ALBERT

Beschreibung: BACKGROUND: Regulatory T cells (Tregs) are non-redundant mediators of immunity and tolerance. Adoptive transfer of CD4+Foxp3+ Tregs has emerged as a promising therapy for graft-versus-host disease (GVHD) following allogeneic HSCT (aHSCT), solid organ transplantation and autoimmune diseases (Hanash AM, Blood 2005; Copsel S, Wolf D, Haematologica 2019; Marek-Trzonkowska N, Diabetes Care 2012; Tang Q, J Mol Cell Biol 2012). Our prior work was the first to demonstrate a two-pathway in vivo strategy targeting TNFRSF25 (with TL1A-Ig fusion protein) and CD25 (with low dose IL-2, IL-2LD) receptors can elicit a rapid and strong increase in Treg numbers and function (Wolf D, BBMT 2017). In fact, very low numbers of these in vivo expanded donor Tregs exhibited effective GVHD suppression in recipients following aHSCT (Copsel S, BBMT 2018). Based on these findings and the success of IL-2LD in pre-clinical and clinical studies, we asked: are there phenotypic/functional differences between Tregs expanded via IL-2LD, TL1A-Ig or their combined application (TL1A-Ig+IL-2LD)? METHODS: Mice were administered IL-2LD, TL1A-Ig or TL1A-Ig+IL-2LD over 6 days. Splenic and lymph node (LN) Treg phenotype was determined by flow cytometry. Treg functionality was assessed with sorted populations using an in vivo MHC-mismatched aHSCT. RESULTS: Treatment of C57BL/6-FoxP3RFP mice with TL1A-Ig+IL-2 LD versus IL-2LD only treatment resulted in significantly higher levels of activation/differentiation and functional markers on Tregs including KLRG1, CD103, Nrp1, ICOS (Fig. 1A). Ki67 expression was higher in two-pathway versus IL-2LD stimulation alone (Fig.1B). These data suggested a key role for TNFRSF25 stimulation. Notably, Treg stimulation with TL1A-Ig alone drove the above phenotype indicating a pathway difference between the TNFRSF25 and IL-2 receptors. This difference was further apparent as high affinity IL-2 receptor (CD25) expression was reduced after TL1A-Ig +/- IL-2LD -mediated expansion compared with IL-2 LD alone stimulated Tregs (Fig. 1A). Results were corroborated using a second independent mouse strain, BALB/c, following use of these protocols. To begin addressing if the observed phenotypic differences between CD25 vs. TNFRSF25 + CD25 expanded Tregs could be related to a more potent Treg in vivo suppressive activity, an initial MHC-mismatched aHSCT (donor/recipient = C57BL/6-BALB/c) was performed. We employed 200,000 sorted Tregs (〉98% purity by CD4+FoxP3+ selection from C57BL/6-FoxP3RFP reporter mice) from donor unexpanded, IL-2LD, or TL1A-Ig+IL-2LD treated mice combined with 1.0 x106 T cells. As anticipated, transfer 200,000 TL1A-Ig+IL-2LD stimulated Tregs ameliorated acute GVHD (Fig. 1C). Remarkably, lower GVHD clinical scores were obtained using the same number of IL-2LD only expanded Tregs compared with TL1A-Ig+IL-2LD stimulated Tregs (Fig. 1C). Moreover, early post-transplant, higher LN and splenic CD4/CD8 ratios were detected in aHSCT recipients treated with IL-2LD expanded Tregs vs. TL1A-Ig+IL-2LD (Fig. 1D). CONCLUSION: Our donor TL1A-Ig+IL-2LD Treg expansion protocol promotes a more activated/differentiated and proliferative phenotype versus IL-2LD stimulation alone. This finding may have accounted for their initial effectiveness - but less efficient long-term GVHD amelioration compared to IL-2LD only stimulated Tregs. Multiple variables are associated with the application of Tregs for therapy including numbers, persistence, and suppressive capacity. Our findings suggest a rationale that one-pathway and / or two-pathway stimulated Tregs may be beneficial for use in aHSCT recipients dependent on whether there is a perceived need for prolonged Treg presence and the stage of GVHD. Disclosures Levy: Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

