Lethal GVHD Research Articles

Galectin-3 expression in donor T cells reduces GvHD severity and lethality after allogeneic hematopoietic cell transplantation

Abundant donor cytotoxic Tcells that attack normal host organs remain a major problem for patients receiving allogeneic hematopoietic cell transplantation (allo-HCT). Despite an increase in our knowledge of the pathobiology of acute graft versus host disease (aGvHD), the mechanisms regulating the proliferation and function of donor Tcells remain unclear. Here, we show that activated donor Tcells express galectin-3 (Gal-3) after allo-HCT. In both major and minor histocompatibility-mismatched models of murine aGvHD, expression of Gal-3 is associated with decreased Tcell activation and suppression of the secretion of effector cytokines, including IFN-γ and GM-CSF. Mechanistically, Gal-3 results in activation of NFAT signaling, which can induce Tcell exhaustion. Gal-3 overexpression in human Tcells prevents severe disease by suppressing cytotoxic Tcells in xenogeneic aGvHD models. Together, these data identify the Gal-3-dependent regulatory pathway in donor Tcells as a critical component of inflammation in aGvHD.

Differentiation of a CD4/CD8αβ Double Positive T Cell Population from the CD8 Pool Is Both Predictive and Sufficient to Mediate Graft-Vs-Host Disease

Acute graft-vs-host disease (aGVHD) and cancer relapse remain the primary complications following an allogeneic hematopoietic stem cell transplantation (allo-HSCT) for malignant blood disorders. While post-transplant cyclophosphamide combined with standard GVHD prophylaxis has greatly reduced the overall prevalence and severity of aGVHD, relapse rates remain a concern. There is thus a need to identify the specific human T cell populations mediating GVHD vs GVL activity as a means to develop targeted therapeutics capable of controlling aGVHD without inhibiting GVL activity. In this study, we identify a novel human T cell population that develops after transplant that is predictive and sufficient for GVHD pathology.To determine the role of human T cell populations in aGVHD, we performed xenogeneic transplantation studies using primary human graft tissue from a variety of sources (peripheral blood, G-CSF mobilized peripheral blood, bone marrow and umbilical cord blood) in addition to collecting primary human aGVHD blood samples from our clinic. Using the LD50 dose of human graft tissue, we identified a novel mature CD4 +/CD8αβ + double positive (DP) T cell population that only developed after transplantation. The development of this population was further confirmed in aGVHD patients from our clinic. The presence of DP T cells, irrespective of graft source, was also predictive of lethal GVHD in as early as one week after xenogeneic transplantation.To identify the origin of DP T cells, we transplanted isolated human CD4 or CD8 T cells into mice which showed that DP T cells only arise from the CD8 pool. Furthermore, re-transplantation of flow-sorted CD8 T cells from GVHD mice did not reveal a 2nd wave of DP T cell differentiation. This data, in addition to their highly proliferative state, suggests that DP T cells represent highly activated CD8 T cell clones. The ability of these CD8-derived DP T cells to gain CD4 expression coincides with their co-expression of both RUNX3 and THPOK, the master transcription factors of the CD8 and CD4 lineages respectively, that classically repress each other. Intracellular cytokine staining also revealed that DP T cells are the primary activated T cell population in xenogeneic GVHD, secreting both modulatory and cytotoxic cytokines (e.g. IFNγ, IL-17A, IL-22, perforin and granzyme). Ex vivo re-stimulation or re-transplantation of flow-sorted DP T cells showed that this T cell population is capable of dividing and expanding independent of CD4 and CD8 single positive T cells with the majority of the isolated DP T cells retaining their co-expression of CD4 and CD8.Finally, transplantation of either isolated human peripheral blood CD4 or CD8 T cell populations were capable of causing lethal GVHD. Conversely, re-transplantation of flow-sorted DP, CD8 or CD4 T cells from GVHD mice revealed that DP and CD4 T cells are sufficient to mediate GVHD pathology but re-transplanted CD8 T cell are not. This correlates with the absence of DP T cell differentiation in that re-transplanted CD8 population. The differentiation of DP T cells from chronically activated CD8 T cells represents a novel mechanism of GVHD pathology not previously described. The presence of DP T cells in other chronic inflammatory human diseases also suggests a broader pathology mediated by DP T cells. Further understanding of DP T cell differentiation and pathology may lead to targeted prophylaxis and/or treatment regimens for aGVHD and other human chronic inflammatory diseases. [Display omitted] DisclosuresCapitini: Nektar Therapeutics: Honoraria; Novartis: Honoraria.

Longitudinal Analysis of Gut Bacteriome, Mycobiome, Virome and Metabolome in Allogeneic Stem Cell Transplantation Reveals Susceptibility for Acute Graft-Versus-Host Disease

The intestinal microbiome directly effects outcomes in patients undergoing allogeneic stem cell transplantation (ASCT) (Peled et al., N Engl J Med, 2020). Recent developments in microbiome research allow us to look beyond bacterial communities at the composition of the intestinal mycobiome and virome. However, only few studies have addressed these fungal (Zhang et al., Nat Commun, 2021; Legoff et al., Nat Med, 2017) and viral communities (Van Der Velden et al., Biol Blood Marrow Transplant, 2013) in the ASCT setting.Loss of bacterial diversity and domination of single taxa was linked to increased incidence of graft-versus-host-disease (aGvHD) and mortality. However, the functional consequences of intestinal dysbiosis remain poorly understood. Recently, microbial-derived metabolites such as short-chain fatty acids (SCFAs), indoles and secondary bile acids (BA) have been proposed to explain how microbiota exert effects on epithelial and immune responses and shown to protect from GVHD in animal models.To assemble a complete picture of the phylogenetic and metabolic changes which occur as patients undergo ASCT we designed a multiomics approach and identified patterns in bacteriome, mycobiome, virome and metabolome dynamics in relation to clinical factors and GvHD.353 stool samples from 80 patients undergoing ASCT were obtained at two different transplantation centers (Munich and Regensburg) at defined timepoints before and after transplantation. Changes in metabolite composition were evaluated via mass spectrometry using a targeted metabolomic profiling approach, accompanied by bacterial 16S rDNA-, fungal 18S-ITS- and deep virome sequencing. Results were linked among each other and with clinical metadata such as GvHD, response to steroids, antimicrobial therapy and mortality.Longitudinal profiling of ASCT patients revealed a significant loss of bacterial, fungal and viral diversity following ASCT. At both centers, the effect was most pronounced in the early post-transplant period. Patients that developed GI-GVHD had the lowest diversity scores (Panel 1). GVHD patient samples were dominated by Enteroccocus and Candida, and loss of Caudovirales species, indicative of deep dysbiosis effecting all intestinal communities.ASCT had a strong impact on microbiome function and their ability to produce metabolites. We assayed each sample for production of metabolites with reported protective effects: SCFAs (acetate, butyrate and propionate), indoles (indole-3-carboxylaldehyde, ICA) and BAs (deoxycholic and lithocholic acid). When admitted to the transplantation unit (timepoint “conditioning”) most patients had rich metabolite expression profiles. In the early post-transplant period, metabolite levels became progressively more depleted. GVHD patients displayed a nearly complete loss of metabolites (Panel 2a). Importantly, levels of the metabolite ICA at early timepoints could predict the occurrence of GVHD, leading us to consider prophylactic administration in future studies.We performed beta diversity analysis to identify clinical factors that had the highest impact on microbial communities. As expected, we noted that administration of broad-spectrum antibiotics had a profound impact of bacteria, fungi and viruses; but also had a significant negative effect on metabolites levels.Lastly, we report on a case of successful treatment of severe GI-GVHD with fecal microbiota transplant. Response to treatment was characterized by the recipient adopting the FMT donor's rich metabolite expression profile (Panel 2b) .To our knowledge, this is the first multicenter study that combines bacteriome, mycobiome, virome and metabolome analyses a longitudinal fashion in matched patient cohorts. Our results are consistent with previous findings that there are major alterations in gut microbiome and metabolite composition after ASCT. We expand upon those findings by characterizing the time course of phylogenetic alterations and their functional consequences. Those alterations play an important role in GvHD pathogenesis and might serve as potential biomarkers to identify patients at high risk for developing lethal GvHD early on. Our results highlight two strategies for targeted microbiome-based interventions: (1) preventing loss of microbial communities by restrictive use of antibiotics and (2) prophylactic administration of metabolite cocktails. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Early Administration of Low-Dose IL-2 Intensify Graft-Versus-Leukemia Effect without Worsening GVHD through Sequential Enhancement of Effector T Cell and CD62L+ Regulatory T Cell Subset

