⁶ WT splenocytes (n=7) or 2×10⁶ CD70^-/- BM (n=4) ± 3.5×10⁶ CD70^-/- splenocytes (n=8). Mice were then monitored for weight loss (B) and survival (C). (D-E) BALB/c mice were given 792 cGy irradiation on day -1 and transplanted with 2×10⁶ WT BM (n=5) ± 0.4×10⁶ WT or CD70^-/- CD25- PanT (n=5). Mice were then monitored for weight loss (D) and survival (E). In (A, C, E) data are presented as percent survival. In (B, D) data are presented as mean ± SEM. Statistical significance evaluated by log rank (Mantel-Cox) test. *P<0.05.

Figure 2. Absence of host CD70 significantly increases GVHD

(A-D) WT or CD70^-/- C57BL/6 mice received 965 cGy irradiation on day -1 and were subsequently transplanted day 0 with (A-B) 4×10⁶ BALB/c BM (n=5-8) ± 2×10⁶ PanT (n=17-20), or (C-D) 4×10⁶ BALB/c BM (n=5-8) ± 3×10⁶ PanT (n=13-14). Data are pooled from 2 individual experiments. Weight loss (A, C) and survival (B, D) are shown. (E-F) WT or CD70^-/- C57BL/6 mice received 965 cGy irradiation on day -1 and were subsequently transplanted day 0 with 4×10⁶ BALB/c TCD-BM (n=4) ± 1×10⁶ CD25- PanT (n=8). Mice were then monitored for weight loss (E) and survival (F). In (A, C, E) data are presented as mean ± SEM. Statistical significance determined by ANOVA in (A, C, E) and log-rank (Mantel Cox) in (B, D, F); *P<.05; **P<.01;***P<.001.

Figure 3. Absence of host CD70 results in significantly increased pathologic GVHD

WT or CD70^-/- C57BL/6 mice received 965 cGy irradiation on day -1 and were subsequently transplanted day 0 with 4×10⁶ BALB/c BM + 2×10⁶ PanT. 65 days following allo-HCT mice were sacrificed and small, large intestines, and liver formalin fixed, embedded in paraffin, and stained with hematoxylin and eosin. Samples were scored for GVHD on a scale of 0-4 by a blinded pathologist using an established scoring system (39-41), with mice chosen from one of two individual experiments described in Fig. 2A-B. (A) Scores from all three organs were added to create a combined score. Scores of the (B) large intestine, (C) small intestine, and (D) liver are shown. Each point represents an individual sample and data were analyzed by student's t test. *P<.05. (E) Representative areas of liver tissue (Scale bar 100μm) from WT (left) and CD70^-/- (right) hosts. Arrows indicate damaged bile ducts.

Figure 4. Serum levels of pro-inflammatory cytokines are significantly increased following allo-HCT in CD70^-/- hosts

WT or CD70^-/- C57BL/6 mice received 965 cGy irradiation on day -1 and were subsequently transplanted on day 0 with 4×10⁶ BALB/c BM ± 5×10⁶ PanT. 5 days post allo-HCT serum was collected via retro-orbital eye bleed and frozen immediately. Luminex was performed to evaluate (A) TNFα, (B) IL-2, (C) IFN-γ, and (D) IL-17A as per manufacturer's instructions. Shown are representative data from one of two individual experiments. Each point represents an individual sample and data were analyzed by student's t test. *P<.05; **P<.01.

Figure 5. Donor T cell expansion is increased in CD70^-/- hosts

WT or CD70^-/- C57BL/6 mice received 965 cGy irradiation on day -1 and were subsequently transplanted day 0 with 4×10⁶ BALB/c BM ± PanT. For early time points (days 3, 4, and 5), 5×10⁶ PanT were injected in order to harvest sufficient T cells for reliable analysis. For the late time point (day 7), 2×10⁶ PanT were injected to minimize untimely host lethality. At days 3, 4, 5, and 7 following allo-HCT, spleens were harvested, counted and stained for flow cytometry. Absolute numbers of donor-derived CD4⁺ (A) and CD8⁺ (B) T cells in WT and CD70^-/- hosts receiving BM + PanT were acquired via the following equation: (total number of splenocytes) × (percentage of live H-2K^d+CD3⁺CD4⁺ or H-2K^d+CD3⁺CD8⁺ T cells). Summary data from three individual experiments are shown (n=2-5 for each time point). Statistical significance was analyzed by student's t test. **P<.01; ***P<.001.

