Alloreactive T-cell Responses Research Articles

Human MAIT cells inhibit alloreactive T cell responses and protect against acute graft-versus-host disease.

Adoptive transfer of immunoregulatory cells can prevent or ameliorate graft-versus-host disease (GVHD), which remains the main cause of nonrelapse mortality after allogeneic hematopoietic stem cell transplantation. Mucosal-associated invariant T (MAIT) cells were recently associated with tissue repair capacities and with lower rates of GVHD in humans. Here, we analyzed the immunosuppressive effect of MAIT cells in an in vitro model of alloreactivity and explored their adoptive transfer in a preclinical xenogeneic GVHD model. We found that MAIT cells, whether freshly purified or short-term expanded, dose-dependently inhibited proliferation and activation of alloreactive T cells. In immunodeficient mice injected with human PBMCs, MAIT cells greatly delayed GVHD onset and decreased severity when transferred early after PBMC injection but could also control ongoing GVHD when transferred at delayed time points. This effect was associated with decreased proliferation and effector function of human T cells infiltrating tissues of diseased mice and was correlated with lower circulating IFN-γ and TNF-α levels and increased IL-10 levels. MAIT cells acted partly in a contact-dependent manner, which likely required direct interaction of their T cell receptor with MHC class I-related molecule (MR1) induced on host-reactive T cells. These results support the setup of clinical trials using MAIT cells as universal therapeutic tools to control severe GVHD or mucosal inflammatory disorders.

Interindividual HLA Evolutionary Divergence in Single HLA-Mismatched Unrelated Donor Hematopoietic Cell Transplantation for Malignant Hematological Disorders: A Report on Behalf of the Cellular Therapy and Immunobiology Working Party of the EBMT

In allogeneic hematopoietic cell transplantation (allo-HCT), alloreactive T-cell responses (graft versus leukemia - GvL and graft versus host disease - GvHD) are mediated by the interaction between recipient antigens and donor lymphocytes via human leukocyte antigen (HLA) molecules. HLA evolutionary divergence (HED) is a metric recently implemented as a functional proxy for the structural variability between two homologous HLA alleles at their peptide binding sites, mirroring quantitative differences in pathogen and cancer-related immunopeptidomes ( A). 1 Here, we hypothesize that differences in and between donor and recipient HEDs at given loci may modulate the direction and the intensity of alloreactivity, informing on post-transplant outcomes. We tested this hypothesis in onco-hematologic patients undergoing allo-HCT from a single HLA-A, -B, -C, -DRB1, or -DQB1 (9/10) mismatched unrelated donor (MMUD) at EBMT centers. We included patients with selected hematological malignancies, receiving a thymoglobulin-based T cell depletion, for whom complete clinical and HLA information was available in the EBMT database. We deployed individual locus-specific recipient/donor HED (HED-R, HED-D) metrics as previously described 1, and conceptualized two novel interindividual HED scores accounting for functional differences at the mismatched HLA loci ( A). The latter were considered as: 1) DeltaHED, quantifying the relative directional difference between HED-D and HED-R and informing on differences in immunopeptidome sizes, potentially capturing the direction and the intensity of the alloreactive response (GvH or HvG); 2) HEDmismatch, describing the HED of the mismatched locus as a proxy for the qualitative differences in immunopeptidomes between donor and recipient ( B). Using multivariable Cox regression (cause-specific) models adjusted for relevant clinical variables, we investigated the associations of HED-R, DeltaHED and HEDmismatch, analyzed as continuous variables, with relapse free survival (RFS), overall survival (OS), non-relapse mortality (NRM), relapse and grade II-IV acute and chronic GvHD. For each outcome and in each mismatched subgroup, we built separate models for these immunogenetic parameters and checked for proportional hazards assumption (PHA). To account for multiple comparisons, only p-values <0.01 were considered significant. Overall, 4639 adult patients, with available HLA data, received a 9/10 MMUD allo-HCT between 2010 and 2019 for acute leukemia (N=3853, 83%), myelodysplastic syndromes or myeloproliferative neoplasms (N=786, 17%). Median recipient age was 53.5 (IQR 40.2-61.9) with a male/female ratio of 1.28, whilst median donor age was 33.1 (IQR 24.5-40). Majority of patients (90%) received peripheral blood stem cells and were mismatched with their donors for locus A (33%), followed by C (21%), B (15%), DQB1 (14%) and DRB1 (6%). When analyzing the impact of HED-related parameters in each model, we observed that most recurrent associations with outcomes occurred with DRB1-related metrics. In the DRB1-mismatched subgroup (N=276), a time-varying association was found for HEDmismatch of DRB1 locus with higher HEDmismatch leading to worse RFS [HR:1.25 at t=0, (95% CI 1.1-1.42), p<0.001] and lower OS [HR:1.23 at t=0, (95%CI 1.08-1.41), p=0.002]. The strength of this associations declined over time for both endpoints (respectively RFS: HR:0.91, 95% CI 0.86-0.97, p=0.005 and OS: HR:0.93, 95% CI 0.88-0.98, p=0.013). The (directional) DeltaHED DRB1 measure instead did not display any impact on outcomes. In the same subgroup, higher HED-R in the locus C showed also a time-varying association with NRM at transplant [HR:1.89, (95% CI 1.29-2.85), p=0.002], progressively declining over time (HR:0.77, 95% CI 0.65-0.92, p=0.003). Furthermore, DRB1 HED-R modulated the probability of survival in B-mismatched subgroup, being higher scores associated with reduced OS [HR:1.03, (95%CI 1.01-1.06), p=0.009]. Here, we provide an immunogenetic benchmark to explore donor-recipient interactions in MMUD allo-HCT, demonstrating how different HLA configurations within the mismatched loci, in particular for DRB1 genes, can influence post-transplant outcomes. Given the peculiar associations observed, HED may have a role in driving early GvL effects, informing on future donor selection strategies in high-risk patients.