Allogeneic hematopoietic stem cell transplantation (HSCT) is the curative treatment of many hematological malignancies, but the relapse is one of the major causes of treatment failure and mortality. Previous clinical studies have shown that low-dose IL-2 was well tolerated and could lower the relapse rate in human T cell depleted allogeneic BMT. On the other hand, we also demonstrated that low-dose IL-2 could preferentially enhance regulatory T cell (Treg) and improve the symptoms of chronic GVHD (NEJM 2011,Sci Trans Med 2013). In murine bone marrow transplantation (BMT) model showed that Treg can protect from lethal GVHD while preserving graft-versus-leukemia (GVL) effect. Especially, CD62L+ Tregs has a higher capacity in suppressing GVHD than their CD62L- counterpart. Our recent study reported that exogenous low-dose IL-2 induced the expression of PD-1 on Treg preferentially and it was most evident in CD44+CD62L+central-memory Treg (Blood 2016). However, the impact of CD62L+ Treg enhanced by low-dose IL-2 on GVHD and GVL has not been well investigated. In this study, we studied the effect of exogenous IL-2 on the lymphocyte phenotypic profiles including naïve- versus memory-type balance and also examined donor-derived immunity, especially GVHD and GVL, by using leukemia-bearing BMT model. Lethally irradiated B6D2F1 recipients were transplanted spleen cells and BM cells from B6 donors and 5000 IU Teceleukin (recombinant IL-2) was injected once a day for 14 days. The number of CD4+CD25+Foxp3+ Treg, CD4+CD25-Foxp3- conventional T cell (Tcon) and CD8+ T cell was compared between IL2- and vehicle-treated groups. The expressions of CD44, CD62L, CCR4, CCR7, Ki-67, PD-1, CTLA-4, LAG3, GITR, ICOS and TIM-3 in each subset were also examined. Lymphocytes profiling analysis showed that IL-2 treatment in the first 7 days resulted in the dominant expansion of effector T cells without Treg increase (Treg 0.10 vs 0.16,p=0.35, Tcon 2.83 vs 4.20, p=0.01, CD8 T cell 5.52 vs 10.4, p=0.01, million). On the contrary, the extended IL-2 treatment in the next 7 days increased CD62L+ Treg (21.5 vs 48.2 % of total Treg, p<0.001, 0.02 vs 0.07 million, p=0.006) without increasing effector T cells. PD-1 on Treg was very high in BMT-recipients even without receiving IL-2, so further enhancement of PD-1 expression by IL-2 was not observed. However, the expression of other inhibitory molecules including CTLA-4 (MFI, 6.61 vs 9.92, p=<0.001), LAG-3 (1.73 vs 2.17, p=0.04) and ICOS (2.41 vs 3.84, P<0.001) was enhanced by the IL-2 treatment. To elucidate the mechanisms of the increase in CD62L+ Treg, the in vivo violet dye dilution assay was performed. We first sorted out CD62L+ T cells by micro-beads from spleen cells, and then adaptively transferred into recipients. Five days treatment of low-dose IL-2 resulted in the enhancement of the division of CD62L+ Treg (% divided cells; 19.5 vs 29.8 % of Treg, p=0.005, 62.3 vs 108 in number, p=0.002). Interestingly, when CD62L- T cells were transferred, IL-2 treatment also induced the division of the cells and, of note, induced the phenotypic change of CD62L- into CD62L+ Treg (7.63 vs 19.8 % of Treg, p=0.009, 91.3 vs 361, p=0.01). These results suggested that IL-2 have the possibility to enhance the proliferative activity of Treg with the up-regulation of CD62L expression. Lastly, we transplanted lucipherase+P815 leukemia cells into this BMT model and evaluated the clinical impact of low-dose IL-2 on GVL by using in vivo bioluminescence system. To assesse the GVL and GVHD independently, we used two different tumor burden models. In the high tumor burden model, IL-2 injection decreased bioluminescence intensity (BLI) of P815 (BLI at week3, 8.3x10E7 vs 8.1x10E6 photon/second, p<0.001) and significantly extends overall survival. On the other hand, in the low tumor burden model, both treatment groups did not die from leukemia but vehicle-treated control group showed a significant higher clinical GVHD score and died from GVHD. These data demonstrated the clinical benefits on anti-tumor effect of IL-2, which could enhance the GVL activity without the detrimental effect on GVHD. In conclusion, our data indicate multiple effects of IL-2 on post-transplant immunity can coordinate to intensify GVL separately from GVHD on the mechanism of sequential enhancement of effector T cell and CD62L+ Treg in the different phases. These data provide the important information for developing Treg-targeted therapy. DisclosuresMaeda:Toko Pharmaceutical Industries: Other: Investigational drug is provided free of charge.

