Acute graft versus host disease (GVHD) of the gastrointestinal (GI) tract is a major complication of allogeneic hematopoietic stem cell marrow transplantation (HSCT). CD4+ T cells that produce GM-CSF have emerged as central mediators of inflammation in the GI tract as GM-CSF serves as a critical proinflammatory cytokine link between the adaptive and innate arms of the immune system. Notably, there exists cellular heterogeneity within the CD4+ GM-CSF+ T cell population due to the concurrent production of other inflammatory cytokines that has raised questions as to whether these cell populations are regulated by different transcription factors. To examine this question, we employed a major histocompatibility mismatched murine GVHD model (C57BL/6 [H-2b]→Balb/c [H-2d]) and performed single cell RNA sequencing analysis on T cells isolated from the GI tract which identified two CD4+ GM-CSF+ T cell populations that were distinguishable by the presence or absence of IFN-γ co-expression. Bioinformatic analysis of gene regulatory networks in CD4+ GM-CSF+ T cells revealed that CD4+ GM-CSF+ IFN-γ+ T cells had increased expression of Batf and Bhlhe40, whereas CD4+ GM-CSF+ IFN-γ- T cells had preferential expression of Gata3. To determine the functional significance of these transcriptional factors in CD4+ GM-CSF+ T cell biology, we employed CRISPR Cas9-mediated gene editing to create a GM-CSF-Cre mouse that we crossed to Batffl/fl, Bhlhe40fl/fl or Gata3fl/fl animals so that each transcription factor could be individually deleted from GM-CSF expressing cells. We observed that irradiated Balb/c mice reconstituted with B6 bone marrow and splenocytes from either GM-CSF-Cre Batffl/fl or GM-CSF-Cre Bhlhe40fl/fl animals had significantly less GVHD-induced mortality when compared to non-Cre floxed control mice. In addition, these cohorts had a significant reduction in both CD4+ GM-CSF+ IFN-γ- and CD4+ GM-CSF+ IFN-γ+ T cells in the colon, demonstrating that Batf and Bhlhe40 regulated both CD4+ GM-CSF T cell populations. In contrast, there was significantly increased GVHD lethality in mice transplanted with marrow grafts from GM-CSF-Cre Gata3fl/fl animals. Strikingly, this was accompanied by a marked reduction in CD4+ GM-CSF+ IFN-γ+ T cells and a near complete absence of CD4+ GM-CSF+ IFN-γ- T cells, while there was a significant increase in CD4+ GM-CSF- IFN-γ+ T cells in the colon. These data indicated that Gata3 was critical for expression of GM-CSF in CD4+ T cells and that absence of this transcription factor in CD4+ T cells poised to make GM-CSF markedly skewed these cells towards a TH1 cytokine phenotype. In addition, we observed that there was a significant increase in serum IFN-γ and IL-17 levels and augmented pathological damage in the colon of these mice, demonstrating that deletion of Gata3 in poised CD4+ GM-CSF+ T cells augmented their pathogenicity. To verify that Cre excision of GATA3 occurred and was directly responsible for the absence of GM-CSF production in CD4+ T cells we crossed GM-CSF-Cre Gata3fl/fl animals with mT/mG mice to create GM-CSF-Cre Gata3fl/fl mT/mG double fluorescent animals. In these mice, Cre-mediated excision of Gata3 in CD4+ T cells poised to make GM-CSF results in GFP expression thereby validating effective excision in the CD4+ T cell population of interest. Analysis of CD4+ GFP+ T cells in the colon confirmed the near compete absence of GM-CSF production in Gata3-deleted CD4+ T cells. As further validation of a pivotal role for Gata3 in driving GM-CSF production in CD4+ T cells, we stimulated these cells in vitro with anti-CD3 and anti-CD28 antibodies along with culture in IL-7 and soluble anti-IFN-γ antibody to polarize these cells towards a CD4+ GM-CSF+ IFN-γ- T cell population. Culture of naive CD4+ T cells derived from GM-CSF-Cre Gata3fl/fl animals resulted in near complete elimination of CD4+ GM-CSF+ IFN-γ- T cells with a corresponding increase in CD4+ GM-CSF- IFN-γ+ T cells thereby confirming in vivo results. A requisite role for IL-7 as well was corroborated by transplantation of GM-CSF Cre Il7rfl/fl marrow grafts into lethally irradiated recipients which phenocopied results observed with CD4+ T cells from GM-CSF-Cre Gata3fl/fl animals. In summary, these studies identify a critical and nuanced role for Gata3 in driving GM-CSF production in CD4+ T cells while also preventing reversion of these cells to more proinflammatory TH1 cytokine phenotype in the GI tract during GVHD.