Beschreibung: The capacity of CD4+CD25+Foxp3+ (Treg) cells to regulate adaptive and innate immune responses has led to studies investigating their application to regulate allogeneic T cell responses arising during hematopoietic stem cell transplants (HCT). With respect to HCT, a fundamental clinical concern is the reconstitution of the lymphoid compartment in recipients, particularly T cells since this process can be exceedingly delayed. We have previously found that host Treg cells can regulate resistance to engraftment following HCT, demonstrating that such cells survive and function at least transiently in recipients. The present studies investigated the residual host Treg compartment following HCT. Utilizing varying levels of total body irradiation (5.0 – 14Gy), we observed: recipient CD4+CD25+Foxp3+ cells can survive ablative as well as reduced intensity conditioning, the surviving, i.e. residual Treg cells undergo expansion as assessed by BrdU uptake and cell numbers, and these cells comprise the majority of the Treg compartment in recipients for several months post-HCT during which time donor derived Treg cells gradually arise and cede this compartment. Residual Tregs also dominated the compartment following allogeneic HCT of MHC-matched bone marrow depleted of T cells. To assess the functional capacity of the residual Treg cell compartment, the development of autoimmune disease following transplant of IL-2Rβ −/− (CD122−/−) bone marrow into syngeneic recipients with and without residual Tregs was examined. Autoimmune disease symptoms and T cell alterations were prevented in B6-wt but not T cell deficient recipients. Interestingly, the failure to transfer autoimmune disease following IL-2Rβ −/− HCT into lethally conditioned B6-CD4−/− recipients was associated with the presence of a peripheral CD8+FoxP3+ population not detected in B6-wt mice or the B6-wt mice transplanted with IL-2Rβ −/− BM. This finding indicates that in the genetic absence of CD4+ T cells, a CD8 regulatory population appears to emerge. In total, our observations support the notion that functioning host Tregs initially occupy a niche in the transplant recipient permitting lymphopenic expansion and an extended period of contribution to this compartment. Notably, this contribution reflected much greater levels than conventional T cell populations - even in aggressively conditioned recipients. Finally, these findings imply that the presence of host regulatory cells may be important to consider with respect to eliciting anti-tumor responses and vaccination in recipients during the early period post -hematopoietic cell transplantation.

Beschreibung: Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (allo-HSCTs). Pre-HSCT conditioning typically consists of irradiation and drug administration resulting in the death of rapidly dividing cells. Damage to host tissues initiates a cytokine storm, promoting activation and expansion of donor anti-host alloreactive T cells. Cell death following conditioning has promoted the hypothesis that sensors of cytoplasmic DNA damage in GVHD target tissues contribute to cytokine production. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched (MUD) allo-HSCT models. Our studies show that the STING pathway rapidly regulates cytokine production in the intestinal tract and non-hematopoietic cells can contribute to these responses. Using mice expressing a human STING allele associated with decreased STING activity (Patel S, et al, J Immunol. 2016), we demonstrate its potential clinical importance. To assess STING involvement immediately post-HSCT, cytokine mRNA expression was examined 48 hrs after transplant of C3H.SW bone marrow (BM) + T cells into irradiated B6-WT or STING-/- recipients. Colonic tissue from STING-/- recipients had 〉2x reduction in IFNβ, TNFα and IL-6 mRNA vs. WT. On day 10 post-transplant, colons from STING-/- recipients exhibited reduced inflammation and overall pathology scores than WT. MHC-matched STING-/- HSCT recipients also experienced decreased weight loss, GVHD scores and skin pathology 6 weeks post-HSCT vs. WT. Chimeric studies demonstrated that