Increased Naive, Stem Cell Memory and Terminally Differentiated CD8+ T Cells in Chronic Graft Versus Host Disease

BACKGROUND AND AIMSDespite improved clinical outcomes of patients undergoing Hematopoietic Stem Cell Transplantation (HSCT) in recent years, chronic GVHD (cGVHD) and its associated complications have a significant impact on non-relapse mortality and quality of life. Loss of immune tolerance in the post-transplant period plays an important role in the emergence of cGVHD, and while imbalances in CD4+ T cell subsets, such as regulatory T cells, are known to contribute to its pathobiology, the role of different subsets of CD8+ T cells is poorly characterized.PATIENTS AND METHODSWe prospectively monitored a group of 56 patients who underwent unrelated donor HSCT after reduced intensity conditioning containing ATG. GVHD prophylaxis consisted of cyclosporine plus mycophenolate mofetil for all patients. 15 patients were excluded due to early disease relapse and/or death secondary to infection or acute GVHD in the first 9 months post-HSCT. cGVHD diagnosis and staging was performed according to the 2014 NIH criteria. Patients were divided into 2 groups according to cGVHD status: 17 patients (41%) with cGVHD (cGVHD+) and 24 patients (59%) without cGVHD (cGVHD-).Peripheral blood was analyzed by flow cytometry to characterize T cell subsets at fixed time points over a 2 year period. CD8+ T subsets were identified as follows: True Naïve cells as CD45RA+CD62L+CD95- (TN), Stem Cell Memory cells as CD45RA+CD62L+CD95+ (SCM), Central Memory cells as CD45RA-CD62L+ (CM), Effector Memory cells as CD45RA-CD62L- (EM) and terminally differentiated effector cells as CD45RA+CD62L- (EMRA).Frozen PBMC from patients at months 9 and 12 post-HSCT were used to purify CD8+ T cells by cell sorting to quantify Signal-Joint T Cell Receptor Excision Circles (TREC) by Real-Time PCR and to evaluate TCR diversity by TCR Vbeta complementarity-determining region 3 spectratyping.RESULTSPatients with cGVHD had significantly increased percentages and absolute counts of TN CD8 in the first 6 months post-HSCT compared with cGVHD- patients (p≤0.04 at months 1 and 3 for percentages; p≤0.02 for months 1-6 in absolute counts).Furthermore, cGVHD+ patients had increased SCM CD8 cells throughout the post-HSCT period (significances of p<0.05 at months 6 and 12 for percentages and 18 and 24 for counts). The increased prevalence of these two subsets was not associated with increased proliferation as assessed by Ki67, which was reduced in cGVHD+ in the total CD45RA+CD62L+ gate (TN plus SCM cells) (p≤0.04 at months 6, 12 and 18). No significant differences were observed in susceptibility to apoptosis as measured by BCL2 or CD95 expression levels.CM and EM subset counts were reduced in cGVHD+ as compared to cGVHD- (p=0.04 and 0.02 at month 12). EM CD8 were the dominant population in cGVHD- patients throughout the follow up. Significant increases in the percentage of EM CD8 cells in cGVHD- were observed at months 2, 9, 12 and 18 (p≤0.04).cGVHD+ patients had increased percentages of EMRA (p<0.04 at months 9, 12 and 18) and absolute counts (p<0.04 in months 2, 9 and 24). This was not correlated to altered Ki67, Bcl-2 or CD95 levels.The increase in TN in cGVHD+ patients was not associated with increased TREC numbers within CD8+ T cells. TREC content was significantly reduced in both patient groups at month 9 as compared healthy controls (HC) (p<0.05). This could be due to the low prevalence of these cells and/or to rapid differentiation into other memory subsets such as the SCM and EMRA. Despite the differences in subset prevalence in the two patient groups, we did not observe any significant differences in TCR Vbeta diversity in CD8+T cells. Reduced TCR diversity was only observed when patients were compared to HC (p=0.0001).DISCUSSIONOur observations of increased TN, SCM and EMRA CD8 subsets very early after HSCT in patients who subsequently develop cGVHD suggest the possibility that these may be relevant biomarkers for cGVHD development. SCM T cells have previously been shown to cause lethal GVHD in mouse models and to represent a reservoir of antigen-specific T cells capable of differentiating in vivo into other memory subsets with effector function. We speculate that thymic tissue damage resulting from conditioning regimens and/or acute GVHD, together with other yet unidentified factors, may lead to the emergence of self-reactive naïve CD8+ T cells that differentiate to SCM and later to EMRA cells potentially mediating cGVHD. DisclosuresNo relevant conflicts of interest to declare.

Stat3-/- Alloreactive Donor T Cells Reduce FAO but Increase Leakage of ROS in Response to PD-1 Signaling Triggered By Host-Tissue PD-L1, Leading to Prevention of Gvhd While Preserving GVL Effect

The GVHD pathogenicity of alloreactive T cells is associated with enhanced glycolysis or enhanced fatty acid oxidation (FAO). PD-1 signaling in T cells inhibits glycolysis but augments FAO. Host tissue PD-L1 interaction with PD-1 on activated alloreactive T cells is not able to effectively prevent acute GVHD mediated by donor CD4+ and CD8+ T cells together, although it can reduce the severity of the disease. Using a murine HCT model of C57BL/6 donor to BALB/c recipient, we show that although WT T cells (CD4+ and CD8+ T cells together) induced lethal GVHD at 1-5 x106/mouse, the same dose of Stat3-/- donor T cells induced little signs of acute or chronic GVHD because of induction of T cell anergy, exhaustion and apoptosis, but with retention of a strong GVL effect. This induction of tolerance depends on donor T cell PD-1 interaction with host-tissue PD-L1, because Stat3-/- T cells induce severe lethal GVHD in the PD-L1-/- recipients and in WT recipients treated with blocking anti-PD-1 mAb. The tolerance of Stat3-/- donor T cells induced by PD-1 signaling is associated with lower FAO and mitochondria membrane potential (MMP) but higher ROS leakage, with little difference in glycolysis, as compared to non-tolerant WT T cells. In the absence of PD-1 signaling, Stat3-/- T cells manifest markedly enhanced glycolysis and loss of tolerance. These results indicate that activated donor T cell PD-1 interaction with host-tissue PD-L1 reduces glycolysis and reduces their GVHD capacity. In addition, absence of mitochondrial pStat3 in Stat3-/- donor T cells leads to reduced FAO and MMP but increased ROS leakage, resulting in effective anergy, exhaustion, and apoptosis of tissue infiltrating donor T cells and prevention of GVHD. At the same time, the reduced expression of PD-L1 in recipient lymphoid tissues allows donor T cell expansion and strong GVL activity. Our studies have revealed novel roles of mitochondria pStat3 in regulating PD-1 signaling, T cell metabolism and tolerance. (Grant support: R01AI066008 to Zeng) . Disclosures No relevant conflicts of interest to declare.

Activation of Regulatory T Cells with IL-2 and TNF-α to Promote Homing to the Gut and to Improve Acute GvHD Prevention

