Calcineurin inhibition rescues alloantigen-specific central memory T cell subsets that promote chronic GVHD.

Calcineurin inhibitors (CNIs) constitute the backbone of modern acute graft-versus-host disease (aGVHD) prophylaxis regimens but have limited efficacy in the prevention and treatment of chronic GVHD (cGVHD). We investigated the effect of CNIs on immune tolerance after stem cell transplantation with discovery-based single-cell gene expression and T cell receptor (TCR) assays of clonal immunity in tandem with traditional protein-based approaches and preclinical modeling. While cyclosporin and tacrolimus suppressed the clonal expansion of CD8+ T cells during GVHD, alloreactive CD4+ T cell clusters were preferentially expanded. Moreover, CNIs mediated reversible dose-dependent suppression of T cell activation and all stages of donor T cell exhaustion. Critically, CNIs promoted the expansion of both polyclonal and TCR-specific alloreactive central memory CD4+ T cells (TCM) with high self-renewal capacity that mediated cGVHD following drug withdrawal. In contrast to posttransplant cyclophosphamide (PT-Cy), CSA was ineffective in eliminating IL-17A-secreting alloreactive T cell clones that play an important role in the pathogenesis of cGVHD. Collectively, we have shown that, although CNIs attenuate aGVHD, they paradoxically rescue alloantigen-specific TCM, especially within the CD4+ compartment in lymphoid and GVHD target tissues, thus predisposing patients to cGVHD. These data provide further evidence to caution against CNI-based immune suppression without concurrent approaches that eliminate alloreactive T cell clones.

IL-6-mediated endothelial injury impairs antiviral humoral immunity after bone marrow transplantation.

Endothelial function and integrity are compromised after allogeneic bone marrow transplantation (BMT), but how this affects immune responses broadly remains unknown. Using a preclinical model of CMV reactivation after BMT, we found compromised antiviral humoral responses induced by IL-6 signaling. IL-6 signaling in T cells maintained Th1 cells, resulting in sustained IFN-γ secretion, which promoted endothelial cell (EC) injury, loss of the neonatal Fc receptor (FcRn) responsible for IgG recycling, and rapid IgG loss. T cell-specific deletion of IL-6R led to persistence of recipient-derived, CMV-specific IgG and inhibited CMV reactivation. Deletion of IFN-γ in donor T cells also eliminated EC injury and FcRn loss. In a phase III clinical trial, blockade of IL-6R with tocilizumab promoted CMV-specific IgG persistence and significantly attenuated early HCMV reactivation. In sum, IL-6 invoked IFN-γ-dependent EC injury and consequent IgG loss, leading to CMV reactivation. Hence, cytokine inhibition represents a logical strategy to prevent endothelial injury, thereby preserving humoral immunity after immunotherapy.

Regulatory T cells suppress myeloma-specific immunity during autologous stem cell mobilization and transplantation.

ABSTRACT: Autologous stem cell transplantation (ASCT) is the standard of care consolidation therapy for eligible patients with myeloma but most patients eventually progress, an event associated with features of immune escape. Novel approaches to enhance antimyeloma immunity after ASCT represent a major unmet need. Here, we demonstrate that patient-mobilized stem cell grafts contain high numbers of effector CD8 T cells and immunosuppressive regulatory T cells (Tregs). We showed that bone marrow (BM)-residing T cells are efficiently mobilized during stem cell mobilization (SCM) and hypothesized that mobilized and highly suppressive BM-derived Tregs might limit antimyeloma immunity during SCM. Thus, we performed ASCT in a preclinical myeloma model with or without stringent Treg depletion during SCM. Treg depletion generated SCM grafts containing polyfunctional CD8 T effector memory cells, which dramatically enhanced myeloma control after ASCT. Thus, we explored clinically tractable translational approaches to mimic this scenario. Antibody-based approaches resulted in only partial Treg depletion and were inadequate to recapitulate this effect. In contrast, a synthetic interleukin-2 (IL-2)/IL-15 mimetic that stimulates the IL-2 receptor on CD8 T cells without binding to the high-affinity IL-2Ra used by Tregs efficiently expanded polyfunctional CD8 T cells in mobilized grafts and protected recipients from myeloma progression after ASCT. We confirmed that Treg depletion during stem cell mobilization can mitigate constraints on tumor immunity and result in profound myeloma control after ASCT. Direct and selective cytokine signaling of CD8 T cells can recapitulate this effect and represent a clinically testable strategy to improve responses after ASCT.