Gastrointestinal (GI) tract graft-versus-host disease (GVHD) is a major complication after allogeneic hematopoietic stem cell transplantation and is attributable to dysregulation that occurs between the effector and regulatory arms of the immune system. Whereas regulatory T cells have a primary role in counterbalancing GVHD-induced inflammation, identifying and harnessing other pathways that promote immune tolerance remain major goals in this disease. Herein, we identified interleukin-34 (IL-34) as an intestinal epithelium-derived cytokine that was able to mitigate the severity of GVHD within the GI tract. Specifically, we observed that the absence of recipient IL-34 production exacerbated GVHD lethality, promoted intestinal epithelial cell death, and compromised barrier integrity. Mechanistically, the absence of host IL-34 skewed donor macrophages toward a proinflammatory phenotype and augmented the accumulation of pathogenic CD4+ granulocyte-macrophage colony-stimulating factor (GM-CSF)+ T cells within the colon. Conversely, the administration of recombinant IL-34 substantially reduced GVHD mortality and inflammation, which was dependent on the expression of apolipoprotein E in donor macrophages. Complementary genetic and imaging approaches in mice demonstrated that intestinal epithelial cells were the relevant source of IL-34. These results were supported by colonic biopsies from patients with GVHD, which displayed IL-34 expression in intestinal epithelial cells and apolipoprotein E in lamina propria macrophages, validating similar cellular localization in humans. These studies indicate that IL-34 acts as a tissue-intrinsic cytokine that regulates GVHD severity in the GI tract and could serve as a potential therapeutic target for amelioration of this disease.