INTRODUCTION Adoptive immunotherapy with donor conventional T cells (Tcons) and CD4+CD25+FOXP3+regulatory T cells (Tregs) is used to enhance immune reconstitution in clinical trials of haploidentical transplantation. Donor Tregs are infused to prevent adverse alloreactions mediated by Tcons against normal tissues, which cause GvHD, in the absence of any post transplant immune suppression. This strategy proved to be effective in preventing leukemia relapse and chronic GvHD, allowing for ~70% event-free survival in patients with acute leukemia (Pierini et al. submitted). However, acute GvHD occurs in ~30% of the patients. We are investigating if ex-vivo stimulation of Tregs with a combination of low-dose IL-2 and TNF-α before adoptive transfer could improve acute GvHD prevention. Tregs express higher levels of the TNF receptor 2 (TNFR2) compared with Tcons and TNFR2 expression defines a Treg subpopulation with maximal suppressive capacity. Recent studies showed that activation of TNFR2 signaling in mouse Tregs reduced GvHD lethality in mouse models. METHODS Human CD4+or CD4+CD25+T cells were incubated for 60 hours with IL-2 (10 UI/ml) with or without TNF-α (20 ng/ml). Treg proliferation in the presence of allogeneic irradiated peripheral blood mononuclear cells (PBMC), and Treg-mediated suppression of Tcon proliferation were evaluated by CFSE assay. Colon biopsies from patients with suspected GvHD were stained with anti-CD3 and anti-FOXP3 antibodies. RESULTS We developed a translatable in vitro system for improving human Treg function by adding a short term stimulation with low-dose IL-2 and TNF-α (IL-2/TNF) in the selection process. CD4+T cells were primed with IL-2/TNF before CD25 positive selection. When total CD4+T cells were stimulated with IL-2/TNF, CD25 expression was selectively upregulated on CD4+FOXP3+Tregs, but not on CD4+FOXP3- T cells. IL-2/TNF-primed Tregs maintained TNFR2 expression and expressed higher levels of FOXP3 and CD25 compared with Tregs stimulated with IL-2 alone (p&lt;.05). They also had enhanced proliferation in response to allogeneic PBMC (p&lt;.05) and suppressed Tcon proliferation more efficiently (p&lt;.01). Whole transcript expression analyses showed IL-2/TNF priming increased the expression of genes associated with Treg activation, proliferation and function compared to IL-2 alone, such us OX-40, 4-1BB, CD69, CCR8, CD39 genes and components of the NF-κB signaling pathway. We analyzed GvHD clinical signs of the last 20 patients who were diagnosed with grade II-IV acute GvHD after Treg/Tcon-based haploidentical transplantation at our institution. Nineteen presented with early-onset gut GvHD and 40% of those patients did not have any skin involvement. We hypothesized that preferential targeting to the gut was the consequence of defective homing of Tregs to the gut. Compared with Tcons, Tregs express higher levels of skin homing receptors (such as CCR4 and CLA-4), but lower levels of the gut homing receptor α4β7-integrin. We analyzed the infiltration of Tregs and Tcons in 31 colon biopsies from patients with suspected GvHD. The number of Tregs was higher in patients who were not diagnosed with acute GvHD compared with those who were (158.3/mm2 vs 86.3/mm2, p&lt;.05). A similar trend was observed for the Treg:Tcon ratio (0.27 vs 0.15, p&lt;.05). These results suggest that homing of Tregs to the gut plays an important role in the effectiveness of gut GvHD prevention. We also found that IL-2/TNF priming selectively upregulated α4β7-integrin expression on Tregs compared with freshly isolated cells (p&lt;.01). Experiments in humanized mouse models are underway to ascertain if IL-2/TNF priming improves Treg homing to the gut and site-specific GvHD prevention. CONCLUSION The present study shows that activation of TNFR2 signaling in human Tregs enhances their proliferation and suppressive function. It also suggests that Treg suppressive function exerted in periphery could be critical for tissue-specific GvHD control. Moreover, IL-2/TNF-priming could promote homing of Tregs to the gut and their capability to inhibit gut GvHD. Thus, IL-2/TNF-priming may be critical to optimize the ongoing clinical trial of adoptive immunotherapy with Tregs for GvHD prevention. Future studies will ascertain if IL-2/TNF-priming of Tregs can also allow for the infusion of higher doses of Tcons to further enhance immune reconstitution and leukemia killing without increasing the risk of GvHD. Disclosures No relevant conflicts of interest to declare.

IL-33 signaling in the barrier tissues sustains alloimmune responses mediating graft vs. host disease

Abstract Objective Graft vs. host disease (GVHD), where donor T cells recognize and destroy alloantigen-expressing host tissues, is a major complication after allogeneic bone marrow transplantation (alloBMT). To create space for donor stem cells and remove recipient immune cells, alloBMT protocols rely on conditioning regimens involving irradiation and/or chemotherapy. Interleukin (IL)-33 is augmented in recipient barrier tissues by conditioning before alloBMT and is a required signal to donor T cells for GVHD. In the current studies, we tested the hypothesis that the dominant role for IL-33 in GVHD lethality is to act as a sustaining signal to allogeneic T helper Type 1 (Th1) effectors in the barrier tissues. Methods To establish the role of IL-33 in CD4+ T cell effector function in the target tissue during GVHD, we used two CD4+-driven GVHD models. In the first, irradiated Il33−/− and Il33+/+ B6 recipients were given BALB/c alloBMT and T cells. In the second, irradiated Il33−/− and Il33+/+ bm12 recipients were given BMT and CD90.1+ B6 T cells. Donor T cell responses were characterized by flow cytometry at day 7, 14, and 28 post alloBMT. Results B6 Il33−/− recipients had significantly reduced frequency of IL-33 receptor-expressing CD4+Tbet+ cells in the small intestine lamina propria (SI LP), but not in the spleen (SPN) at day 7. In the MHCII-disparate B6 to bm12 GVHD model, IL-33 deficient recipients had profoundly reduced overall CD4+ donor T cells in the SI LP, but not in the SPN or lymph nodes (LN) at both day 14 and 28. Conclusions Our data reveal that IL-33 is not critical for donor Th1 responses in the SPN and LN, but necessary for Th1 cells to persist in barrier tissues such as the SI LP after alloBMT.

Vasoactive intestinal polypeptide synthesis by somatic tissues in transplant recipients limits GVHD in allo-BMT

Abstract Both donor and recipient hematopoietic cells regulate GvHD in allo-BMT, but the role of somatic host tissues in the induction and maintenance of GvHD is less clear. Vasoactive Intestinal peptide (VIP) is a neuropeptide with potent immune-regulatory activity. Our goal was to examine the role of VIP synthesis by recipient tissues on donor T cell activation and GvHD in allo-BMT. We used MHC mis-matched allo-BMT models, B10.BR--&amp;gt;B6 and Balb/c--&amp;gt;B6, irradiating WT and VIP-KO B6 mice with 11Gy on day −1 and transplanting with 5 × 10E6 T cell depleted (TCD) bone marrow (BM) plus 0, 1 × 10E6, or 3 × 10E6 T cells from MHC mis-matched donors. B6--&amp;gt;B6 radiation chimeras lacking VIP in either hematopoietic or non-hematopoietic cells were created by performing B6 WT--&amp;gt;VIP-KO and VIP-KO--&amp;gt;WT syngeneic transplants. Production of VIP in transplant recipients was visualized with immune-fluorescent staining of tissues using an anti-VIP antibody. Transplanting TCD BM plus 3 × 10E6 donor T cells from B10.BR or Balb/c donors in VIP KIO mice led significantly increased GVHD-mortality compared with WT recipients. Donor-derived T cells in VIP-KO recipients had significantly higher Th1 and Th17 polarization. B10.BR--&amp;gt;B6 second allogeneic transplantation of radiation chimeras caused lethal GVHD mortality in VIP-KO-- &amp;gt;VIP-KO and WT--&amp;gt;VIP-KO mice, but not in WT--&amp;gt;WT or VIP-KO--&amp;gt;WT B6 mice, demonstrating the protective effect of VIP was due to synthesis by somatic recipient cells. Immunofluorescent imaging of allo-BMT recipients showed up-regulation of VIP in lungs post-transplant and high VIP production within neurons innervating the lungs. Short-term administration of VIP from D0 to D10 prevented GvHD-mortality in VIP-KO recipients transplanted BM &amp; T cells.