Figure 6. Residual host lymphocyte number and anti-donor function are not affected by CD70

(A) WT or CD70^-/- C57BL/6 mice were transplanted and spleen cells were analyzed as described in Fig. 5 legend on days 3, 4, 5 and 7 after allo-HCT. Absolute numbers of host-derived CD4⁺ and CD8⁺ T cells in WT and CD70^-/- hosts were acquired via the following equation: (total number of splenocytes) × (percentage of live H-2K^b+CD3⁺CD4⁺ or H-2K^b+CD3⁺CD8⁺ T cells). Summary data from three individual experiments are shown (n=2-12 for each time point). Host T cells (A) and NK cells (B) in non-irradiated WT or CD70^-/- C57BL/6 mice were also provided. (C) One dose of 200μg NK1.1 antibody (PK136) or IgG control was injected on day -2. The host mice then received 965 cGy irradiation on day -1 and were subsequently transplanted on day 0. Survival data of hosts receiving 4×10⁶ BALB/c BM ± 3×10⁶ PanT are pooled from two individual experiments (n=8-14 for each group). Statistical significance evaluated by log rank (Mantel-Cox) test. *P<0.05; ***P<.001.

Figure 7. Donor T cells in CD70^-/- hosts have similar proliferation, but significantly decreased caspase 8-dependent activation induced cell death

(A-B) WT or CD70^-/- C57BL/6 mice received 965 cGy irradiation on day -1 and were subsequently transplanted on day 0 with 4×10⁶ BALB/c BM + 5×10⁶ CFSE labeled PanT. (A) Represented histograms of CFSE dilution in donor-derived live H-2K^d+CD8⁺ T cells in WT hosts at the indicated hours after allo-HCT. (B) 69 hours after allo-HCT, CFSE dilution was assessed in donor-derived live H-2K^d+CD4⁺ and H-2K^d+CD8⁺ T cells. Summary data from 4 mice of each genotype. (C-D) WT or CD70^-/- C57BL/6 mice received 965 cGy irradiation on day -1 and were subsequently transplanted day 0 with 4×10⁶ BALB/c BM + 5×10⁶ PanT. 4 days post allo-HCT spleens were harvested and active caspase-8 was evaluated in H-2K^d+H-2K^b-TCRβ⁺CD4⁺ and H-2K^d+H-2K^b-TCRβ⁺CD8⁺ T cells. (C) Flow diagrams depicting T cell expression of live/dead viability dye versus active caspase-8 in WT and CD70^-/- hosts. (D) Summary data from 5 mice of each genotype. Representative data from one of three individual experiments is shown. Data were analyzed by student's t test. *P<0.05; **P<0.01.

Figure 8. CD70 expressed by host hematopoietic APCs suppresses GVHD by inducing AICD in alloreactive donor T cells

(A) Chimeras were generated between C57BL/6 WT and CD70^-/- mice through syngeneic transplants with 5×10⁶ BM plus 5×10⁶ splenocytes. After 3 months, the chimeric hosts were lethally irradiated with 800 cGy for allo-HCT. Survival data of chimeras receiving 4×10⁶ BALB/c BM ± 3×10⁶ PanT are pooled from two individual experiments (n=7-14 for each group). Statistical significance evaluated by log rank (Mantel-Cox) test. * P<0.05. (B) WT and CD70^-/- BMDCs were cultured in RPMI medium containing GM-CSF for 6 days. LPS was added to culture on day 6 to fully mature the BMDC culture. On day 7 CD70 expression on CD11b⁺CD11c⁺ BMDCs was assessed by flow cytometry. (C) Active caspase-8 was analyzed on day 5 after culture of WT or CD70^-/- BMDCs mixed at a 1:5 ratio with BALB/c PanT cells. Shown are representative data from three experiments. Statistical significance for caspase-8 activation was analyzed by student's t test. **P<0.01; ***P<0.001.

Figure 9. Donor effector T cells are significantly increased in CD70^-/- hosts

WT or CD70^-/- C57BL/6 mice received 965 cGy irradiation on day -1 and were subsequently transplanted day 0 with 4×10⁶ BALB/c BM + 5×10⁶ PanT. On day 5 post allo-HCT mice were injected with 250μg of BFA. 6 hrs post-injection mice were sacrificed, spleens were harvested and assessed for IFN-γ production by flow cytometry. (A) Depicted is IFN-γ expression within live H-2K^d+CD4⁺ and live H-2K^d+CD8⁺ T cells. (B) The percentage and absolute numbers of donor CD4⁺ and CD8⁺ producing IFN-γ. Representative data from one of two individual experiments is shown. Data were analyzed by student's t test. **P<0.01.