Effects of HLA Mismatches on Cytokine Release Syndrome and Associated Non-Relapse Mortality in Allogeneic Stem Cell Transplantation with Post-Transplant Cyclophosphamide

Background Post-transplant cyclophosphamide (PTCy) is an effective method for graft versus host disease prevention in allogeneic stem cell transplantation (alloSCT). The alloreactive T-cell response in the first days after PTCy-alloSCT can induce a systemic inflammatory response known as cytokine release syndrome (CRS). The incidence of CRS appears to increase with higher degrees of HLA mismatch between patient and donor. In haplo-identical PTCy-alloSCT, CRS occurs frequently (73-94%) and has a negative impact on non-relapse mortality (NRM) and survival. In 10/10 HLA matched related and unrelated allogeneic stem cell transplantation (MRD/MUD PTCy-alloSCT), CRS is less frequently observed (14-23%) without effects on NRM and survival. The specific contributions of different HLA mismatches in CRS development are not well known. Aim To study the effects of HLA mismatching in MRD/MUD PTCy-alloSCT transplantation on development of CRS and CRS-associated NRM and survival. Methods We retrospectively studied CRS, survival and NRM in a group of 97 MRD/MUD PTCy-alloSCT patients with different degrees of HLA matching (20% of patients were 12/12, 50% 10/10 and 30% 9/10 HLA-matched). Results CRS developed in 69% of patients, mostly grade 1 (85%), 12% grade 2 and 3% grade 3. CRS developed significantly more in patients transplanted with a 9/10 or 10/10 match compared to a 12/12 match (90% and 75% versus 26%). By focusing on the maximum grade of fever developing during CRS, CRS severity grading was refined, revealing effects of specific HLA mismatches on CRS. In 10/10 matched patients, HLA-DPB1 non-permissiveness significantly increased grade 2-3 fever CRS incidence from 10 to 62%. Although regarded to be unimportant in HLA matching, single HLA-DRB3/4/5 mismatches could induce grade 3 fever CRS. Strongest effects on fever developed in 9/10 matched patients (35% grade 3 fever CRS), showing differences between various HLA mismatch groups. HLA-DRB1 mismatches induced significantly higher fever than HLA-C mismatches. The total number of HLA mismatches (combined HLA-A, B, C, DRB1, DRB3/4/5, DQ and DP non-permissive mismatches) was found to correlate with the height of fever developing. Importantly, patients with grade 3 fever CRS showed significantly increased NRM and significantly inferior survival compared to patients with grade 0-2 fever CRS. Conclusions In contrast to earlier publications in which CRS was demonstrated mainly in haplo-identical PTCy-alloSCT, we show that also in HLA matched related and unrelated PTCy-alloSCT incidence of CRS can be high and influence transplantation results. Development of CRS is dependent on the degree of HLA matching (90% in 9/10, 75% in 10/10 and 26% in 12/12 matched patients). By focusing on the maximum grade of fever developing during CRS, CRS severity grading can be refined, revealing effects of specific HLA mismatches on CRS. The height of fever during CRS is dependent on the number and types of HLA mismatches. CRS with grade 3 fever increases NRM and decreases survival. HLA matching decisions may be used to prevent grade 3 fever CRS, and thereby possibly also NRM. Alternatively, anti-inflammatory interventions may be considered in patients developing grade 3 fever early after alloSCT.

A novel RIPK1 inhibitor reduces GVHD in mice via a nonimmunosuppressive mechanism that restores intestinal homeostasis

AbstractIntestinal epithelial cells (IECs) are implicated in the propagation of T-cell–mediated inflammatory diseases, including graft-versus-host disease (GVHD), but the underlying mechanism remains poorly defined. Here, we report that IECs require receptor-interacting protein kinase-3 (RIPK3) to drive both gastrointestinal (GI) tract and systemic GVHD after allogeneic hematopoietic stem cell transplantation. Selectively inhibiting RIPK3 in IECs markedly reduces GVHD in murine intestine and liver. IEC RIPK3 cooperates with RIPK1 to trigger mixed lineage kinase domain-like protein-independent production of T-cell–recruiting chemokines and major histocompatibility complex (MHC) class II molecules, which amplify and sustain alloreactive T-cell responses. Alloreactive T-cell–produced interferon gamma enhances this RIPK1/RIPK3 action in IECs through a JAK/STAT1-dependent mechanism, creating a feed-forward inflammatory cascade. RIPK1/RIPK3 forms a complex with JAK1 to promote STAT1 activation in IECs. The RIPK1/RIPK3-mediated inflammatory cascade of alloreactive T-cell responses results in intestinal tissue damage, converting the local inflammation into a systemic syndrome. Human patients with severe GVHD showed highly activated RIPK1 in the colon epithelium. Finally, we discover a selective and potent RIPK1 inhibitor (Zharp1-211) that significantly reduces JAK/STAT1-mediated expression of chemokines and MHC class II molecules in IECs, restores intestinal homeostasis, and arrests GVHD without compromising the graft-versus-leukemia (GVL) effect. Thus, targeting RIPK1/RIPK3 in IECs represents an effective nonimmunosuppressive strategy for GVHD treatment and potentially for other diseases involving GI tract inflammation.