Comparison of Gvhd Biomarker Algorithms for Predicting Lethal Gvhd and Non-Relapse Mortality

Acute GVHD biomarkers predict long-term outcomes after GVHD diagnosis. Our group has validated the MAGIC algorithm which uses the concentrations of 2 GVHD biomarkers, ST2+REG3a, to predict lethal GVHD (defined as death from GVHD without relapse) [Hartwell 2017]. Other published biomarker combinations that predict GVHD outcomes include ST2+ REG3a+TNFR1 [Levine 2015], ST2+TIM3 [Abu Zaid 2017], ST2+TNFR1 [McDonald 2015], TIM3+TNFR1+IL6 [McDonald 2015], as well as AREG alone [Holtan 2018]. It is not clear which biomarker combination best predicts lethal GVHD because these algorithms were developed using different patient cohorts with different endpoints. To answer this question, we compared the predictive accuracy of different biomarker combinations as well as these six specific combinations in the same patient cohort. We studied 522 patients with serum samples at GVHD diagnosis who were transplanted at 19 Mount Sinai Acute GVHD International Consortium (MAGIC) centers between January 1, 2004 and April 30, 2017. Patients were divided into training (n = 253) and validation (n = 269) sets; validation patients were transplanted after November 1, 2015 and had not previously been used to generate an algorithm. Biomarkers were measured by ELISA and log-transformed values were used to predict 1-year lethal GVHD by competing risk regression. Four of the 6 biomarkers (ST2, REG3a, TNFR1, and TIM3) independently predicted lethal GVHD in the training set. We then developed algorithms that predicted lethal GVHD using all possible combinations of these 4 biomarkers as well as the published combination of TIM3+TNFR1+IL6 and AREG alone. The best algorithms of 1-4 biomarkers for predicting lethal GVHD were REG3a alone, ST2+REG3a, ST2+REG3a+TNFR1, and ST2+REG3a+TNFR1+TIM3. While ST2+REG3a was the most accurate combination based on the lowest Akaike Information Criterion (AIC), the other published biomarker combinations produced similar AICs. Therefore, we used the independent validation set to compare all six published algorithms. We generated area under the receiver operating characteristic curves (AUC) for each algorithm (FIG 1). We next determined the threshold that maximized sensitivity and specificity for each algorithm and calculated the cumulative incidence of lethal GVHD in the resulting high and low risk strata (FIG 2). Highly similar results were obtained when the cumulative incidence of non-relapse mortality was used as the endpoint. In a validation set of previously unanalyzed patients, several biomarker combinations reproducibly stratified patients with GVHD for risk of death. The best 4 algorithms, all of which included ST2, produced comparable outcomes. Adding TNFR1 to ST2+ REG3a did not improve accuracy. The ST2+REG3a algorithm best identifies patients at onset of GVHD for high risk of lethal GVHD and NRM, and thus remains a standard for biomarker-based risk prediction.

Network Pharmacology-Guided Development of a Novel Integrative Regimen to Prevent Acute Graft-vs.-Host Disease.

Lapses in the graft-vs.-host disease (GVHD) prophylaxis and side effects of current standard care following allogeneic hematopoietic stem cell transplantation (allo-HSCT) call for novel regimens. Traditional approaches targeting T cells showed limited success in preventing acute GVHD (aGVHD). System medicine showed promising results treating complex diseases such as sepsis and multi-organ dysfunction syndrome (MODS). Adapting established network pharmacology analysis methods, we aimed to develop novel integrative regimens to prevent aGVHD. Our network pharmacology analysis predicted that Xuebijing injection (XBJ) targets a series of key node proteins in aGVHD network. It also unveiled that Salviae miltiorrhizae (Danshen), an herb in Xuebijing formula, which prevented aGVHD in rats, shares five out of six key GVHD node proteins targeted by XBJ. Interestingly, network pharmacology analysis indicated Xuebijing may share multiple aGVHD targets with Cyclosporin A (CsA), a first-line drug for preventing aGVHD in the clinic. Based on current information, we hypothesized that combination of XBJ and CsA may yield superior results in aGVHD prevention than either drug alone. We performed in vitro and in vivo assays to validate the predictions by the network pharmacology analysis. In vitro assays revealed XBJ prevented platelet aggregation and NF-κB nuclear translocation in macrophages. XBJ also promoted angiogenesis in tube-formation assay. Importantly, the combination of CsA and XBJ was effective in rescuing mice subjected to lethal GVHD. XBJ contributed to the rescue through preventing NF-κB nuclear translocation, attenuating inflammation and maintaining viability of macrophages. Overall, network pharmacology is a powerful tool to develop novel integrative regimens. Combination of XBJ and CsA may shed light on preventing aGVHD.

Intestinal Enterococcus Is a Major Risk Factor for the Development of Acute Gvhd

Introduction:Increasing evidence suggests that the intestinal microbiota is involved in the development of acute graft-vs.-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT). We previously reported in single center studies that Enterococcus a) is associated with GVHD (Holler et al., BBMT 2014) and b) can dominate the post-transplant gut microbiota in up to 50% of allo-HCT patients resulting in a 9-fold increased risk of bacteremia (Taur et al, CID 2012). To further investigate the hypothesis that Enterococcus can trigger the development of GVHD, we studied both allo-HCT patients and pre-clinical mouse transplant models.Methods and Results:Stool samples from 1240 allo-HCT patients at 4 different transplant centers in the U.S., Germany and Japan were collected approximately weekly during inpatient hospitalization. The V4-V5 region of the bacterial 16S rRNA genes from 6718 samples was sequenced at one central site on the Illumina platform. We observed Enterococcus mono-domination (relative abundance > 30%) in post-transplant samples ranging from 20 to 60% of patients at different centers (Fig. A, left). This mono-domination was primarily attributable to E. faecium, and was associated with a significantly increased risk for grade 2-4 acute GVHD (Fig. A, right). In three different mouse models we found a transient bloom of E. faecalis around 7 days after transplant in allo-HCT recipients with GVHD (Fig. B; C57BL/6 -> 129SV model). This bloom did not occur in allo-HCT recipients of a T cell depleted allograft without GVHD. To further investigate this Enterococcus bloom, we treated mice with an experimental E. faecalis-strain on days 4 to 6 after transplant and found significantly increased lethal GVHD. Colonizing germ-free mice with a minimal gut flora also lead to increased lethal GVHD when enterococci were added to the gnotobiotic flora (Fig. C). The allo-HCT recipients with Enterococcus-containing flora had also increased serum IFNg levels. Short chain fatty acids (SCFA) can be protective against GVHD and gut inflammation through maintenance of epithelial homeostasis and increases in anti-inflammatory regulatory T cells in the gut. In BMT mouse models, we found that Enterococcus-dominated allo-HCT recipients with GVHD have significantly less cecal butyrate, a major SCFA. Similarly, Enterococcus domination after allo-HCT also leads to a decrease in fecal SCFAs in patients (Fig. D). Next, we hypothesized that intestinal IgA might have a protective role against this pathogen in mice. 16S sequencing of flow sorted IgA-coated vs. non-coated bacteria from fecal samples of allo-HCT patients and transplanted mice revealed no specific IgA-coating pattern of enterococci both before or after transplant rather excluding the hypothesis that IgA might have a protective role against Enterococci. E. faecalis and E. faecium use the disaccharide lactose as a major carbohydrate source for growth and expansion as observed by analyses of the Enterococcus genome and in vitro growth experiments. In mice, we observed that a lactose-free diet significantly decreases the Enterococcus bloom after transplant in allo-T cell recipients and in first survival experiments attenuates lethal GVHD (Fig. E).Conclusion:Our studies in mouse and man demonstrate that the abundance of Enterococcus in the intestinal flora plays a role in the development of GVHD and the prevention of Enterococcus growth with a lactose-free diet can ameliorate GVHD. [Display omitted] DisclosuresPeled:Seres Therapeutics: Research Funding.