Background Post-transplant cyclophosphamide (PTCy) at 50mg/kg on days (D) +3/+4, along with tacrolimus and mycophenolate mofetil (MMF), represents standard of care (SOC) for graft-versus-host disease (GVHD) prophylaxis in adults undergoing reduced intensity conditioning (RIC) allogeneic transplantation (HCT) from an HLA-matched donor. This is based on results of the BMT CTN 1703 study (Bolaños-Meade et al. NEJM 2023). Nearly 25% of patients randomized to PTCy on BMT CTN 1703 developed severe acute GVHD or chronic GVHD by 1 year, and, compared to tacrolimus/methotrexate, these patients experienced significantly more grade 2 infections, and numerically higher rates of severe organ toxicity. Recent studies point to the feasibility of administering PTCy at a reduced dose, 25mg/kg on D+3/+4, preserving GVHD prevention effects and potentially lowering infection rates and organ toxicity. Furthermore, recent studies also suggest that ruxolitinib, a treatment for GVHD, may also be effective for GVHD prophylaxis. Taken together, we initiated a Phase II study where older adults undergoing RIC, HLA-matched HCT, receive PTCy at a lower dose, along with ruxolitinib, to potentially build upon current SOC. Methods This is a prospective phase II study (NCT05622318) to de-escalate PTCy and add ruxolitinib for GVHD prophylaxis in adults 60 years and older, with hematologic malignancies, undergoing RIC peripheral blood allogeneic HCT from an HLA-matched donor. GVHD prophylaxis consisted of PTCy, 25mg/kg on D+3/4, tacrolimus (D+5-180), MMF (D+5-35), and ruxolitinib 5mg twice daily (post-engraftment - 1 year). In this analysis, we report on early safety and feasibility parameters on this novel prophylactic GVHD regimen, including primary engraftment, neutrophil and platelet engraftment kinetics, ruxolitinib compliance and dose modifications through the first 100 days, incidence of grade 2-3 infections by D+100, incidence of Grade 2-4 and 3-4 acute GVHD by day +100, and non-relapse mortality (NRM) by D+100. Results 20 of 54 planned patients have initiated treatment on study. Median age is 67 years (range 61-78 years), 11 patients had AML, 9 patients had MDS. RIC regimens include fludarabine/busulfan (N=19) or fludarabine/melphalan (N=1). Donors included matched related (N=1) or unrelated donors (N=19). Median follow up of survivors is 162 days (range: 20-278 days). Median time to neutrophil and platelet engraftment was 13 days (range: 12-15 days), and 13 days (range: 10-25 days) post-HCT, respectively. Ruxolitinib was initiated at a median 35 days (range: 28-58 days) post-HCT. Median neutrophil and platelet count at initiation was 4,600/µL (range: 1,900-9,200/µL) and 168,000/µL (range: 71,000-340,000/µL) respectively. Zero patients required dose modifications or holds due to ruxolitinib related AEs within the first 100 days. By day 100, the cumulative incidence of grade 2-3 infections was 11.5%, one patient developed bacteremia within the first 30 days; one additional patient developed CMV colitis between day 30 and 100. This case represents the only CMV reactivation/infection event on study. The cumulative incidence of grade 2-4 and 3-4 acute GVHD at D100 was 11.5% and 0%, respectively. Both Grade 2 acute GVHD events were skin only disease. NRM at day 100 is 0%. Discussion Among older adults undergoing HLA-matched RIC allo HCT, de-escalated PTCy and ruxolitinib appears feasible, with rapid and reliable engraftment, low rates of infectious complications, and no NRM in the first 100 days. Early and robust engraftment from lower Cy dose along with lower ruxolitinib dosing (5mg twice daily) both facilitate successful continuous ruxolitinib administration in patients thus far. These results support continued investigation on study as currently written.

The reduced risk of chronic graft-versus-host-disease (GVHD) with posttransplant cyclophosphamide (ptCy) in the setting of haploidentical related donor and more recently, with HLA-matched related and matched and mismatched unrelated donor allogeneic transplantation has been established. There is, however, paucity of data to show if ptCy impacts chronic GVHD pathogenesis, its phenotype and evolution after HCT regardless of the donor status. We examined the differences in chronic GVHD incidence and presentation in 314 consecutive patients after receiving their first allogeneic transplantation (HCT) using ptCy-based GVHD prophylaxis (ptCy-HCT; n = 120; including 95 with haploidentical related donor) versus conventional calcineurin inhibitor-based prophylaxis (CNI-MUD; n = 194) between 2012 and 2019. The 1-year cumulative incidence of all-grade chronic GVHD and moderate/severe chronic GVHD was 24% and 12%, respectively, after ptCy-HCT and 40% and 23% in the CNI-MUD recipients (p = 0.0003 and 0.007). Multivariable analysis confirmed that use of CNI-based GVHD prophylaxis and peripheral blood stem cell graft as the risk factors for chronic GVHD. The cumulative incidence of visceral (involving ≥1 of the following organs: liver, lungs, gastrointestinal tract, serous membranes) chronic GVHD was significantly higher with CNI-MUD vs. ptCy-HCT (27% vs. 15% at 1 year, p = 0.009). The incidence of moderate/severe visceral chronic GVHD was 20% in CNI-MUD group vs. 7.7% in the ptCy-HCT group at 1 year (p = 0.002). In addition, significantly fewer ptCy-HCT recipients developed severe chronic GVHD in ≥3 organs (0.8%) vs. 8.8% in the CNI-MUD group at 1-year posttransplant (p = 0.004). There was no significant different in relapse, non-relapse mortality, and relapse-free and overall survival between the two groups. Further investigation is needed to confirm that reduced risk and severity of chronic GVHD, less visceral organ distribution with ptCy-HCT leads to improved quality of life.

