Blocking MHC class II on human endothelium mitigates acute rejection

Abstract

Acute allograft rejection is mediated by host CD8⁺ cytotoxic T lymphocytes (CTL) targeting graft class I major histocompatibility complex (MHC) molecules. In experimental rodent models, rejection requires differentiation of naive CD8⁺ T cells into alloreactive CTL within secondary lymphoid organs, whereas in humans, CTL may alternatively develop within the graft from circulating CD8⁺ effector memory T cells (T_EM) that recognize class I MHC molecules on graft endothelial cells (EC). This latter pathway is poorly understood. Here, we show that host CD4⁺ T_EM, activated by EC class II MHC molecules, provide critical help for this process. First, blocking HLA-DR on EC lining human artery grafts in immunodeficient mice reduces CD8⁺ CTL development within and acute rejection of the artery by adoptively transferred allogeneic human lymphocytes. Second, siRNA knockdown or CRISPR/Cas9 ablation of class II MHC molecules on EC prevents CD4⁺ T_EM from helping CD8⁺ T_EM to develop into CTL in vitro. Finally, implanted synthetic microvessels, formed from CRISPR/Cas9-modified EC lacking class II MHC molecules, are significantly protected from CD8⁺ T cell-mediated destruction in vivo. We conclude that human CD8⁺ T_EM-mediated rejection targeting graft EC class I MHC molecules requires help from CD4⁺ T_EM cells activated by recognition of class II MHC molecules.

Figure 7. Genetic ablation of CIITA in EC by CRISPR/Cas9 reduces T cell–mediated destruction of synthetic vessels in vivo.

Synthetic human microvessels formed from WT or CIITA^null EC were implanted s.c. in the abdominal wall of C.B-17 SCID/bg mice. After 2 weeks, mice were adoptively transferred with peripheral blood mononuclear cells (PBMC). In the absence of circulating T cells, WT and CIITA^null EC form equivalent numbers of vessels (n = 3 each). In the presence of circulating T cells, however, there is a significantly higher loss of perfused vessels formed from WT compared with CIITA^null EC (n = 3 each). Scale bar: 100 μm. *P < 0.05, 2-tailed Student’s t test. CIITA, class II MHC transactivator; bg, beige; EC,endothelial cells.

Figure 6. CD4⁺ T_EM enhancement of CD8⁺ T_EM responses are mediated by secreted IL-2.

(A) EC precultured with CD4⁺ T_EM and subsequently isolated by FACS have not increased their capacity to activate CD8⁺ T_EM. (B) When separated by a semipermeable transwell membrane, CD4⁺ T_EM enhance CD8⁺ T_EM alloresponses. (C) CM generated by EC-activated CD4⁺ T_EM is sufficient to enhance proliferation of CD8⁺ T_EM. Immunoabsorption of IL-2 removes that capacity, whereas addition of IL-2 to control CM mimics activated CM. (D) Similarly, CM induces phosphorylates STAT5 in CD25⁺ but not CD25^–CD8⁺ T_EM in an IL-2–dependent manner. (E) In addition, CM enhances expression of perforin and promotes CTL killing in an IL-2–dependent manner. Similar results were seen in three independent experiments. *P < 0.05, by 2-tailed Student’s t test (B) or by 1-way ANOVA with Bonferroni post-hoc test (A and C–E). T_EM, effector memory T cell; CIITA, class II MHC transactivator; CM, conditioned medium; EC,endothelial cells; CTL, cytotoxic T cell.

Figure 5. Activation of CD4⁺ T_EM by allogeneic EC is necessary to enhance CD8⁺ T_EM responses to the same EC.

(A and B) Addition of CD4⁺ T_EM to EC/CD8⁺ T_EM cocultures enhances CD8⁺ T_EM expansion as measured by CFSE dilution and flow cytometry at 7 days (A), but CD4⁺ T_EM do not enhance survival of CD8⁺ T_EM (B) as measured by viability. (C) Knockdown of CIITA expression by siRNA, to prevent CD4⁺ T_EM activation, inhibits CD4⁺ T_EM enhancement of CD8⁺ T_EM expansion at 7 days as measured by CFSE dilution and flow cytometric analysis at 7 days. Note that the inclusion of activated CD4⁺ T_EM increases the rounds of replication of CD8⁺ T_EM (inset boxes). Similar results were seen in three independent experiments. *P < 0.05, 2-tailed Student’s t test. T_EM, effector memory T cell; CIITA, class II MHC transactivator; FVD, fixable viability dye; EC,endothelial cells.

Figure 4. siRNA-mediated knockdown of CIITA in EC directly inhibits CD4⁺ but not CD8⁺ T_EM alloresponses.

(A and B) Anti-CIITA siRNA inhibits activation of CD4⁺ T_EM (10:1 T cell/EC ratio) (A) but not activation of CD8⁺ T_EM (30:1 T cell/EC ratio) (B), as measured by ELISA at 24 hours for IL-2 and IFN-γ and proliferation by CFSE dilution and flow cytometric analysis at 7 days. Note that the majority of CD8⁺ T_EM have divided only once, whereas CD4⁺ T_EM undergo multiple rounds of proliferation. Similar results were seen in three independent experiments. *P < 0.05, 2-tailed Student’s t test. CIITA, class II MHC transactivator; T_EM, effector memory T cell; EC, endothelial cells.

Figure 3. anti-CIITA siRNA selectively inhibits induction of CIITA and class II MHC molecules in human EC.

Cultured human EC were transfected with anti-CIITA siRNA or control siRNA on day 0, treated with IFN-γ at 24 hours, and analyzed on day 3. (A and B) Specific transcripts were measured by qPCR (A), and protein was assessed by FACS (B). Similar results were seen in four independent experiments. ***P < 0.0005, 2-tailed Student’s t test. CIITA, class II MHC transactivator; MHC, major histocompatibility complex; EC, endothelial cells.

Figure 2. HLA-DR blockade reduces CD8⁺ T cell infiltration into and CTL development within implanted allogeneic vessel segments in vivo.

(A and B) Anti–HLA-DRα F(ab)′₂ fragment significantly (n = 6 per group) reduces total intimal T cell infiltration as detected by immunofluorescence (scale bar: 20 μm) (A) and CD8⁺ T cell–associated transcripts of cytokines and CTL effector molecules in rejected artery grafts at 21 days (B) as detected by qPCR normalized to either total mRNA or T cell–specific mRNA as indicated on the y axis. Arrows indicate infiltrating intimal CD45RO⁺ T cells. *P < 0.05, 2-tailed Student’s t test. CTL, cytotoxic T cell.

Figure 1. HLA-DR blockade reduces acute T cell–mediated injury to implanted allogeneic vessel segments in vivo.

(A and B) In an MHC-mismatched model of arterial rejection by allogeneic T cells (n = 6 per group), blockade of class II MHC by anti–HLA-DRα F(ab)′₂ fragment reduces intimal area expansion and increases lumen area as measured by H&E and EVG staining (scale bar: 50 μm) (A) and reduces disruption of EC lining the vessel lumen, a hallmark of intimal arteritis/endothelialitis, as measured by percent of circumferential coverage (scale bar: 20 μm) (B), both at 21 days. Arrows indicate areas of intact endothelial lining. *P < 0.05, 2-tailed Student’s t test. EVG, Elastia-van Gieson; MHC, major histocompatibility complex; EC, endothelial cells.