Sphingolipid metabolism in T cell responses after allogeneic hematopoietic cell transplantation.

Allogeneic hematopoietic cell transplantation (allo-HCT) is an effective immunotherapy against hematopoietic malignancies. The infused donor lymphocytes attack malignant cells and normal tissues, termed a graft-verse-leukemia (GVL) effect and graft-verse-host (GVH) response or disease (GVHD), respectively. Although engineering techniques toward donor graft selection have made HCT more specific and effective, primary tumor relapse and GVHD are still major concerns post allo-HCT. High-dose systemic steroids remain to be the first line of GVHD treatment, which may lead to steroid-refractory GVHD with a dismal outcome. Therefore, identifying novel therapeutic strategies that prevent GVHD while preserving GVL activity is highly warranted. Sphingolipid metabolism and metabolites play pivotal roles in regulating T-cell homeostasis and biological functions. In this review, we summarized the recent research progress in this evolving field of sphingolipids with a focus on alloreactive T-cell responses in the context of allo-HCT. We discussed how sphingolipid metabolism regulates T-cell mediated GVH and GVL responses in allo-HCT and presented the rationale and means to target sphingolipid metabolism for the control of GVHD and leukemia relapse.

Permissive HLA-DPB1 mismatches in HCT depend on immunopeptidome divergence and editing by HLA-DM

AbstractIn hematopoietic cell transplantation (HCT), permissive HLA-DPB1 mismatches between patients and their unrelated donors are associated with improved outcomes compared with nonpermissive mismatches, but the underlying mechanism is incompletely understood. Here, we used mass spectrometry, T-cell receptor-β (TCRβ) deep sequencing, and cellular in vitro models of alloreactivity to interrogate the HLA-DP immunopeptidome and its role in alloreactive T-cell responses. We find that permissive HLA-DPB1 mismatches display significantly higher peptide repertoire overlaps compared with their nonpermissive counterparts, resulting in lower frequency and diversity of alloreactive TCRβ clonotypes in healthy individuals and transplanted patients. Permissiveness can be reversed by the absence of the peptide editor HLA-DM or the presence of its antagonist, HLA-DO, through significant broadening of the peptide repertoire. Our data establish the degree of immunopeptidome divergence between donor and recipient as the mechanistic basis for the clinically relevant permissive HLA-DPB1 mismatches in HCT and show that permissiveness is dependent on HLA-DM–mediated peptide editing. Its key role for harnessing T-cell alloreactivity to HLA-DP highlights HLA-DM as a potential novel target for cellular and immunotherapy of leukemia.

Vitamin D3 combined with antibody agents suppresses alloreactive memory T-cell responses to induce heart allograft long-term survival

BackgroundThe pre-stored memory T cells in organ transplant patient carry a high risk of allograft rejection. The current study aimed to determine whether the allogenic response of adoptively transferred memory T cells in mice was suppressed by vitamin D3 monotherapy alone or in combination with monoclonal antibody treatment. MethodsPrior to vascularized heterotopic heart transplantation, naïve C57BL/6 mice were primed with memory T cells. Recipient mice were administered vitamin D3 alone or in combination with monoclonal antibodies (anti-CD40L/ anti-LFA-1). Memory T cells and CD4+ forkhead box P3+ T cells in recipient spleens were measured using flow cytometry. Additionally, the expression of cytokines was measured by ELISA and quantitative PCR. Inflammatory factors in the grafts were identified by hematoxylin and eosin staining. ResultsVitamin D3 in conjunction with anti-CD40L/ anti-LFA-1 antibodies were administered according to the median survival time from 6.5 to 80 days. The results revealed that grafts were protected through the prevention of inflammatory cell infiltration. Combined treatment decreased the mRNA levels of IL-2, IFN-γ and IL-10 and increased the mRNA levels of IL-4, Foxp3 and TGF-β in the allograft. Rejection was suppressed by a reduction of CD4+CD44high CD62L+ and CD8+ CD44high CD62L+ memory T cells, the induction of regulatory T cells in the recipient spleen and a reduction of serum IL-2, IFN-γ and IL-10 levels. ConclusionVitamin D3 efficiently protected allografts from memory T-cell allo-responses when combined with anti-CD40L/anti-LFA-1 antibodies therapy.