Graft versus host disease (GVHD) is a proinflammatory syndrome driven by alloreactive donor T cells and inflammatory cytokine production which results in tissue destruction and immunological impairment. A critical element of the pathophysiology of GVHD is the failure to reconstitute the regulatory T cell (Treg) compartment. Re-establishment of an effective regulatory network to counterbalance the pro inflammatory milieu by enhancing Treg survival and augmenting suppressive capability therefore remains a major goal in the field. To unveil therapeutically targetable inflammatory cytokine-driven pathways that adversely impact Treg reconstitution during GVHD, our studies focused on the role of interleukin 27 (IL-27) which is produced by activated antigen presenting cells. To examine this question, we employed major (C57BL/6 [H-2b]→Balb/c [H-2d]) and minor (B6 [H-2b]→Balb.B [H-2b]) histocompatibility mismatched murine bone marrow transplantation models and observed that blockade of IL-27 signaling using an IL-27p28-specific antibody significantly improved survival and reduced pathological damage in the liver, lung, and colon when compared to isotype control antibody-treated mice. Similarly, transplantation with IL-27R-/- marrow grafts significantly reduced GVHD lethality and decreased the number of proinflammatory T cells in GVHD target tissues, supporting findings observed after p28 antibody administration. Notably, we observed that the genetic deletion of the IL-27R as well as p28-specfic antibody blockade augmented the reconstitution of peripherally induced and thymically derived CD4+ Tregs in recipient animals, and stabilized Foxp3 expression, preventing reversion to a more pathogenic cellular phenotype. Furthermore, adoptive transfer of IL-27R-/- Tregs was more potent at suppressing GVHD lethality than wild type (WT) Tregs. To identify downstream IL-27-dependent pathways that regulated CD4+ Treg function, donor CD4+ Foxp3+ EGFP+ T cells were flow sorted from pooled livers and colons of mice transplanted with either WT (B6) or IL-27R-/- Foxp3+ EGFP+ marrow grafts and subjected to bulk RNA sequencing. Bioinformatic analysis of RNA sequencing results revealed that the gene IFI30, which encodes the protein known as GILT, a gamma interferon inducible lysosomal thiol reductase, was significantly over expressed in WT when compared to IL-27R-/- Tregs in both tissue sites. To confirm a direct linkage between IL-27 and GILT expression, in vitro-induced CD4+ Foxp3+ Tregs cultured in IL-27 were subjected to western blot analysis which demonstrated a linear correlation between increasing IL-27 concentrations and GILT expression. In vivo studies also confirmed that GILT expression was constitutively increased in naïve splenic WT when compared to IL-27R-/- CD4+ Tregs. Competitive repopulation studies revealed that GILT-/- CD4+ Tregs were present in significantly higher frequencies (9:1 ratio) than WT Tregs in all GVHD target organs, indicating that these cells had superior in vivo survival. To determine if GILT expression was functionally significant, WT or GILT-/- CD4+ Foxp3+ EGFP+ Tregs were adoptively transferred into transplant recipients to prevent GVHD. These studies demonstrated increased Treg persistence in GVHD target organs and improved survival in mice reconstituted with GILT-/- CD4+ Tregs, confirming that these cells were more potent at suppressing GVHD lethality and that the functional inhibitory effects of IL-27/IL-27R signaling were mediated by downstream expression of GILT. Since Treg function and survival is critically dependent upon mitochondrial metabolism, we comparatively assessed metabolic function in WT versus GILT-/- Tregs and observed that polyclonally activated GILT-/- CD4+ Tregs had superior glycolytic activity, oxidative phosphorylation, and fatty acid oxidation when compared to WT CD4+ Tregs. Mechanistically, augmented mitochondrial fitness was attributable to increased mTor signaling leading to inhibition of autophagy. Additionally, GILT-/- CD4+ Tregs had a significant reduction in mitochondrial reactive oxygen species, indicating that GILT exacerbated oxidative stress in these cells. Thus, these results demonstrate that IL-27/IL-27R signaling inhibits CD4+ Treg survival, suppressive capability, and mitochondrial fitness by regulating intracellular lysosomal thiol reductase expression.