Intestinal microbiota controls graft-versus-host disease independent of donor-host genetic disparity.

Acute graft-versus-host disease (aGVHD) remains a major limitation of allogeneic stem cell transplantation (SCT), and severe intestinal manifestation is the major cause of early mortality. Intestinal microbiota control MHC class II (MHC-II) expression by ileal intestinal epithelial cells (IECs) that promote GVHD. Here, we demonstrated that genetically identical mice of differing vendor origins had markedly different intestinal microbiota and ileal MHC-II expression, resulting in discordant GVHD severity. We utilized cohousing and antibiotic treatment to characterize the bacterial taxa positively and negatively associated with MHC-II expression. A large proportion of bacterial MHC-II inducers were vancomycin sensitive, and peri-transplant oral vancomycin administration attenuated CD4+ T cell-mediated GVHD. We identified a similar relationship between pre-transplant microbes, HLA class II expression, and both GVHD and mortality in a large clinical SCT cohort. These data highlight therapeutically tractable mechanisms by which pre-transplant microbial taxa contribute to GVHD independently of genetic disparity.

TIGIT inhibition and lenalidomide synergistically promote antimyeloma immune responses after stem cell transplantation in mice.

Autologous stem cell transplantation (ASCT) with subsequent lenalidomide maintenance is standard consolidation therapy for multiple myeloma, and a subset of patients achieve durable progression-free survival that is suggestive of long-term immune control. Nonetheless, most patients ultimately relapse, suggesting immune escape. TIGIT appears to be a potent inhibitor of myeloma-specific immunity and represents a promising new checkpoint target. Here we demonstrate high expression of TIGIT on activated CD8+ T cells in mobilized peripheral blood stem cell grafts from patients with myeloma. To guide clinical application of TIGIT inhibition, we evaluated identical anti-TIGIT antibodies that do or do not engage FcγR and demonstrated that anti-TIGIT activity is dependent on FcγR binding. We subsequently used CRBN mice to investigate the efficacy of anti-TIGIT in combination with lenalidomide maintenance after transplantation. Notably, the combination of anti-TIGIT with lenalidomide provided synergistic, CD8+ T cell-dependent, antimyeloma efficacy. Analysis of bone marrow (BM) CD8+ T cells demonstrated that combination therapy suppressed T cell exhaustion, enhanced effector function, and expanded central memory subsets. Importantly, these immune phenotypes were specific to the BM tumor microenvironment. Collectively, these data provide a logical rationale for combining TIGIT inhibition with immunomodulatory drugs to prevent myeloma progression after ASCT.

Adults with Plasmodium falciparum malaria have higher magnitude and quality of circulating T-follicular helper cells compared to children.

BACKGROUND: Protective malarial antibodies are acquired more rapidly in adults than children, independently of cumulative exposure, however the cellular responses mediating these differences are unknown. CD4 T-follicular helper (Tfh) cells have key roles in inducing antibodies, with Th2-Tfh cell activation associated with antibody development in malaria. Whether Tfh cell activation in malaria is age dependent is unknown and no studies have compared Tfh cell activation in children and adults with malaria. METHODS: We undertook a comprehensive study of Tfh cells, along with B cells and antibody induction in children and adults with malaria. Activation and proliferation of circulating Tfh (cTfh) cell subsets was measured ex vivo and parasite-specific Tfh cell frequencies and functions studied with Activation Induced Marker (AIM) assays and intracellular cytokine staining. FINDINGS: During acute malaria, the magnitude of cTfh cell activation was higher in adults than in children and occurred across all cTfh cell subsets in adults but was restricted only to the Th1-cTfh subset in children. Further, adults had higher levels of parasite-specific cTfh cells, and cTfh cells which produced more Th2-Tfh associated cytokine IL-4. Consistent with a role of higher Tfh cell activation in rapid immune development in adults, adults had higher activation of B cells during infection and higher induction of antibodies 7 and 28 days after malaria compared to children. INTERPRETATION: Our data provide evidence that age impacts Tfh cell activation during malaria, and that these differences may influence antibody induction after treatment. Findings have important implications for vaccine development in children. FUNDING: This word was supported by the National Health and Medical Research Council of Australia, Wellcome Trust, Charles Darwin University Menzies School of Health Research, Channel 7 Children's Research Foundation, and National Health Institute.

IFN-λ therapy prevents severe gastrointestinal graft-versus-host disease.

