Microbiota dictate T cell clonal selection to augment graft-versus-host disease after stem cell transplantation.

Allogeneic T cell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a closed genetic system within which donor T cells interact with peptide-major histocompatibility complexes (MHCs), though clonal interrogation remains challenging due to the sparseness of the T cell repertoire. We developed a Bayesian model using donor and recipient T cell receptor (TCR) frequencies in murine stem cell transplant systems to define limited common expansion of T cell clones across genetically identical donor-recipient pairs. A subset of donor CD4+ T cell clonotypes differentially expanded in identical recipients and were microbiota dependent. Microbiota-specific T cells augmented GVHD lethality and could target microbial antigens presented by gastrointestinal epithelium during an alloreactive response. The microbiota serves as a source of cognate antigens that contribute to clonotypic T cell expansion and the induction of GVHD independent of donor-recipient genetics.

Infection induces tissue-resident memory NK cells that safeguard tissue health.

Tissue health is dictated by the capacity to respond to perturbations and then return to homeostasis. Mechanisms that initiate, maintain, and regulate immune responses in tissues are therefore essential. Adaptive immunity plays a key role in these responses, with memory and tissue residency being cardinal features. A corresponding role for innate cells is unknown. Here, we have identified a population of innate lymphocytes that we term tissue-resident memory-like natural killer (NKRM) cells. In response to murine cytomegalovirus infection, we show that circulating NK cells were recruited in a CX3CR1-dependent manner to the salivary glands where they formed NKRM cells, a long-lived, tissue-resident population that prevented autoimmunity via TRAIL-dependent elimination of CD4+ T cells. Thus, NK cells develop adaptive-like features, including long-term residency in non-lymphoid tissues, to modulate inflammation, restore immune equilibrium, and preserve tissue health. Modulating the functions of NKRM cells may provide additional strategies to treat inflammatory and autoimmune diseases.

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.

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.

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.

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.

Cytokines and costimulation in acute graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (alloSCT) is an important curative therapy for high-risk hematological malignancies, but the development of severe and/or steroid-refractory acute graft-versus-host disease (aGVHD) remains a significant limitation to optimal outcomes. New approaches to prevent and treat aGVHD remain an unmet need that can be best addressed by understanding the complex disease pathophysiology. It is now clear that chemoradiotherapy used prior to alloSCT induces the release of endogenous alarmins (eg, HMGB-1, ATP, IL-1α, IL-33) from recipient tissue. Exogenous pathogen-derived molecules (eg, lipopolysaccharide, nucleic acids) also translocate from the gastrointestinal tract lumen. Together, these danger signals activate antigen-presenting cells (APCs) to efficiently present alloantigen to donor T cells while releasing cytokines (eg, interleukin-12 [IL-12], IL-23, IL-6, IL-27, IL-10, transforming growth factor-ß) that expand and differentiate both pathogenic and regulatory donor T cells. Concurrent costimulatory signals at the APC-T-cell interface (eg, CD80/CD86-CD28, CD40-CD40L, OX40L-OX40, CD155/CD112-DNAM-1) and subsequent coinhibitory signals (eg, CD80/CD86-CTLA4, PDL1/2-PD1, CD155/CD112-TIGIT) are critical to the acquisition of effector T-cell function and ensuing secretion of pathogenic cytokines (eg, IL-17, interferon-γ, tissue necrosis factor, granulocyte-macrophage colony-stimulating factor) and cytolytic degranulation pathway effectors (eg, perforin/granzyme). This review focuses on the combination of cytokine and costimulatory networks at the T-cell surface that culminates in effector function and subsequent aGVHD in target tissue. Together, these pathways now represent robust and clinically tractable targets for preventing the initiation of deleterious immunity after alloSCT.

The primacy of gastrointestinal tract antigen-presenting cells in lethal graft-versus-host disease.

Allogeneic stem cell transplantation is a cornerstone of curative therapy for high-risk and/or advanced hematological malignancies but remains limited by graft-versus-host disease (GVHD). GVHD is initiated by the interaction between recipient antigen-presenting cells (APCs) and donor T cells, culminating in T-cell differentiation along pathogenic type-1 and type-17 paradigms at the expense of tolerogenic regulatory T-cell patterns. Type-1 and type-17 T cells secrete cytokines (eg, granulocyte-macrophage colony-stimulating factor and interferon-γ) critical to the cytokine storm that amplifies expansion of donor APCs and their alloantigen presentation. It has become increasingly clear that pathogenic donor T-cell differentiation is initiated by both professional recipient APCs (eg, dendritic cells [DCs]) and nonprofessional APCs (eg, epithelial and mesenchymal cells), particularly within the gastrointestinal (GI) tract. In the immediate peritransplantation period, these APCs are profoundly modified by pathogen-associated molecular pattern (PAMP)/damage-associated molecular pattern (DAMP) signals derived from conditioning and intestinal microbiota. Subsequently, donor DCs in the GI tract are activated by DAMP/PAMP signals in the colon that gain access to the lamina propria once the mucosal barrier mucosa is compromised by GVHD. This results in donor DC expansion and alloantigen presentation in the colon and subsequent migration into the mesenteric lymph nodes. Here, new donor T cells are primed, expanded, differentiated, and imprinted with gut-homing integrins permissive of migration into the damaged GI tract, resulting in the lethal feed-forward cascade of GVHD. These new insights into our understanding of the cellular and molecular factors initiating GVHD, both spatially and temporally, give rise to a number of logical therapeutic targets, focusing on the inhibition of APC function in the GI tract.