Neuroinflammation is a recognized complication of immunotherapeutic approaches such as immune checkpoint inhibitor treatment, chimeric antigen receptor therapy, and graft versus host disease (GVHD) occurring after allogeneic hematopoietic stem cell transplantation. While T cells and inflammatory cytokines play a role in this process, the precise interplay between the adaptive and innate arms of the immune system that propagates inflammation in the central nervous system remains incompletely understood. Using a murine model of GVHD, we demonstrate that type 2 cannabinoid receptor (CB2R) signaling plays a critical role in the pathophysiology of neuroinflammation. In these studies, we identify that CB2R expression on microglial cells induces an activated inflammatory phenotype which potentiates the accumulation of donor-derived proinflammatory T cells, regulates chemokine gene regulatory networks, and promotes neuronal cell death. Pharmacological targeting of this receptor with a brain penetrant CB2R inverse agonist/antagonist selectively reduces neuroinflammation without deleteriously affecting systemic GVHD severity. Thus, these findings delineate a therapeutically targetable neuroinflammatory pathway and has implications for the attenuation of neurotoxicity after GVHD and potentially other T cell-based immunotherapeutic approaches.

In response to acute infection, naive CD4+ T cells primarily differentiate into T helper 1 (Th1) or T follicular helper (Tfh) cells that play critical roles in orchestrating cellular or humoral arms of immunity, respectively. However, despite the well established role of T-bet and BCL-6 in driving Th1 and Tfh cell lineage commitment, respectively, whether additional transcriptional circuits also underlie the fate bifurcation of Th1 and Tfh cell subsets is not fully understood. In this article, we study how the transcriptional regulator Bhlhe40 dictates the Th1/Tfh differentiation axis in mice. CD4+ T cell-specific deletion of Bhlhe40 abrogates Th1 but augments Tfh differentiation. We also assessed an increase in germinal center B cells and Ab production, suggesting that deletion of Bhlhe40 in CD4+ T cells not only alters Tfh differentiation but also their capacity to provide help to B cells. To identify molecular mechanisms by which Bhlhe40 regulates Th1 versus Tfh lineage choice, we first performed epigenetic profiling in the virus specific Th1 and Tfh cells following LCMV infection, which revealed distinct promoter and enhancer activities between the two helper cell lineages. Furthermore, we identified that Bhlhe40 directly binds to cis-regulatory elements of Th1-related genes such as Tbx21 and Cxcr6 to activate their expression while simultaneously binding to regions of Tfh-related genes such as Bcl6 and Cxcr5 to repress their expression. Collectively, our data suggest that Bhlhe40 functions as a transcription activator to promote Th1 cell differentiation and a transcription repressor to suppress Tfh cell differentiation.

Supplementary Figure 2 from Instability of Foxp3 Expression Limits the Ability of Induced Regulatory T Cells to Mitigate Graft versus Host Disease

Paneth cell metaplasia (PCM) typically arises in pre-existing gastrointestinal (GI) diseases; however, the mechanistic pathway that induces metaplasia and whether PCM is initiated exclusively by disorders intrinsic to the GI tract is not well known. Here, we describe the development of PCM in a murine model of chronic myelogenous leukemia (CML) that is driven by an inducible bcr-abl oncogene. Mechanistically, CML induces a proinflammatory state within the GI tract that results in the production of epithelial-derived IL-33. The binding of IL-33 to the decoy receptor ST2 leads to IL-9 production by type 2 innate lymphoid cells (ILC2) which is directly responsible for the induction of PCM in the colon and tissue remodeling in the small intestines, characterized by goblet and tuft cell hyperplasia along with expansion of mucosal mast cells. Thus, we demonstrate that an extra-intestinal disease can trigger an ILC2/IL-9 immune circuit, which induces PCM and regulates epithelial cell fate decisions in the GI tract.