Immunopathology and intestinal stem cell (ISC) loss in the gastrointestinal (GI) tract is the prima facie manifestation of graft-versus-host disease (GVHD) and is responsible for significant mortality after allogeneic bone marrow transplantation (BMT). Approaches to prevent GVHD to date focus on immune suppression. Here, we identify interferon-λ (IFN-λ; interleukin-28 [IL-28]/IL-29) as a key protector of GI GVHD immunopathology, notably within the ISC compartment. Ifnlr1-/- mice displayed exaggerated GI GVHD and mortality independent of Paneth cells and alterations to the microbiome. Ifnlr1-/- intestinal organoid growth was significantly impaired, and targeted Ifnlr1 deficiency exhibited effects intrinsic to recipient Lgr5+ ISCs and natural killer cells. PEGylated recombinant IL-29 (PEG-rIL-29) treatment of naive mice enhanced Lgr5+ ISC numbers and organoid growth independent of both IL-22 and type I IFN and modulated proliferative and apoptosis gene sets in Lgr5+ ISCs. PEG-rIL-29 treatment improved survival, reduced GVHD severity, and enhanced epithelial proliferation and ISC-derived organoid growth after BMT. The preservation of ISC numbers in response to PEG-rIL-29 after BMT occurred both in the presence and absence of IFN-λ-signaling in recipient natural killer cells. IFN-λ is therefore an attractive and rapidly testable approach to prevent ISC loss and immunopathology during GVHD.

CMV exposure drives long-term CD57+ CD4 memory T-cell inflation following allogeneic stem cell transplant.

Donor and recipient cytomegalovirus (CMV) serostatus correlate with transplant-related mortality that is associated with reduced survival following allogeneic stem cell transplant (SCT). Prior epidemiologic studies have suggested that CMV seronegative recipients (R-) receiving a CMV-seropositive graft (D+) experience inferior outcomes compared with other serostatus combinations, an observation that appears independent of viral reactivation. We therefore investigated the hypothesis that prior donor CMV exposure irreversibly modifies immunologic function after SCT. We identified a CD4+/CD57+/CD27- T-cell subset that was differentially expressed between D+ and D- transplants and validated results with 120 patient samples. This T-cell subset represents an average of 2.9% (D-/R-), 18% (D-/R+), 12% (D+/R-), and 19.6% (D+/R+) (P < .0001) of the total CD4+ T-cell compartment and stably persists for at least several years post-SCT. Even in the absence of CMV reactivation post-SCT, D+/R- transplants displayed a significant enrichment of these cells compared with D-/R- transplants (P = .0078). These are effector memory cells (CCR7-/CD45RA+/-) that express T-bet, Eomesodermin, granzyme B, secrete Th1 cytokines, and are enriched in CMV-specific T cells. These cells are associated with decreased T-cell receptor diversity (P < .0001) and reduced proportions of major histocompatibility class (MHC) II expressing classical monocytes (P < .0001), myeloid (P = .024), and plasmacytoid dendritic cells (P = .0014). These data describe a highly expanded CD4+ T-cell population and putative mechanisms by which prior donor or recipient CMV exposure may create a lasting immunologic imprint following SCT, providing a rationale for using D- grafts for R- transplant recipients.

A phase 3 double-blind study of the addition of tocilizumab vs placebo to cyclosporin/methotrexate GVHD prophylaxis.

We determined the efficacy of tocilizumab (TCZ) in preventing grade 2-4 acute graft-versus-host disease (aGVHD) in patients with acute leukemia or myelodysplasia undergoing matched sibling donor (MSD) or volunteer unrelated donor (VUD) allogeneic stem cell transplantation after myeloablative or reduced-intensity conditioning across 5 Australian centers. A total of 145 patients (50 MSD, 95 VUD) were randomly assigned to placebo or TCZ on day -1. All patients received T-cell-replete peripheral blood stem cell grafts and graft-versus-host disease (GVHD) prophylaxis with cyclosporin/methotrexate. A planned substudy analyzed the VUD cohort. With a median follow-up of 746 days, the incidence of grade 2-4 aGVHD at day 100 for the entire cohort was 36% for placebo vs 27% for TCZ (hazard ratio [HR], 0.69; 95% confidence interval [CI], 0.38-1.26; P = .23) and 45% vs 32% (HR, 0.61; 95% CI, 0.31-1.22; P = .16) for the VUD subgroup. The incidence of grade 2-4 aGVHD at day 180 for the entire cohort was 40% for placebo vs 29% for TCZ (HR, 0.68; 95% CI, 0.38-1.22; P = .19) and 48% vs 32% (HR, 0.59; 95% CI, 0.30-1.16; P = .13) for the VUD subgroup. Reductions in aGVHD were predominantly in grade 2 disease. For the entire cohort, transplant-related mortality occurred in 8% vs 11% of placebo-treated vs TCZ-treated patients, respectively (P = .56), and overall survival was 79% vs 71% (P = .27). Median day to neutrophil and platelet engraftment was delayed by 2 to 3 days in TCZ-treated patients, whereas liver toxicity and infectious complications were similar between groups. In this phase 3 randomized double-blind trial, TCZ showed nonsignificant trends toward reduced incidence of grade 2-4 aGVHD in recipients from HLA-matched VUDs but no improvements in long term-survival.