Phenotypic Myeloid-Derived Suppressor Cells (MDSCs) are expanded after Post-Transplantation Cyclophosphamide (PTCy), but are not necessary for Graft-Versus-Host Disease Prevention by PTCy

The use of PTCy substantially lowers the rates of severe acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT), but the mechanisms of its action are incompletely understood. Recently, we showed in a T-cell–replete, MHC-haploidentical, murine HCT model (B6C3F1→B6D2F1) that PTCy prevents GVHD not by eliminating alloreactive T cells but instead by inducing alloreactive T-cell dysfunction and suppression. Depletion of CD4+regulatory T cells (Tregs) early post-transplant did not result in substantially worsening GVHD for ∼4 weeks. Surprisingly, we found that alloreactive T cells could be infused in extremely high numbers early after PTCy but not cause GVHD. Here, we used this same MHC-haploidentical murine HCT model to test the role of Tregs in preventing GVHD induced by infusion of new splenocytes after PTCy; depleting Tregs in the initial graft but not the reinfused splenocytes did not result in worsening GVHD in these mice. However, when Tregs were depleted both in the initial graft and in the reinfused splenocytes, universally fatal GVHD ensued. These results suggested that Tregs were necessary to prevent GVHD but a direct effect of PTCy on Tregs was not required. Thus, we hypothesized that other suppressive cell populations may be playing a critical role early after PTCy. We examined the frequency of MDSCs after PTCy in our model and found that both granulocytic and monocytic MDSCs were increased at post-transplant days +7 and +21 compared with vehicle-treated mice. Next we examined the role of MDSCs in GVHD prevention after PTCy by using a Gr-1-depleting antibody. Gr-1 depletion immediately post-transplant led to decreased percentages of Tregs and inferior survival, clinical scores, and weights in PTCy-treated mice. Conversely, Gr-1 depletion at days +28 or +150 had a transient effect only on weights but no impact on clinical scores or survival. Yet, histopathologic evaluation revealed that mice depleted of MDSCs early post-transplant did not have worse GVHD; instead the worse survival in those mice appeared related to infection presumably as a result of neutrophil depletion by the anti-Gr-1 antibody in mice with mucosal disruption by radiation and GVHD. We also tested the suppressive functionality of MDSCs on alloreactive T cells in in vitro mixed lymphocyte cultures. We found neither population of MDSCs from either vehicle-treated or PTCy-treated mice suppressed the proliferation of vehicle-treated T cells nor was inflammatory cytokine secretion reduced. Overall, these findings confirm that Tregs are required for GVHD prevention by PTCy but a direct effect of PTCy on Tregs is not required; although phenotypic MDSCs are expanded after PTCy and may contribute to Treg expansion, these MDSCs do not appear functionally capable of suppressing alloreactive T-cell response to alloantigen in vitro or decreasing GVHD in vivo.

263 - Protmune Reduces the Incidence and Severity of Acute Gvhd by Transiently Dampening T-Cell Signaling

Acute graft-versus-host disease (aGvHD) is a serious complication of allogeneic hematopoietic cell transplantation (allo-HCT), occurring in up to half of all patients and remains the leading cause of morbidity and non-relapse mortality. ProTmune was developed as a next-generation graft by modulating the properties of mobilized peripheral blood with a combination of two small molecules, designed to simultaneously reduce the incidence and severity of aGvHD, while maintaining immune protection against infections and relapse. Mechanistically, we have demonstrated that modulated T cells have: (i) dampened T cell receptor (TCR) and (ii) inflammatory cytokine receptor signaling that manifests in reduced overall activation and expansion. Alloreactive T-cell responses typically initiate during the first 48-96 hours after allo-HCT, driven by coordinated signals from the TCR, co-stimulation, and inflammatory cytokines that lead lead to unwanted expansion, proliferation and differentiation of T effector cells that migrate and attack aGvHD target organs. ProTmune modulated T cells, when immediately activated by allo-antigen, TCR crosslinking, or cognate antigen, expand poorly in vitro compared to unmodulated T cells. We next assessed the relative strength of TCR signaling within naïve T cells. Both ERK phosphorylation and CD25 expression, two immediate outcomes of TCR signaling, were dramatically abrogated within ProTmune modulated T cells. The duration of this effect was evaluated by transferring modulated and unmodulated naïve T cells into congenic mice. Relative TCR signal strength was reassessed 14 days later, and, unlike the input T cells, no deficits in pERK, CD25, and TNF expression were observed, demonstrating that ProTmune transiently restricts the relative strength of TCR signaling, thereby limiting their activation and eventual expansion. The JAK-STAT signaling pathway mediates inflammatory cytokine signaling and has been implicated in aGVHD pathogenesis; we therefore assessed whether ProTmune would impact these pathways in human donor T cells. Both Stat1 and Stat3 phosphorylation was significantly limited within ProTmune modulated T cells compared to unmodulated T cells, suggesting that ProTmune modulation also limits the sensitivity of T cells to activating inflammatory cytokines. In summary, these data suggest that T cells within ProTmune modulated grafts are transiently less responsive to TCR signals and inflammatory cytokines, resulting in reduced overall activation and expansion immediately following infusion. These observations provide a rationale for the simultaneously reduced incidence and severity of aGvHD and intact GvL responses previously observed in preclinical animal models. ProTmune is currently undergoing clinical testing in PROTECT, an ongoing Phase 1-2 clinical trial in adult patients with hematologic malignancies.

T-Cell Composition of the Lymph Node Is Associated with the Risk for Early Rejection after Renal Transplantation.