Supplementary Figure 1 from Instability of Foxp3 Expression Limits the Ability of Induced Regulatory T Cells to Mitigate Graft versus Host Disease

Acute graft versus host disease (GVHD) of the gastrointestinal (GI) tract is a major complication of allogeneic bone marrow transplantation. While donor T cells are the proximate trigger of GVHD, innate cells also contribute to disease pathogenesis, although the role of these cell populations is not as well defined. Macrophages, for example, are known contributors in the pathophysiology of chronic GVHD, but their role in acute GVHD is not well delineated. Fundamentally, macrophages are dependent upon exposure to m-CSF and IL-34 which serve as critical trophic factors necessary for their development through binding to the CSF-1R. IL-34 is a more recently discovered cytokine produced by macrophages, but whose role in GVHD pathogenesis is unknown. To examine this question, we employed a murine bone marrow transplantation model (B10.BR [H-2k]®B6[H-2b]) and observed that that there was no difference in GVHD-induced lethality when recipients were reconstituted with marrow grafts from wild type (WT) or IL-34 -/- donors. In contrast, recipient IL-34 -/- animals had significantly worse survival compared to WT mice indicating that host IL-34 expression protected animals from lethal GVHD. We observed no difference in the absolute number of donor macrophages in the colons of WT versus IL-34 -/- mice; however, IL-34 -/- recipient mice had a significant increase in the number of inflammatory Ly6C + macrophages, and a corresponding decrease in Ly6C - cells. Single cell RNA sequence (scRNAseq) analysis of donor macrophages from the colons of WT and IL-34 -/- recipients revealed that macrophages from WT animals had increased expression of anti-inflammatory genes (i.e., CD83, ApoE, Selenop, Lyz1 and Mrc1), whereas macrophages from IL-34 -/- recipients had increased expression of pro-inflammatory genes (i.e., Ifitm1, S100A8, S100A9 and GOs2), indicating that absence of host IL-34 skewed donor macrophages towards an inflammatory phenotype. Since the CSF-1R is not expressed on T cells, we sought to determine the effect of IL-34/CSF-1R signaling on proinflammatory T cells in GVHD target organs. scRNAseq analysis of colonic CD4+ T cells revealed that Csf2 (GM-CSF) was the most differentially expressed gene in IL-34 -/- CD4+ T cells. Functionally, this was confirmed by the observation of a significantly increased number of CD4 + GM-CSF+ T cells in the colons of these mice. Notably, there was no difference in proinflammatory CD8 + T cells, nor any effect on T cell phenotype in the liver or lung, demonstrating that the mitigating effect of host IL-34 expression was confined to the GI tract. To determine the cellular source of IL-34 production, we employed IL-34 LacZ reporter mice and showed that β-Gal co-localized with the epithelial marker, E-cadherin, indicating IL-34 is produced primarily by intestinal epithelial cells. Additional studies using chimeric mice in which IL-34 was restricted to either the hematopoietic or non-hematopoietic compartmentrevealed that absence of IL-34 in the non-hematopoietic compartment phenocopied findings observed with global IL-34 -/- animals, confirming that intestinal epithelial cells were the primary source of this cytokine. The administration of exogenous IL-34 prolonged survival, abrogated pathological damage in the colon and resulted in a corresponding decrease in pro-inflammatory macrophages and T cells, demonstrating this pathway could be pharmacologically targeted. Re-analysis of the scRNAseq data on donor macrophages from the colons of WT and IL-34 -/- mice revealed that apolipoprotein E ( Apoe) was the most differentially expressed gene in macrophages from WT GVHD animals. To confirm a functional role for ApoE, mice were transplanted with T cell depleted (TCD) BM from either WT or ApoE -/- mice and treated with IL-34. These studies revealed that the protective effect conferred by IL-34 was abrogated in animals that received TCD ApoE -/- BM grafts, indicating that ApoE expression in donor macrophages was necessary for agonist driven IL-34-mediated regulation of GVHD. Finally, we confirmed these studies have translational relevance by demonstrating IL-34 and Apoe have increased expression in intestinal epithelial cells and macrophages, respectively, in the colons of humans with GVHD. Thus, these results identify IL-34 as a novel intestinal epithelial cell-derived cytokine that regulates macrophage polarization and mitigates GVHD in an ApoE-dependent manner.