Single-cell transcriptomics of alloreactive CD4+ T cells over time reveals divergent fates during gut graft-versus-host disease.

Acute gastrointestinal (GI) graft-versus-host disease (GVHD) is a primary determinant of mortality after allogeneic hematopoietic stem cell transplantation (alloSCT). The condition is mediated by alloreactive donor CD4+ T cells that differentiate into pathogenic subsets expressing IFN-γ, IL-17A, or GM-CSF and is regulated by subsets expressing IL-10 and/or Foxp3. Developmental relationships between Th cell states during priming in mesenteric lymph nodes (mLNs) and effector function in the GI tract remain undefined at genome scale. We applied scRNA-Seq and computational modeling to a mouse model of donor DC-mediated GVHD exacerbation, creating an atlas of putative CD4+ T cell differentiation pathways in vivo. Computational trajectory inference suggested emergence of pathogenic and regulatory states along a single developmental trajectory in mLNs. Importantly, we inferred an unexpected second trajectory, categorized by little proliferation or cytokine expression, reduced glycolysis, and high tcf7 expression. TCF1hi cells upregulated α4ß7 before gut migration and failed to express cytokines. These cells exhibited recall potential and plasticity following secondary transplantation, including cytokine or Foxp3 expression, but reduced T cell factor 1 (TCF1). Thus, scRNA-Seq suggested divergence of alloreactive CD4+ T cells into quiescent and effector states during gut GVHD exacerbation by donor DC, reflecting putative heterogeneous priming in vivo. These findings, which are potentially the first at a single-cell level during GVHD over time, may assist in examination of T cell differentiation in patients undergoing alloSCT.

ASC Modulates CTL Cytotoxicity and Transplant Outcome Independent of the Inflammasome.

The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1ß and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8+ T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11-deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8+ T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation.

Continuous pre- and post-transplant exposure to a disease-associated gut microbiome promotes hyper-acute graft-versus-host disease in wild-type mice.

OBJECTIVE: The gut microbiome plays a key role in the development of acute graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation. Here we investigate the individual contribution of the pre- and post-transplant gut microbiome to acute GVHD using a well-studied mouse model. DESIGN: Wild-type mice were cohoused with IL-17RA-/ - mice, susceptible to hyperacute GVHD, either pre- or post-transplant alone or continuously (i.e., pre- and post-transplant). Fecal samples were collected from both WT and IL-17RA-/ - mice pre- and post-cohousing and post-transplant and the microbiome analyzed using metagenomic sequencing. RESULTS: Priming wild-type mice via cohousing pre-transplant only is insufficient to accelerate GVHD, however, accelerated disease is observed in WT mice cohoused post-transplant only. When mice are cohoused continuously, the effect of priming and exacerbation is additive, resulting in a greater acceleration of disease in WT mice beyond that seen with cohousing post-transplant only. Metagenomic analysis of the microbiome revealed pre-transplant cohousing is associated with the transfer of specific species within two as-yet-uncultured genera of the bacterial family Muribaculaceae; CAG-485 and CAG-873. Post-transplant, we observed GVHD-associated blooms of Enterobacteriaceae members Escherichia coli and Enterobacter hormaechei subsp. steigerwaltii, and hyperacute GVHD gut microbiome distinct from that associated with delayed-onset disease (>10 days post-transplant). CONCLUSION: These results clarify the importance of the peri-transplant microbiome in the susceptibility to acute GVHD post-transplant and demonstrate the species-specific nature of this association.