The T-cell composition within the lymph node (LN) of end-stage renal disease (ESRD) patients differs from the composition within the circulation. Activation of the alloreactive T-cell response within secondary lymphoid organs is important after organ transplantation. However, to date no data are present on LN T-cell subsets and the risk for acute rejection after kidney transplantation. T cells from LNs of ESRD patients were analyzed for frequency of recent thymic emigrants, relative telomere length, expression of differentiation markers, and were related to the development of early acute rejection (EAR), occurring within 3 months after renal transplantation (RT). Furthermore, the alloreactive potential of mononuclear cells isolated from the LN and peripheral blood of 10 patients was analyzed. Measures of alloreactive potential included proliferation, cytokine production, frequencies of interferon-gamma-producing cells, and the presence of cytotoxic molecules. Patients with EAR were younger (p = 0.019), cytomegalovirus-seropositive (p = 0.037) and usually received dialysis prior to RT (p = 0.030). Next to this, patients with EAR showed a lower CD4:CD8 ratio (p = 0.027) within the LN. T cells from the LN were similar with regard to alloreactive capacity compared with those within the circulation. Univariate regression analysis showed that the CD4:CD8 ratio (OR: 0.67, p = 0.039), patient age (OR: 0.93, p = 0.024), and preemptive RT (OR: 0.11, p = 0.046) were associated with EAR. After a multivariate analysis, only the CD4:CD8 ratio (OR: 0.58, p = 0.019) and preemptive RT (OR:0.05, p = 0.012) were associated with EAR. A lower CD4:CD8 ratio in the LN is associated with a higher risk for the development of rejection within 3 months after RT.

Clinical and immunologic impact of CCR5 blockade in graft-versus-host disease prophylaxis

AbstractGraft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Lymphocyte trafficking via chemokine receptors such as CCR5 plays a critical role in alloreactive responses, and previous data suggest that CCR5 blockade with maraviroc results in a low incidence of visceral GVHD. However, the full scope of clinical and immunologic effects of CCR5 blockade in HSCT has not been described. We compared a cohort of patients enrolled on a trial of reduced-intensity allo-HSCT with standard GVHD prophylaxis plus maraviroc to a contemporary control cohort receiving standard GVHD prophylaxis alone. Maraviroc treatment was associated with a lower incidence of acute GVHD without increased risk of disease relapse, as well as reduced levels of gut-specific markers. At day 30, maraviroc treatment increased CCR5 expression on T cells and dampened T-cell activation in peripheral blood without impairing early immune reconstitution or increasing risk for infections. Patients who developed acute GVHD despite maraviroc prophylaxis showed increased T-cell activation, naive T-cell skewing, and elevated serum CXCL9 and CXCL10 levels. Collectively, these data suggest that maraviroc effectively protects against GVHD by modulating alloreactive donor T-cell responses, and that CXCR3 signaling may be an important resistance mechanism to CCR5 blockade in GVHD.

Impact of hyperlipidemia on alloimmunity.

Hyperlipidemia is a comorbidity affecting a significant number of transplant patients despite treatment with cholesterol lowering drugs. Recently, it has been shown that hyperlipidemia can significantly alter T-cell responses to cardiac allografts in mice, and graft rejection is accelerated in dyslipidemic mice. Here, we review recent advances in our understanding of hyperlipidemia in graft rejection. Hyperlipidemic mice have significant increases in serum levels of proinflammatory cytokines, and neutralization of interleukin 17 (IL-17) slows graft rejection, suggesting that IL-17 production by Th17 cells was necessary but not sufficient for rejection. Hyperlipidemia also causes an increase in alloreactive T-cell responses prior to antigen exposure. Analysis of peripheral tolerance mechanisms indicated that this was at least in part due to alterations in FoxP3 T cells that led to reduced Treg function and the expansion of FoxP3 CD4 T cells expressing low levels of CD25. Functionally, alterations in Treg function prevented the ability to induce operational tolerance to fully allogeneic heart transplants through costimulatory-molecule blockade, a strategy that requires Tregs. These findings highlight the importance of considering the contribution of inflammatory comorbidities to cardiac allograft rejection, and point to the potential importance of managing hyperlipidemia in the transplant population.

Porcine antigen-specific IFN-γ ELISpot as a potentially valuable tool for monitoring cellular immune responses in pig-to-non-human primate islet xenotransplantation.

Recent progress in xenotransplantation of porcine islets to non-human primates (NHPs) gives hope for human clinical trials in the near future. Thus, implementation of an appropriate monitoring method to detect the development of detrimental porcine antigen-specific cellular immune responses is necessary. The enzyme-linked immunospot (ELISpot) assay has been widely used to monitor antigen-specific alloreactive T-cell responses in humans; however, the utility of porcine islet-specific ELISpot assay has not yet been thoroughly evaluated for pig-to-NHPs intraportal islet xenotransplantation. The optimal ELISpot assay conditions, including the number of responder and stimulator cells and the provision of costimulation, were determined. Then, ELISpot assays were conducted on serial stocks of peripheral blood mononuclear cell (PBMC) samples previously isolated from NHP recipients transplanted with porcine islets. Either splenocytes from donor pigs or pancreatic islets from third-party pigs were used for antigen stimulation. At the same time, the ratio of CD4(+) /CD8(+) T cells and the percentage of CD4(+) FoxP3(+) T cells in the peripheral blood were evaluated. Finally, liver biopsy samples were evaluated to assess the immunopathology of the grafts. The optimal conditions for the ELISpot assay were defined as 2.5 × 10(5) responder cells incubated with 5.0 × 10(5) stimulator cells in 96-well, flat-bottom plates without further costimulation. Using donor splenocytes as stimulators, a serial interferon-gamma (IFN-γ) ELISpot assay with PBMCs from the monkeys with prolonged porcine islet grafts (>180 days) demonstrated that the number of donor antigen-specific IFN-γ-producing cells significantly increased upon overt graft rejection. However, use of third-party porcine islets as stimulators did not reflect graft rejection, suggesting that the use of donor-specific PBMCs, and not tissue (porcine islet)-specific cells, as stimulators could better serve the purpose of this assay in adult porcine islet transplantation. IFN-γ spot number was neither influenced by the peripheral blood CD4(+) /CD8(+) T-cell ratio nor the percentage of CD4(+) FoxP3(+) T cells. Finally, in cases of overt graft rejection, the number of IFN-γ spots and the graft-infiltrating T cells in biopsied liver samples increased simultaneously. Use of PBMCs in a porcine antigen-specific IFN-γ ELISpot assay is a reliable method for monitoring T-cell-mediated rejection in pig-to-NHP islet xenotransplantation.