Not available.

Inflammatory physiology has been linked to behavioral and emotional symptoms in a variety of contexts and experimental paradigms. Hematopoietic cell transplantation (HCT) represents an intersection of significant immune dysregulation and psychosocial stress, and this biobehavioral relationship can influence important clinical outcomes. For those undergoing HCT with inflammation-related neuropsychiatric symptoms, using targeted agents such as the IL-6 receptor antagonist tocilizumab may be an effective therapeutic approach. We conducted an observational cohort study to explore patient reported outcomes (PROs) and inflammatory biomarkers among allogeneic HCT recipients who received tocilizumab compared to those who did not. Individuals on a larger trial of tocilizumab for prevention of graft-versus-host disease received a single dose of tocilizumab 24 h prior to stem cell infusion. Measures of anxiety, depression, pain, fatigue, and sleep quality and parallel blood samples for inflammatory cytokines were collected from participants and an analogous comparison cohort at baseline and Day 28 after stem cell infusion. Demographic and medical characteristics were reported; an analysis of covariance regression model was fitted to evaluate differences in PROs and distance correlation t-tests assessed for associations between biomarkers and PRO measures. For n = 18 tocilizumab-treated and n = 22 comparison patients, there were no significant differences between patient demographics, but the tocilizumab cohort had a different distribution of primary diagnoses (p = 0.009) with more patients with leukemias and a higher proportion of patients in their first remission (64% vs 28%, p = 0.024). Depression was higher at Day 28 compared to baseline in both groups (comparison group: +5.1 [95% CI 0.14–10, p = 0.045], tocilizumab: +8.6 [95% CI 2.3–15, p = 0.011]), though the difference between groups did not reach statistical significance. The tocilizumab group had significantly increased circulating IL-6 and decreased CRP at Day 28 (all p < 0.05). There was an association between collective baseline biomarkers and PROs (distance correlation dCor = 0.110, p = 0.005), but this same association was not present at Day 28 (dCor = −0.001, p = 0.5). In univariate analyses, a 10-fold increase in plasma IL-6 was associated with a 3.6-point higher depression score (95% CI 1.0–6.2, p = 0.008). In this exploratory analysis of PROs and inflammatory biomarkers in patients undergoing HCT, tocilizumab was not associated with favorable patient-reported symptom profiles. This finding is aligned with our prior work in the HCT population but diverges from hypothesized therapeutic effects of tocilizumab on depressive symptoms, thus highlighting the need for larger prospective translational studies in biobehavioral HCT research.