How Adoptive Immunotherapy with Conventional T and Regulatory T Cells Exerts a Gvl Effect without GvHD, after Haploidentical Hematopoietic Transplantation

▪Post-transplant relapse is still a major cause of treatment failure in high-risk acute leukemia (AL) patients. Attempts to manipulate donor T cell alloreactivity to spare normal tissues while killing leukemic cells have been largely unsuccessful. In the search for strategies to separate the GvL effect from GVHD, we investigated the role of a thymic-derived CD4+CD25+ FoxP3+ regulatory T-cell subpopulation (Tregs) that physiologically helps maintain immunological self-tolerance and immune homeostasis. Evidence from murine bone marrow (BM) transplantation across major histocompatibility barriers showed co-infusion of conventional T lymphocytes (Tcons) with Tregs suppressed lethal GVHD without impairing Tcon activity against malignant diseases. In 69 high-risk AL patients who had received an HLA haploidentical T cell-depleted hematopoietic transplant and no post-transplant immunosuppressive GvHD prophylaxis, adoptive immunotherapy with donor Tregs (2 × 106/kg) and Tcons (1 × 106/kg) protected patients from GvHD (Di Ianni et al., Blood 2011) and largely prevented post-transplant leukemia relapse. In fact, only 5% of patients relapsed (Martelli et al., Blood 2014). However, also because such patients had advanced stage disease, TRM was still in the range of that of T cell-depleted haplo transplants, (i.e. 40%, Aversa et al., JCO 2005). A current cohort of AL patients was conditioned with total body irradiation (TBI) (8Gy as single dose TBI or 13.5 Gy as fractionated TBI), cyclophosphamide (30mg/kg), thiotepa (8mg/kg) and fludarabine (200mg/m2). To date, 24 high-risk AL patients (7 ALL, 17 AML) have been enrolled. Twenty-two of the 24 patients engrafted, one AML patient relapsed, 4/22 developed aGvHD (4/4 are alive) and one developed cGvHD, TRM was exceptionally low (Fig. 1). At a median follow up of 2 years, 19 patients are alive. Consequently, probabilities of leukemia-free and leukemia/cGVHD-free survivals are good (Fig. 1). Hypotheses have been put forward to explain the ability of Tregs to prevent of GvHD while preserving the GvL effect mediated by Tcons. However, the mechanism is still unknown. In humans, naïve CD45RA+ Tregs express CXCR4 BM homing receptor and preferentially localize to the BM while memory CD45RO+ Tregs display lower CXCR4 expression and home to the periphery (Booth et al., J Immunol. 2010). Since Tregs that are recovered from peripheral blood and are used for adoptive immunotherapy are CD45RO+ and largely CxCR4 negative, we hypothesized that GvL without GvHD might be due to unopposed Tcon alloreactivity in the BM combined with regulated T cell alloreactivity at the periphery. In order to verify such hypothesis, we infused NSG mice with human leukemia and human Tregs and Tcons. Mice that received leukemia and haploidentical Tcons (without Tregs) cleared leukemia but died of GvHD. T cells harvested from their BM, spleen and liver were predominantly CD8+ and displayed alloreactivity against leukemia. Mice that received leukemia and Tcons plus Tregs were rescued from leukemia and survived without GvHD. T cells harvested from spleen and liver were composed of CD8+ T cells (40%) and CD4+T cells (60%). Purified CD4+ T cells had retained their regulatory function as they inhibited mixed lymphocyte reaction. Purified CD8+ T cells displayed no alloreactivity against leukemia. In contrast, T cells harvested from BM were still predominantly CD8+ and still displayed alloreactivity against leukemia suggesting Tcons had retained their alloantigen recognition. Finally, in mice treated with Tregs alone, T cells were recovered only in the spleen and liver, they displayed a CD4+ phenotype and inhibited mixed lymphocyte reaction. No T cells were found in the BM. In contrast, when we infused human purified CD45RA+ naïve Tregs (that display the CXCR4 BM homing receptor), mice died of leukemia progression despite the co-infusion of large doses of Tcons. In the BM we isolated CD8+ T cells that displayed no ability to kill leukemia and CD4+ T cells that displayed regulatory function. Thus, naïve Tregs homed to the BM and blocked the GvL effect of T cons. When CXCR4 was blocked with an anti-CXCR4 antibody, naïve Tregs could not home to the BM and T cons killed leukemia. In conclusion, Treg-Tcon adoptive immunotherapy confines the graft-versus-host alloreaction to the hematopoietic system and, consequently, allows a GvL effect without GvHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Donor Conditioning with Recipient-Derived PD-L1/PD-L2-Expressing B Cells Prevents Lethal Acute GVHD in a Fully Allogeneic Mouse Model

Aim We recently found that the inoculation with Balb/c (H-2d) PD-L1/PD-L2-expressing B cells lead to persistent suppression of anti-Balb/c T cell-responses, and significantly prolonged the survival of subsequently grafted Balb/c hearts in C57BL/6 (B6) (H-2b) mouse recipients. Given the effectiveness of those B cells as regulatory antigen-presenting cells targeting alloreactive T cells, we investigated if donor pre-conditioning with recipient-derived PD-L1/PD-L2 B cells could prevent acute GVHD after fully allogeneic bone marrow transplantation (BMT)/donor lymphocyte infusion (DLI) in mice. Methods To induce acute GVHD, lethally irradiated BALB/c mice were transplanted with 5 × 106 bone marrow cells and 1 × 106 splenic T cells from B6 mice. Twelve days prior to the BMT/DLI, B6 donors were intravenously injected with 10 × 106 BALB/c peritoneal cavity (PerC) B cells. Results Mixed-lymphocyte reaction (MLR) assays using splenocytes from the B6 donor mice at 12 days after the B cell injection revealed the significantly reduced anti-Balb/c T cell-responses in the recipients of PerC B cells, as compared to those in untreated control mice. Since PerC B cells contained MHC class II+ CD80+ CD86+ PD-L1+ PD-L2+ cells among the CD5+ B-1a cell subset, PerC B cells from Balb/c mice were pre-incubated with anti-PD-L1/PD-L2 mAbs prior to injection. This treatment abrogated their immune-regulatory effects on anti-Balb/c T cells in the MLR assays, indicating that the expression of both PD-L1 and PD-L2 on allogeneic PerC B cells is required to inhibit T cell-responses to the cognate allostimulation through engaging PD-1. The difference of T cell repertoire between the conditioned and untreated B6 was investigated using mAbs directed against T cell receptor Vβ regions. The proportion of Vβ5.1/5.2+ and Vβ11+ CD8+ T cells in the spleen of the conditioned B6 mice was significantly diminished, while that of Vβ8.1/8.2+ CD8+ T cells was not, indicating the specificity of the deletion for super-antigens presented by the BALB/c B cells. The donor pre-conditioning with recipient-derived PerC B cells markedly improved clinicopathological manifestations and survival in the lethal acute GVHD model, i.e. the mean survival of recipients of BMT/DLI from the conditioned donor was 27.9 ± 16.0 days (n=8), while that of recipients of BMT/DLI from the untreated donor was 6.6 ± 0.9 days (n=5). Conclusions These findings demonstrate that the PerC B cells, including PD-L1/PD-L2 B-1a cells, suppress T cells responding to allostimulation by clonal deletion. Thus, our study presents what we believe to be the reversed paradigm for the prevention of lethal acute GVHD by donor pre-conditioning with those immune-regulatory B cells derived from recipients.