Different sensitivity of rituximab-treatment to B-cells between ABO-incompatible kidney and liver transplantation

A desensitization protocol with rituximab is currently widely used for kidney transplantation (KT) and liver transplantation (LT) across the ABO blood group-incompatible (ABO-I) barrier. However, it remains to be elucidated whether rituximab is equally effective for B-cell and T-cell immune responses in both KT and LT recipients. To clarify these effects of rituximab, we enrolled 46 KT and 77 LT recipients in this study. The proportion of peripheral blood B-cells was determined at the perioperative period. T-cell responses to allostimulation were evaluated by a mixed lymphocyte reaction (MLR) assay. One week after rituximab administration, peripheral B-cells became undetectable in ABO-I KT recipients but remained detectable in some of the ABO-I LT recipients; B-cells were undetectable in both groups by week 2. B-cells remained below the detection limit throughout the first year in the ABO-I KT recipients, whereas they reappeared in the periphery after 6months in the ABO-I LT recipients. There were no significant differences in alloreactive T-cell responses based on MLR analyses between ABO-I and ABO-compatible groups. This study indicates that rituximab has differing B-cell sensitivity between KT and LT recipients and a minimal effect on the alloreactive T-cell responses in KT and LT recipients.

PD-L1 Deficiency within Islets Reduces Allograft Survival in Mice.

BackgroundIslet transplantation may potentially cure type 1 diabetes mellitus (T1DM). However, immune rejection, especially that induced by the alloreactive T-cell response, remains a restraining factor for the long-term survival of grafted islets. Programmed death ligand-1 (PD-L1) is a negative costimulatory molecule. PD-L1 deficiency within the donor heart accelerates allograft rejection. Here, we investigate whether PD-L1 deficiency in donor islets reduces allograft survival time.MethodsGlucose Stimulation Assays were performed to evaluate whether PD-L1 deficiency has detrimental effects on islet function. Islets isolated from PDL1-deficient mice or wild- type (WT) mice (C57BL/6j) were implanted beneath the renal capsule of streptozotocin (STZ)-induced diabetic BALB/c mice. Blood glucose levels and graft survival time after transplantation were monitored. Moreover, we analyzed the residual islets, infiltrating immune cells and alloreactive cells from the recipients.ResultsPD-L1 deficiency within islets does not affect islet function. However, islet PD-L1 deficiency increased allograft rejection and was associated with enhanced inflammatory cell infiltration and recipient T-cell alloreactivity.ConclusionsThis is the first report to demonstrate that PD-L1 deficiency accelerated islet allograft rejection and regulated recipient alloimmune responses.

Ex Vivo Modulation of Donor Cells Results in Enhanced Survival and Reduced Gvhd Mortality

Allogeneic hematopoietic stem cell transplant (HSCT) represents a potential curative treatment for a number of life-threatening blood malignancies. The utility of this treatment regimen, however, is limited by a number of serious complications including graft versus host disease, which occurs in approximately half of all transplant patients. Standard-of-care for treating acute GvHD has remained unchanged for several decades and consists of high doses of steroids, which are only effective in approximately 35 percent of the cases. Therefore, the reduction of GvHD represents a large unmet medical need, and new approaches are needed to effectively attenuate GvHD. Here we present a fundamentally novel strategy for potentially reducing GVHD - by modulating donor mobilized peripheral blood cells with small molecules prior to HSCT, a programmed mobilized peripheral blood (mPB) allogeneic graft, with reduced T-cell alloreactivity, can be administered as the hematopoietic cell source for HSCT.To this end, we applied our screening platform to identify a combination of small molecule modulators (FT1050, FT4145) that promote the activation of genes implicated in cell cycle, immune tolerance and anti-viral properties of T cells, as well as in the survival, proliferation and engraftment potential of CD34+ cells. Genome-wide expression analysis of the T-cell compartment of mobilized peripheral blood following treatment with FT1050+FT4145 revealed the induction of genes involved in cell cycle (e.g., CCND1, CCNE1), immune tolerance (e.g., ALDH, AREG) and anti-viral properties (e.g., EFNB2). To further assess the therapeutic impact of ex vivo programming with FT1050 and FT4145, a number of T cell assays to assess T cell phenotype and function were conducted on mPB. Overall, ex vivo programming of mPB resulted in reduced allogeneic T cell responses and was accompanied by reduced capacity of modulated T cells to produce Interferon Gamma (IFN-ɣ). Concomitantly, the ability of the modulated T cells to make Interleukin 4 (IL-4) and 10 (IL-10) was enhanced, suggesting a polarization of these cells towards a less inflammatory functional state. This was further evidenced by increased surface expression of an immune-inhibitory molecule, PD1, and reduced expression of the activation markers 41BB and ICOS.We next examined the potential beneficial role of ex vivo programming with FT1050+FT4145 in a major histocompatibility complex (MHC) mis-matched HSCT mouse model. Briefly, lethally irradiated BALB/c mice received bone marrow and splenocytes from C57BL/6 donor mice pulse treated with vehicle or FT1050+FT4145. Significantly less GvHD, as determined by survival, weight loss, GVHD score (diarrhea, inactivity, hunched posture, ruffled fur, eye lesion, snout swelling/skin integrity), cytokine production and histopathology of GvHD target organs was observed in recipients receiving FT1050+FT4145 treated cells as compared to those receiving vehicle treated cells. In addition, we observed increased levels of donor T regulatory cells (Tregs) in secondary lymphoid organs concomitant with decreased levels of circulating IFN-ɣ in recipients receiving FT1050+FT4145 treated cells. Based on the attenuation of alloreactive T-cell responses in these preclinical studies, we believe our findings provide a compelling scientific basis to support the clinical evaluation of ex vivo programmed mobilized peripheral blood in patients undergoing HSCT for the treatment of hematologic malignancies. DisclosuresLevin:Fate Therapeutics, Inc: Employment, Equity Ownership. Shoemaker:Fate Therapeutics Inc: Employment.