ABSTRACT A common feature in the gastrointestinal (GI) tract during allogeneic hematopoietic stem cell transplantation is the loss of microbial diversity and emergence of opportunistic pathogens that can adversely impact survival. Consequently, preventing transplant-associated dysbiosis is an emerging strategy for optimizing treatment outcomes. In this study, we examined the effect of an extended tocilizumab administration schedule in addition to tacrolimus/methotrexate (Tac/MTX) as graft versus host disease (GVHD) prophylaxis on microbial composition in the GI tract along with overall transplant outcomes. Twenty-nine patients received busulfan-based myeloablative conditioning and were transplanted with HLA-matched related or unrelated peripheral blood stem cell grafts. The primary end point of the trial was GVHD-free relapse-free survival (GRFS) at 12 months. The cumulative incidences of grades 2-4 and 3-4 acute GVHD were 10.5% and 7% at day 180, respectively. There was one case of GVHD of the lower GI tract within the first 12 months. Non-relapse mortality and relapse-free survival were 3.4% and 86.2% at one year, respectively. GRFS was 38% at one year which was significantly higher than the pre-specified historical control rate of 20% (p=0.02) and therefore met the primary end point of the trial. Fecal samples from this patient population were sequenced and computationally analyzed centrally along with a demographically matched control cohort that received only Tac/MTX for GVHD prophylaxis. This comparative analysis revealed significantly less loss of α-diversity and reduced emergence of pathogenic organisms such as enterococcus in tocilizumab-treated recipients, demonstrating that loss of microbial diversity and enterococcal domination is attenuated in these patients. (Clinicaltrial.gov Identifier: NCT03699631 ).

Gastrointestinal (GI) tract involvement is a major determinant for subsequent morbidity and mortality arising during graft-versus-host disease (GVHD). CD4+ T cells that produce granulocyte-macrophage colony stimulating factor (GM-CSF) have emerged as central mediators of inflammation in this tissue site as GM-CSF serves as a critical cytokine link between the adaptive and innate arms of the immune system. However, cellular heterogeneity within the CD4+ GM-CSF+ T-cell population due to the concurrent production of other inflammatory cytokines has raised questions as to whether these cells have a common ontology or if a unique CD4+ GM-CSF+ subset exists that differs from other defined T helper subtypes. Using single-cell RNA sequencing analysis (scRNAseq), we identified two CD4+ GM-CSF+ T-cell populations that arose during GVHD and were distinguishable according to the presence or absence of interferon-γ (IFN-γ) coexpression. CD4+ GM-CSF+ IFN-γ- T cells, which emerged preferentially in the colon, had a distinct transcriptional profile, used unique gene regulatory networks, and possessed a nonoverlapping T-cell receptor repertoire compared with CD4+ GM-CSF+ IFN-γ+ T cells as well as all other transcriptionally defined CD4+ T-cell populations in the colon. Functionally, this CD4+ GM-CSF+ T-cell population contributed to pathologic damage in the GI tract that was critically dependent on signaling through the interleukin-17 (IL-7) receptor but was independent of type 1 interferon signaling. Thus, these studies help to unravel heterogeneity within CD4+ GM-CSF+ T cells that arise during GVHD and define a developmentally distinct colitogenic T helper subtype GM-CSF+ subset that mediates immunopathology.

Graft versus host disease (GVHD) is the major non-relapse complication associated with allogeneic hematopoietic stem cell transplantation (HSCT). Unfortunately, GVHD occurs in roughly half of patients following this therapy and can induce severe life-threatening side effects and premature mortality. The pathophysiology of GVHD is driven by alloreactive donor T cells that induce a proinflammatory environment to cause pathological damage in the skin, gastrointestinal (GI) tract, lung, and liver during the acute phase of this disease. Recent work has demonstrated that the GI tract is a pivotal target organ and a primary driver of morbidity and mortality in patients. Prevention of this complication has therefore emerged as an important goal of prophylaxis strategies given the primacy of this tissue site in GVHD pathophysiology. In this review, we summarize foundational pre-clinical studies that have been conducted in animal models to prevent GI tract GVHD and examine the efficacy of these approaches upon subsequent translation into the clinic. Specifically, we focus on therapies designed to block inflammatory cytokine pathways, inhibit cellular trafficking of alloreactive donor T cells to the GI tract, and reconstitute impaired regulatory networks for the prevention of GVHD in the GI tract.