MAIT Cells Promote Tumor Initiation, Growth, and Metastases via Tumor MR1.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that require MHC class I-related protein 1 (MR1) for their development. The role of MAIT cells in cancer is unclear, and to date no study has evaluated these cells in vivo in this context. Here, we demonstrated that tumor initiation, growth, and experimental lung metastasis were significantly reduced in Mr1 -/- mice, compared with wild-type mice. The antitumor activity observed in Mr1 -/- mice required natural killer (NK) and/or CD8+ T cells and IFNγ. Adoptive transfer of MAIT cells into Mr1 -/- mice reversed metastasis reduction. Similarly, MR1-blocking antibodies decreased lung metastases and suppressed tumor growth. Following MR1 ligand exposure, some, but not all, mouse and human tumor cell lines upregulated MR1. Pretreatment of tumor cells with the stimulatory ligand 5-OP-RU or inhibitory ligand Ac-6-FP increased or decreased lung metastases, respectively. MR1-deleted tumors resulted in fewer metastases compared with parental tumor cells. MAIT cell suppression of NK-cell effector function was tumor-MR1-dependent and partially required IL17A. Our studies indicate that MAIT cells display tumor-promoting function by suppressing T and/or NK cells and that blocking MR1 may represent a new therapeutic strategy for cancer immunotherapy. SIGNIFICANCE: Contradicting the perception that MAIT cells kill tumor cells, here MAIT cells promoted tumor initiation, growth, and metastasis. MR1-expressing tumor cells activated MAIT cells to reduce NK-cell effector function, partly in a host IL17A-dependent manner. MR1-blocking antibodies reduced tumor metastases and growth, and may represent a new class of cancer therapeutics.This article is highlighted in the In This Issue feature, p. 1.

Pilot study investigating the effect of enteral and parenteral nutrition on the gastrointestinal microbiome post-allogeneic transplantation.

Nutrition support is frequently required post-allogeneic haematopoietic progenitor cell transplantation (HPCT); however, the impact of mode of feeding on the gastrointestinal microbiome has not been explored. This study aimed to determine if there is a difference in the microbiome between patients receiving enteral nutrition (EN) and parenteral nutrition (PN) post-allogeneic HPCT. Twenty-three patients received either early EN or PN when required. Stool samples were collected at 30 days post-transplant and analysed with shotgun metagenomic sequencing. There was no difference in microbial diversity between patients who received predominantly EN (n = 13) vs. PN (n = 10) however patients who received predominantly EN had greater abundance of Faecalibacterium (P < 0·001) and ruminococcus E bromii (P = 0·026). Patients who had minimal oral intake for a longer duration during provision of nutrition support had a different overall microbial profile (P = 0·044), lower microbial diversity (P = 0·004) and lower abundance of faecalibacterium prausnitzii_C (P = 0·030) and Blautia (P = 0·007) compared to patients with greater oral intake. Lower microbial diversity was found in patients who received additional beta lactam antibiotics (P = 0·042) or had a longer length of hospital stay (P = 0·019). Post-HPCT oral intake should be encouraged to maintain microbiota diversity and, if nutrition support is required, EN may promote a more optimal microbiota profile.

PAG1 limits allergen-induced type 2 inflammation in the murine lung.

BACKGROUND: Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1) is a transmembrane adaptor protein that affects immune receptor signaling in T and B cells. Evidence from genome-wide association studies of asthma suggests that genetic variants that regulate the expression of PAG1 are associated with asthma risk. However, it is not known whether PAG1 expression is causally related to asthma pathophysiology. Here, we investigated the role of PAG1 in a preclinical mouse model of house dust mite (HDM)-induced allergic sensitization and allergic airway inflammation. METHODS: Pag1-deficient (Pag1-/- ) and wild-type (WT) mice were sensitized or sensitized/challenged to HDM, and hallmark features of allergic inflammation were assessed. The contribution of T cells was assessed through depletion (anti-CD4 antibody) and adoptive transfer studies. RESULTS: Type 2 inflammation (eosinophilia, eotaxin-2 expression, IL-4/IL-5/IL-13 production, mucus production) in the airways and lungs was significantly increased in HDM sensitized/challenged Pag1-/- mice compared to WT mice. The predisposition to allergic sensitization was associated with increased airway epithelial high-mobility group box 1 (HMGB1) translocation and release, increased type 2 innate lymphoid cells (ILC2s) and monocyte-derived dendritic cell numbers in the mediastinal lymph nodes, and increased T-helper type 2 (TH 2)-cell differentiation. CD4+ T-cell depletion studies or the adoptive transfer of WT OVA-specific CD4+ T cells to WT or Pag1-/- recipients demonstrated that the heightened propensity for TH 2-cell differentiation was both T cell intrinsic and extrinsic. CONCLUSION: PAG1 deficiency increased airway epithelial activation, ILC2 expansion, and TH 2 differentiation. As a consequence, PAG1 deficiency predisposed toward allergic sensitization and increased the severity of experimental asthma.