A Phase I/II Trial of Intravenous Azacitidine for Acute Gvhd Prophylaxis in Patients Undergoing Matched Unrelated Stem Cell Transplantation: Phase I Results

BackgroundThe negative impact of acute graft-versus-host disease (GvHD) on morbidity and mortality after allogeneic transplant is significant; thus, finding a means to harness the beneficial Graft versus tumor effect (GVT) while reducing or eliminating GvHD is a major goal of transplant trials. Alterations in immune subsets present after transplant can work to suppress allo-reactive T-cell responses by increasing regulatory T-cells and suppressing allo-reactive T-cell proliferation. Azacitidine (AZA) treatment in pre-clincal models resulted in an increase in regulatory T-cells, a decrease in allo-reactive T-cell proliferation and prevention of acute GvHD while preserving GVT effects. (Choi et al. Blood 2010). Based on these results a phase I/II study was designed to test the safety and efficacy of AZA administered shortly after transplant for the prevention/prophylaxis of acute GvHD and relapse in subjects receiving transplants from matched unrelated stem cell donors. We report the results for the Phase I portion of this trial.MethodsPatients with hematologic malignancies in remission age 18 - 70 were eligible. Myeloablative or reduced intensity conditioning without antithymocyte globulin was used. All recipients were required to receive at least 2 x 106 CD34/kg and have at least 1 x 106 CD34/kg cryopreserved as back up in case of primary graft failure. AZA was administered intravenously on day +7 for five consecutive days and repeated every 28 days for a total of 4 cycles after allogeneic transplant from a 10/10 HLA matched unrelated donor. Standard GvHD prophylaxis with mini-methotrexate and tacrolimus was given. A Phase I, 3+3 dose escalation design of 4 cohorts (AZA dose levels 15, 30, 37.5, and 45 mg/m2) was used to determine toxicity and recommended phase II dose. The primary outcome for phase II is the rate of grade II - IV acute GvHD at day 180 after transplant.ResultsWe have transplanted 16 subjects on trial, 15 have received study drug. Recipient characteristics include: median age 57, 67% male, and diagnoses of AML in CR (9), ALL in CR (2), or MDS (4). One DLT was observed in the final cohort of 6 subjects. The DLT experienced in the final cohort was primary graft failure. The subject had developed Clostridium difficile colitis during conditioning and fungemia shortly after transplant. A total CD34 dose of 2 x 106/kg was infused after myeloablative conditioning of Busulfan and Cytoxan. The subject received the cryopreserved back up donor leukocyte infusion at day +28 but died at day 29 of sepsis without evidence of neutrophil engraftment. Contributing causes to the DLT were thought to be the CD34 dose infused, sepsis, severe colitis and possibly AZA administration. For the remainder of subjects treated in phase I the median CD34 dose infused was 5 x 106/kg (range 2 - 5), median ANC engraftment was 14 days (range 10 - 22 days). Median platelet engraftment was 22 days (range 14 - 70). No grade III/IV acute GvHD has been observed. Grades 1 and 2 skin and gut GvHD have been observed, and all cases have responded to steroids except one case of steroid refractory GvHD in cohort 1 (15mg/m2 AZA). At the recommended phase 2 dose of 45mg/m2 AZA, 3 cases of skin GvHD were observed occurring just prior to or at the time of cycle 2 of treatment. All responded to steroids. With a median follow up of 233 days (range 29 - 784), only 2 subjects have relapsed and 11 (73%) remain alive. The most common non-hematologic grade 3 or 4 AEs were gastrointestinal toxicity (mucositis, nausea and diarrhea), electrolyte abnormalities, and infections.In conclusion, AZA can be given safely starting at day +7 after MUD transplant up to a dose of 45mg/m2 tested. Phase II is currently enrolling subjects. Because of the DLT experienced in phase I, the infused CD34 dose will be increased to a minimum of 4 x 106 CD34/kg with 1 x 106 CD34/kg cryopreserved in backup. Correlative studies from banked Phase I biospecimens evaluating dynamics of regulatory T-cells, T-cell subsets and methylation before and after treatment are being analyzed and will be reported. DisclosuresSchroeder:Incyte: Consultancy; Celgene: Other: Azacitidine provided for this trial by Celgene. Off Label Use: Azacitidine for GVHD prophylaxis. Abboud:Gerson Lehman Group: Consultancy; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Pfizer: Research Funding; Merck: Research Funding; Teva Pharmaceuticals: Research Funding. Vij:Onyx: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millennium: Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy; Takeda: Consultancy, Research Funding; Novartis: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Merck: Consultancy.