Effects of IFN-γ on Pulmonary GVHD after Allo-Hematopoietic Stem Cell Transplantation

To explore the effects of IFN-γ on pulmonary GVHD after hematopoietic stem cell transplantation (HSCT). The mouse GVHD models were established by using C57BL/6J, B6D2F1 mice and different HSCT. According to HSCT modes, the mice were divided into 3 groups: syngeneic HSCT group(C57BL/6J→C57BL/6J), allogeneic HSCT group (C57BL/6J→B6D2F1) and IFN-γ -/- allogeneic HSCT group (IFN-γ -/-→C57BL/6J→B6D2F1). The survival time, GVHD clinical seore, pulmonary pathologic changes in 3 groups were compared and analyzed, and the total cell count in BALF and IFN-γ level in serum were detected in 3 groups after transplantation. The mice in syngeneic HSCT group all survived at day 42 after transplantation without GVHD symptoms in lung, the GVHD clinical score was low. The mice in allogeneic HSCT group survived at day 24 after transplantation (survial rate >50%), the GVHD clinical score was higher than that in syngeneic HSCT group, the pathologic changes in lung did not serious, though the GVHD presentation was observed, but the olveola bleeding and lymphocyte infiltration in lung tissue were observed at day 28 after transplantation. The mice in IFN-γ -/- allogeneic HSCT group all died of lethal GVHD within 14 days after transplantation, the GVHD clinical score reached to 6.7± 0.83 at 1st week after tansplantation, which was significantly higher than that in syngeneic and allogeneic HSCT groups. At 1st week after transplantation, the serious GVHD pathological changes occured in lung tissue, total cell count in BALF significanty increased, compared with syngeneic and allogeneic HSCT groups, but IFN-γ level in serum was significantly lower than that in syngeneic and allogeneic HSCT groups (P<0.05). Donor-derived IFN-γ plays an important immunoprotective role for lung tissue after HSCT.

Allogeneic stem cell transplantation in fully MHC-matched Mauritian cynomolgus macaques recapitulates diverse human clinical outcomes

Allogeneic hematopoietic stem cell transplantation (HSCT) is a critically important therapy for hematological malignancies, inborn errors of metabolism, and immunodeficiency disorders, yet complications such as graft-vs.-host disease (GvHD) limit survival. Development of anti-GvHD therapies that do not adversely affect susceptibility to infection or graft-vs.-tumor immunity are hampered by the lack of a physiologically relevant, preclinical model of allogeneic HSCT. Here we show a spectrum of diverse clinical HSCT outcomes including primary and secondary graft failure, lethal GvHD, and stable, disease-free full donor engraftment using reduced intensity conditioning and mobilized peripheral blood HSCT in unrelated, fully MHC-matched Mauritian-origin cynomolgus macaques. Anti-GvHD prophylaxis of tacrolimus, post-transplant cyclophosphamide, and CD28 blockade induces multi-lineage, full donor chimerism and recipient-specific tolerance while maintaining pathogen-specific immunity. These results establish a new preclinical allogeneic HSCT model for evaluation of GvHD prophylaxis and next-generation HSCT-mediated therapies for solid organ tolerance, cure of non-malignant hematological disease, and HIV reservoir clearance.

PD-L1/CD80 interaction differentially regulates CD8+ T cell metabolism, survival and GVHD capacity in the presence or absence of PD-1

Abstract Purified alloreactive CD8+ T cells induce no acute GVHD, but the mechanisms remain unknown. PD-L1 interacts with PD-1 and CD80. Here we show that, sorted C57BL/6 donor CD8+ T cells (1.25 ×106) expanded in wild-type MHC-mismatched BALB/c recipients but induced no acute GVHD. In PD-L1−/− recipients, donor CD8+ T cells expanded and induced lethal acute GVHD. PD-1−/−CD8+ T cells induced lethal acute GVHD in wild-type recipients. Blockade of PD-L1/CD80 interactions on days 0 and 2 prevented expansion of wild-type and PD-1−/− CD8+ T cells in wild type recipients and prevented acute GVHD caused by PD-1−/− CD8+ T cells. On the other hand, blockade of PD-L1/CD80 interaction after CD8+ T activation on day 5 caused lethal acute GVHD in wild-type recipients that usually have no acute GVHD. Naïve CD8+ T cells express much higher levels of CD80 than PD-1, whereas activated CD8+ T cells express higher levels of PD-1 than CD80. PD-L1/CD80 interaction augmented glycolysis in naïve WT and PD-1−/− CD8+T cells. This interaction was associated with increased phosphorylation of PLC-γ and activation of the AKT/mTOR pathway in a CD28-dependent manner. This interaction increased mitochondrial numbers and volume and augmented ROS production and apoptosis at the peak of CD8+ T cell activation, such that CD8+T cells did not over-expand to cause GVHD. Conversely, blockade of PD-L1/CD80 after CD8+ T activation in the presence of high PD-1 expression led to increased mitophagy, reduced apoptosis and overexpansion of CD8+ T cells, resulting in lethal GVHD. These results indicate that PD-L1/ CD80 interaction is dependent on PD-1 expression to differentially regulate glycolysis and mitophagy as well as expansion, survival and acute GVHD capacity of alloreactive CD8+ T cells.