Pegylated interferon-2α invokes graft-versus-leukemia effects in patients relapsing after allogeneic stem cell transplantation.

Allogeneic stem cell transplantation (SCT) is a curative therapy for patients with hematological malignancies related largely to an immunological graft-versus-leukemia (GVL) effect mediated by donor T cells and natural killer cells. Relapse of disease after SCT represents failure of GVL and is now the major cause of treatment failure. We sought to augment GVL effects in patients (n = 29) relapsing after SCT in a prospective phase I/II clinical trial of dose-escalated pegylated interferon-2α (peg-IFNα). The administration of peg-IFNα after reinduction chemotherapy, with or without subsequent donor lymphocyte infusion (DLI), resulted in a 2-year overall survival (OS) of 31% (95% confidence interval, 17.3%-49.2%), which rejects the null hypothesis of 7% generated by observations in an institutional historical cohort. As expected, peg-IFNα was associated with graft-versus-host disease (GVHD) and hematological toxicity, which was manageable with scheduled dose modifications. Progression-free survival (PFS) was greatest in patients who experienced GVHD, although the majority of those patients still eventually progressed. Higher PFS and OS were associated with pretreatment proportions of immune cell populations with regulatory function, including mucosal invariant T cells, regulatory T cells, and plasmacytoid dendritic cells, independent of any association with GVHD. Peg-IFNα administration after relapse thus constitutes a logical strategy to invoke GVL effects and should be studied in a larger, multicenter cohort. This trial was registered at www.anzctr.org.au as #ACTRN12612000728831.

IL-6 dysregulation originates in dendritic cells and mediates graft-versus-host disease via classical signaling.

Graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (alloSCT) is characterized by interleukin-6 (IL-6) dysregulation. IL-6 can mediate effects via various pathways, including classical, trans, and cluster signaling. Given the recent availability of agents that differentially inhibit these discrete signaling cascades, understanding the source and signaling and cellular targets of this cytokine is paramount to inform the design of clinical studies. Here we demonstrate that IL-6 secretion from recipient dendritic cells (DCs) initiates the systemic dysregulation of this cytokine. Inhibition of DC-driven classical signaling after targeted IL-6 receptor (IL-6R) deletion in T cells eliminated pathogenic donor Th17/Th22 cell differentiation and resulted in long-term survival. After engraftment, donor DCs assume the same role, maintaining classical IL-6 signaling-dependent GVHD responses. Surprisingly, cluster signaling was not active after transplantation, whereas inhibition of trans signaling with soluble gp130Fc promoted severe, chronic cutaneous GVHD. The latter was a result of exaggerated polyfunctional Th22-cell expansion that was reversed by IL-22 deletion or IL-6R inhibition. Importantly, inhibition of IL-6 classical signaling did not impair the graft-versus-leukemia effect. Together, these data highlight IL-6 classical signaling and downstream Th17/Th22 differentiation as important therapeutic targets after alloSCT.

MHC Class II Antigen Presentation by the Intestinal Epithelium Initiates Graft-versus-Host Disease and Is Influenced by the Microbiota.

Graft-versus-host disease (GVHD) in the gastrointestinal (GI) tract is the principal determinant of lethality following allogeneic bone marrow transplantation (BMT). Here, we examined the mechanisms that initiate GVHD, including the relevant antigen-presenting cells. MHC class II was expressed on intestinal epithelial cells (IECs) within the ileum at steady state but was absent from the IECs of germ-free mice. IEC-specific deletion of MHC class II prevented the initiation of lethal GVHD in the GI tract. MHC class II expression on IECs was absent from mice deficient in the TLR adaptors MyD88 and TRIF and required IFNγ secretion by lamina propria lymphocytes. IFNγ responses are characteristically driven by IL-12 secretion from myeloid cells. Antibiotic-mediated depletion of the microbiota inhibited IL-12/23p40 production by ileal macrophages. IL-12/23p40 neutralization prevented MHC class II upregulation on IECs and initiation of lethal GVHD in the GI tract. Thus, MHC class II expression by IECs in the ileum initiates lethal GVHD, and blockade of IL-12/23p40 may represent a readily translatable therapeutic strategy.