Biomarkers to assess donor-reactive T-cell responses in kidney transplant patients

Different to antibody-mediated rejection (ABMR), T-cell mediated rejection (TCMR) still unpredictably occurs after kidney transplantation in a great part because of a poor immunologic evaluation of the cellular allogeneic immune response.However, in the last years, important efforts have focused on the development of novel and more sensitive assays to monitor T-cell alloimmune responses at different biological levels that may improve the understanding of the functional status of the cellular immune compartment in patients undergoing organ transplantation. In this direction, immune assays evaluating T-cell proliferation, intracellular ATP release, multiparameter flow cytometry, profiling T-cell receptor repertoires and measurements of frequencies of cytokine-producing T-cells using an IFN-γ enzyme-linked immunospot assay (IFN-γ ELISPOT) have been reported showing interesting associations between the cellular alloimmune response and kidney transplant outcomes.In summary, an important progress has been made in the assessment of alloreactive T-cell responses in the context of organ transplantation using novel immune assays at different biological levels. However, there is an urgent need for prospective, randomized clinical studies to validate these encouraging preliminary data to ultimately introduce them in current clinical practice for refining current immune-risk stratification in kidney transplantation.

Human graft-derived mesenchymal stromal cells potently suppress alloreactive T-cell responses.

After organ transplantation, recipient T cells contribute to graft rejection. Mesenchymal stromal cells from the bone marrow (BM-MSCs) are known to suppress allogeneic T-cell responses, suggesting a possible clinical application of MSCs in organ transplantation. Human liver grafts harbor resident populations of MSCs (L-MSCs). We aimed to determine the immunosuppressive effects of these graft-derived MSCs on allogeneic T-cell responses and to compare these with the effects of BM-MSCs. BM-MSCs were harvested from aspirates and L-MSCs from liver graft perfusates. We cultured them for 21 days and compared their suppressive effects with the effects of BM-MSCs on allogeneic T-cell responses. Proliferation, cytotoxic degranulation, and interferon-gamma production of alloreactive T cells were more potently suppressed by L-MSCs than BM-MSCs. Suppression was mediated by both cell-cell contact and secreted factors. In addition, L-MSCs showed ex vivo a higher expression of PD-L1 than BM-MSCs, which was associated with inhibition of T-cell proliferation and cytotoxic degranulation in vitro. Blocking PD-L1 partly abrogated the inhibition of cytotoxic degranulation by L-MSCs. In addition, blocking indoleamine 2,3-dioxygenase partly abrogated the inhibitive effects of L-MSCs, but not BM-MSCs, on T-cell proliferation. In conclusion, liver graft-derived MSC suppression of allogeneic T-cell responses is stronger than BM-MSCs, which may be related to in situ priming and mobilization from the graft. These graft-derived MSCs may therefore be relevant in transplantation by promoting allohyporesponsiveness.

Pre-transplant donor-specific T-cell alloreactivity is strongly associated with early acute cellular rejection in kidney transplant recipients not receiving T-cell depleting induction therapy.

Preformed T-cell immune-sensitization should most likely impact allograft outcome during the initial period after kidney transplantation, since donor-specific memory T-cells may rapidly recognize alloantigens and activate the effector immune response, which leads to allograft rejection. However, the precise time-frame in which acute rejection is fundamentally triggered by preformed donor-specific memory T cells rather than by de novo activated naïve T cells is still to be established. Here, preformed donor-specific alloreactive T-cell responses were evaluated using the IFN-γ ELISPOT assay in a large consecutive cohort of kidney transplant patients (n = 90), to assess the main clinical variables associated with cellular sensitization and its predominant time-frame impact on allograft outcome, and was further validated in an independent new set of kidney transplant recipients (n = 67). We found that most highly T-cell sensitized patients were elderly patients with particularly poor HLA class-I matching, without any clinically recognizable sensitizing events. While one-year incidence of all types of biopsy-proven acute rejection did not differ between T-cell alloreactive and non-alloreactive patients, Receiver Operating Characteristic curve analysis indicated the first two months after transplantation as the highest risk time period for acute cellular rejection associated with baseline T-cell sensitization. This effect was particularly evident in young and highly alloreactive individuals that did not receive T-cell depletion immunosuppression. Multivariate analysis confirmed preformed T-cell sensitization as an independent predictor of early acute cellular rejection. In summary, monitoring anti-donor T-cell sensitization before transplantation may help to identify patients at increased risk of acute cellular rejection, particularly in the early phases after kidney transplantation, and thus guide decision-making regarding the use of induction therapy.