TIM-1 blockade of the donor graft provides protection against lethal GVHD

Abstract Graft-versus-host disease (GVHD) continues to be a common cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). The T cell immunoglobulin and mucin (TIM) proteins represent a family of molecules that act in concert with T–cell receptor and costimulatory signals to regulate expansion, differentiation and effector function of T and NKT cells. TIM-1 binds to phosphytidylserine, exposed on the surface of apoptotic cells. Given the immunoregulatory role of TIM-1, we explored how TIM-1 influences GVHD in a murine model of allogeneic HSCT which causes massive apoptosis. Here we show that mice treated with an antagonistic anti-TIM-1 mAb that blocks phosphatidylserine recognition are protected from lethal GVHD. Protection against GVHD appears to be mediated by TIM-1 expression on the donor graft as TIM-1 knockout mice showed minimal survival advantage after allogeneic HSCT. Mice treated with anti-TIM-1 mAb cleared the A20 tumor cells suggesting that GVL effects are preserved. By bioluminescent imaging, it was determined that TIM-1 blockade does not alter the homing and expansion of donor T cells in vivo, nor does it affect T or NKT cells proliferation in vitro. Moreover, using microarray analysis, we found that anti-TIM-1 treatment protects from lethal GVHD by promoting an anti-inflammatory environment in the gut tissue. Antagonistic anti-TIM-1 mAb also protects from lethal GVHD in a xenogeneic model of GVHD. Overall, these findings can form the basis for the development of novel therapeutic strategies to prevent GVHD and improve HSCT.

Human MEMs are a distinct high IL-6 producing macrophage subset with anti-inflammatory and radioprotective properties in vivo

Abstract Macrophages (MØs) can polarize into subsets of “pro-inflammatory” (M1) MØs or “anti-inflammatory” (M2) MØs. Previously we described a unique population of MØs, termed human mesenchymal stem cell (MSC)-educated MØs (MEMs). In this study, we hypothesize that MEMs have potential to attenuate inflammation and promote tissue repair in vivo based on their immunophenotype and secretome. MEMs were generated by isolating monocytes from human peripheral blood and culturing them into MØs, followed by a co-culture with bone marrow-derived MSCs. RNA-Seq analysis showed genes associated with epithelial-mesenchymal transition, collagen formation, tissue development, and cell-cell adhesion were positively correlated with MEMs, while genes associated with oxidative phosphorylation were negatively correlated as compared to M1 MØs. We found increased expression of 8 genes implicated in wound healing, as well as genes representing remodeling enzymes, growth factors and the cytoskeleton. RT-PCR verified MEMs have increased CD206 (p&amp;lt;0.0001), IL-1β (p&amp;lt;0.001), TGFβ (p&amp;lt;0.001) and arginase-1 (p&amp;lt; 0.05), as well as increased IL-6 (p&amp;lt;0.0001) and CD73 (p&amp;lt;0.001) expression. MEMs produced over 4x greater amounts of IL-6 as compared to M2 MØs by ELISA (p&amp;lt;0.001), which could be enhanced with LPS stimulation (p&amp;lt;0.0001) or attenuated by inhibition of COX-2 (p&amp;lt;0.05), arginase-1 (p&amp;lt;0.01) or JAK1/JAK2 (p&amp;lt;0.05). In vivo, MEMs improved survival against lethal GVHD (p&amp;lt;0.01), in part by reducing T cell proliferation (p&amp;lt;0.001). MEMs also enhanced survival of mice from lethal radiation injury (p&amp;lt;0.01), in part by inducing host fibroblast proliferation (p&amp;lt;0.01). MEMs are a unique MØ subset with therapeutic potential for inflammatory disorders.

IL-27 Enhances Inducible Foxp3+ Treg Function to Prevent Acute Graft-Versus-Host Disease Lethality

Foxp3+ regulatory T cells (Tregs) are important regulators of graft-versus-host disease (GvHD) pathogenesis and a potential cellular therapy to prevent and treat GvHD. Thymus-derived (t)Tregs, while less frequent, are effective in preventing GvHD by ex vivo expansion, however Ag-non-specificity, stability, and cost-effectiveness are still critical issues to overcome. Peripherally-induced (p)Tregs are readily generated in large numbers in vitro;however, they are not suitable for therapeutic usage due to their rapid loss of Foxp3 expression and suppressive function when infused in the context of GvHD. The development of new approaches to increase in vivo stability/suppressive function of pTregs for clinical use is thus warranted.We recently demonstrated that IL-27 stimulation enhances suppressive function of Tregs (both tTregs and pTregs) in vitro and in vivo (Do, et al. Mucosal Immunol 2016). We hypothesized that IL-27 pre-stimulation would enhance pTreg function to suppress acute GvHD. Naïve CD4 T cells (from Foxp3GFP mice) were stimulated with anti-CD3/CD28 mAbs in the presence of TGFβ1 and IL-2 for 3 days, and >90% of the cells expressed GFP. Cells were then re-stimulated with the Abs for 3 more days with or without IL-27. IL-27 pre-stimulation enhanced Treg suppressive function in vitro, and was superior to that of rapamycin (Figure 1A). Lethally irradiated BALB/c mice received B6 BM cells with or without purified B6 CD3+ T cells. IL-27 pre-stimulated and control GFP+ pTregs were FACS sorted and transferred at the time of reconstitution. GvHD was evaluated by clinical features and survival. BM recipients that received T cells alone succumbed to death within 2 weeks of reconstitution due to lethal acute GvHD. While only ~40% of the control pTreg recipients were protected from lethal GvHD, all IL-27 pre-stimulated pTreg recipients were protected and survived (Figure 1B). CD4 and CD8 T cell expansion and ex vivo IFNγ production was substantially diminished with IL-27 pre-stimulated pTregs (Figure 1C). Next, lymphoma A20 cells transduced with the luciferase were transferred together with BM cells. Tumor growth was monitored. BM alone recipients had progression of tumor growth by day 14. Recipients of T cells alone died of lethal acute GVHD prior to day 14. Recipients of control and IL-27 stimulated pTregs had complete elimination of tumor cells. Of note, only 3 out of 5 recipients of control pTregs survived, with 2 succumbing to lethal GVHD prior to day 14 (Figure 1D). Although the total numbers of IL-27 pre-stimulated pTregs were higher than control pTregs, this was not statistically significant. GFP (Foxp3) expression of transferred pTregs, however, was markedly increased in IL-27 stimulated cells.We previously reported that IL-27 signaling in Tregs upregulates IL-10 and Lag3 (Do, et al. Mucosal Immunol 2016). We thus generated Il10-/- and Lag3-/- pTregs. Foxp3 expression of these pTregs was comparable to that of wild type pTregs, and IL-27 stimulation did not affect Foxp3 expression. IL-27-mediated enhanced in vitro suppression was lost in Lag3-/- but not in Il10-/- Tregs. Following IL-27 pre-stimulation, Il10-/- and Lag3-/- pTregs were transferred into recipients induced for acute GvHD. Foxp3 expression was similar at the time of transfer. IL-27 pre-stimulated Il10-/- pTregs protected recipients from acute GvHD lethality, however, Lag3-/- pTregs failed to protect from GVHD, even after IL-27 pre-stimulation (Figure 2A). IFNγ production of donor T cells was significantly reduced by IL-27 pre-stimulated Il10-/- but not by Lag3-/- pTregs (Figure 2B).In conclusion, we report that IL-27 pre-stimulation greatly enhances pTreg function, preventing acute GvHD lethality, while preserving a graft-versus-leukemia (GvL) effect. Lag3 on pTregs appears to be a key molecule mediating pTreg suppressive function enhanced by IL-27. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.