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.

Neutrophils provide cellular communication between ileum and mesenteric lymph nodes at graft-versus-host disease onset.

Conditioning-induced damage of the intestinal tract plays a critical role during the onset of acute graft-versus-host disease (GVHD). Therapeutic interference with these early events of GVHD is difficult, and currently used immunosuppressive drugs mainly target donor T cells. However, not donor T cells but neutrophils reach the sites of tissue injury first, and therefore could be a potential target for GVHD prevention. A detailed analysis of neutrophil fate during acute GVHD and the effect on T cells is difficult because of the short lifespan of this cell type. By using a novel photoconverter reporter system, we show that neutrophils that had been photoconverted in the ileum postconditioning later migrated to mesenteric lymph nodes (mLN). This neutrophil migration was dependent on the intestinal microflora. In the mLN, neutrophils colocalized with T cells and presented antigen on major histocompatibility complex (MHC)-II, thereby affecting T cell expansion. Pharmacological JAK1/JAK2 inhibition reduced neutrophil influx into the mLN and MHC-II expression, thereby interfering with an early event in acute GVHD pathogenesis. In agreement with this finding, neutrophil depletion reduced acute GVHD. We conclude that neutrophils are attracted to the ileum, where the intestinal barrier is disrupted, and then migrate to the mLN, where they participate in alloantigen presentation. JAK1/JAK2-inhibition can interfere with this process, which provides a potential therapeutic strategy to prevent early events of tissue damage-related innate immune cell activation and, ultimately, GVHD.

Acute graft-versus-host disease is regulated by an IL-17-sensitive microbiome.

Donor T-cell-derived interleukin-17A (IL-17A) can mediate late immunopathology in graft-versus-host disease (GVHD), however protective roles remain unclear. Using multiple cytokine and cytokine receptor subunit knockout mice, we demonstrate that stem cell transplant recipients lacking the ability to generate or signal IL-17 develop intestinal hyper-acute GVHD. This protective effect is restricted to the molecular interaction of IL-17A and/or IL-17F with the IL-17 receptor A/C (IL-17RA/C). The protection from GVHD afforded by IL-17A required secretion from, and signaling in, both hematopoietic and nonhematopoietic host tissue. Given the intestinal-specificity of the disease in these animals, we cohoused wild-type (WT) with IL-17RA and IL-17RC-deficient mice, which dramatically enhanced the susceptibility of WT mice to acute GVHD. Furthermore, the gut microbiome of WT mice shifted toward that of the IL-17RA/C mice during cohousing prior to transplant, confirming that an IL-17-sensitive gut microbiota controls susceptibility to acute GVHD. Finally, induced IL-17A depletion peritransplant also enhanced acute GVHD, consistent with an additional protective role for this cytokine independent of effects on dysbiosis.

Alloantigen presentation and graft-versus-host disease: fuel for the fire.

Allogeneic stem cell transplantation (SCT) is a unique procedure, primarily in patients with hematopoietic malignancies, involving chemoradiotherapy followed by the introduction of donor hematopoietic and immune cells into an inflamed and lymphopenic environment. Interruption of the process by which recipient alloantigen is presented to donor T cells to generate graft-versus-host disease (GVHD) represents an attractive therapeutic strategy to prevent morbidity and mortality after SCT and has been increasingly studied in the last 15 years. However, the immune activation resulting in GVHD has no physiological equivalent in nature; alloantigen is ubiquitous, persists indefinitely, and can be presented by multiple cell types at numerous sites, often on incompatible major histocompatibility complex, and occurs in the context of intense inflammation early after SCT. The recognition that alloantigen presentation is also critical to the development of immunological tolerance via both deletional and regulatory mechanisms further adds to this complexity. Finally, GVHD itself appears capable of inhibiting the presentation of microbiological antigens by donor dendritic cells late after SCT that is mandatory for the establishment of effective pathogen-specific immunity. Here, we review our current understanding of alloantigen, its presentation by various antigen-presenting cells, subsequent recognition by donor T cells, and the potential of therapeutic strategies interrupting this disease-initiating process to modify transplant outcome.

Corruption of dendritic cell antigen presentation during acute GVHD leads to regulatory T-cell failure and chronic GVHD.

Chronic graft-versus-host disease (cGVHD) is a major cause of late mortality following allogeneic bone marrow transplantation (BMT) and is characterized by tissue fibrosis manifesting as scleroderma and bronchiolitis obliterans. The development of acute GVHD (aGVHD) is a powerful clinical predictor of subsequent cGVHD, suggesting that aGVHD may invoke the immunologic pathways responsible for cGVHD. In preclinical models in which sclerodermatous cGVHD develops after a preceding period of mild aGVHD, we show that antigen presentation within major histocompatibility complex (MHC) class II of donor dendritic cells (DCs) is markedly impaired early after BMT. This is associated with a failure of regulatory T-cell (Treg) homeostasis and cGVHD. Donor DC-restricted deletion of MHC class II phenocopied this Treg deficiency and cGVHD. Moreover, specific depletion of donor Tregs after BMT also induced cGVHD, whereas adoptive transfer of Tregs ameliorated it. These data demonstrate that the defect in Treg homeostasis seen in cGVHD is a causative lesion and is downstream of defective antigen presentation within MHC class II that is induced by aGVHD.

Tc17 cells are a proinflammatory, plastic lineage of pathogenic CD8+ T cells that induce GVHD without antileukemic effects.

IL-17-producing cells are important mediators of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). Here we demonstrate that a distinct CD8(+) Tc17 population develops rapidly after SCT but fails to maintain lineage fidelity such that they are unrecognizable in the absence of a fate reporter. Tc17 differentiation is dependent on alloantigen presentation by host dendritic cells (DCs) together with IL-6. Tc17 cells express high levels of multiple prototypic lineage-defining transcription factors (eg, RORγt, T-bet) and cytokines (eg, IL-17A, IL-22, interferon-γ, granulocyte macrophage colony-stimulating factor, IL-13). Targeted depletion of Tc17 early after transplant protects from lethal acute GVHD; however, Tc17 cells are noncytolytic and fail to mediate graft-versus-leukemia (GVL) effects. Thus, the Tc17 differentiation program during GVHD culminates in a highly plastic, hyperinflammatory, poorly cytolytic effector population, which we term "inflammatory iTc17" (iTc17). Because iTc17 cells mediate GVHD without contributing to GVL, therapeutic inhibition of iTc17 development in a clinical setting represents an attractive approach for separating GVHD and GVL.

Lung parenchyma-derived IL-6 promotes IL-17A-dependent acute lung injury after allogeneic stem cell transplantation.

Idiopathic pneumonia syndrome (IPS) is a relatively common, frequently fatal clinical entity, characterized by noninfectious acute lung inflammation following allogeneic stem cell transplantation (SCT), the mechanisms of which are unclear. In this study, we demonstrate that immune suppression with cyclosporin after SCT limits T-helper cell (Th) 1 differentiation and interferon-γ secretion by donor T cells, which is critical for inhibiting interleukin (IL)-6 generation from lung parenchyma during an alloimmune response. Thereafter, local IL-6 secretion induces donor alloantigen-specific Th17 cells to preferentially expand within the lung, and blockade of IL-17A or transplantation of grafts lacking the IL-17 receptor prevents disease. Studies using IL-6(-/-) recipients or IL-6 blockade demonstrate that IL-6 is the critical driver of donor Th17 differentiation within the lung. Importantly, IL-6 is also dysregulated in patients undergoing clinical SCT and is present at very high levels in the plasma of patients with IPS compared with SCT recipients without complications. Furthermore, at the time of diagnosis, plasma IL-6 levels were higher in a subset of IPS patients who were nonresponsive to steroids and anti-tumor necrosis factor therapy. In sum, pulmonary-derived IL-6 promotes IPS via the induction of Th17 differentiation, and strategies that target these cytokines represent logical therapeutic approaches for IPS.

Cross-dressing by donor dendritic cells after allogeneic bone marrow transplantation contributes to formation of the immunological synapse and maximizes responses to indirectly presented antigen.

The stimulation of naive donor T cells by recipient alloantigen is central to the pathogenesis of graft-versus-host disease after bone marrow transplantation (BMT). Using mouse models of transplantation, we have observed that donor cells become "cross-dressed" in very high levels of recipient hematopoietic cell-derived MHC class I and II molecules following BMT. Recipient-type MHC is transiently present on donor dendritic cells (DCs) after BMT in the setting of myeloablative conditioning but is persistent after nonmyeloablative conditioning, in which recipient hematopoietic cells remain in high numbers. Despite the high level of recipient-derived alloantigen present on the surface of donor DCs, donor T cell proliferative responses are generated only in response to processed recipient alloantigen presented via the indirect pathway and not in response to cross-dressed MHC. Assays in which exogenous peptide is added to cross-dressed MHC in the presence of naive TCR transgenic T cells specific to the MHC class II-peptide combination confirm that cross-dressed APC cannot induce T cell proliferation in isolation. Despite failure to induce T cell proliferation, cross-dressing by donor DCs contributes to generation of the immunological synapse between DCs and CD4 T cells, and this is required for maximal responses induced by classical indirectly presented alloantigen. We conclude that the process of cross-dressing by donor DCs serves as an efficient alternative pathway for the acquisition of recipient alloantigen and that once acquired, this cross-dressed MHC can assist in immune synapse formation prior to the induction of full T cell proliferative responses by concurrent indirect Ag presentation.