the absence of STING in non-hematopoietic cells was responsible for the amelioration of GVHD. Therefore, to test STING signaling in non-hematopoietic intestinal cells, we generated intestinal organoid cultures. Intestinal organoids upregulated IFNβ, TNFα, IL-6 and CXCL10 mRNA 6hrs after stimulation with the highly specific STING agonist DMXAA, supporting the notion that STING in intestinal tissues can contribute to inflammation in vivo. Interestingly, expression of these cytokines returned to baseline levels 24 hrs after stimulation (Fig. 1A). Next, we posited that if the absence of the STING pathway in recipients ameliorated GVHD after MHC-matched HSCT, pathway stimulation would exacerbate GVHD. B6-WT mice were injected with DMXAA immediately prior to HSCT with donor C3H.SW BM + T cells. Administration of a single dose of DMXAA increased expression of IFNβ, TNFα and IL-6 mRNA in colon tissue 48 hrs after transplant (Fig. 1B). Importantly, DMXAA treatment of WT - but not STING-/- - recipients significantly increased GVHD scores and lethality post-HSCT. To evaluate the potential impact of STING in the clinical setting, we evaluated recipients after transplant of C3H.SW BM + T cells into mice homozygous for a human allele associated with diminished STING activity (HAQ-MPYS knock-in mice, termed B6N-STINGHAQ/HAQ here) and found that STINGHAQ/HAQ mice contained a lower frequency of donor T cells expressing an activated phenotype (CD44hiCD62Llo) vs. WT recipients and the former also exhibited diminished GVHD (Fig. 1C,D). In contrast to STING knock-out recipients completely lacking protein, these results indicate that reduced STING activity can also affect GVHD. Interestingly, our findings that STING deficiency ameliorates GVHD in MHC-matched allo-HSCT contrast reported observations that STING activation can exacerbate GVHD after MHC-mismatched HSCT (Fischer J, et al, Sci. Transl. Med. 2017). We are currently investigating how the STING pathway regulates CD4+ and CD8+ T cell mediated GVHD and initial findings may provide insight into understanding the pathway's involvement in MHC-matched vs. mismatched allo-HSCT. In total, our studies demonstrate that STING plays an important role in regulating allo-HSCT and suggest this pathway can provide a target for new therapeutic strategies to ameliorate GVHD. Disclosures Levy: Allergan: Consultancy; Capricor Therapeutics: Consultancy; HEAT Biologics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pelican Therapeutics: Consultancy; OccuRx: Research Funding; Shire: Research Funding.

Beschreibung: We evaluated the role of Fas ligand and perforin, the major T cell-mediated cytotoxic pathways that regulate T cell homeostasis, in a CD8+ T cell mediated model of graft-versus-host disease (GVHD) where donor and recipients differ at a single MHC class I antigen (B6 → bm1). Lethally irradiated (11Gy) bm1 mice were transplanted with T cell depleted BM and CD8+ T cells from either wild type (wt) or cytotoxic double deficient (cdd, deficient in both pathways) B6 donors. We hypothesized that cdd CD8+ T cells would be unable to mediate significant GVHD. Contrary to our hypothesis, recipients of cdd donor CD8+ T cells demonstrated significantly greater histopathologic damage from GVHD and increased serum levels of IFN-gamma and TNF-alpha compared to controls (Table 1). In order to understand this increase, we evaluated the in vivo expansion of donor T cells in these recipients as well as in BMT recipients of allogeneic CD8+ T cells from FasL deficient (gld) and perforin deficient (pfp−/−) donors. CD8+ wt T cells expanded until at day 10 after BMT, followed by a rapid decline. By contrast, cdd CD8+ T cells expanded continuously up to day 30 after BMT, peaking at almost one hundred times the number of wt T cells. gld T cells showed kinetics similar to wt T cells, whereas the pfp−/− T cells showed a significantly greater peak on day 10 but a similar contraction by day 30. Percentages of annexin+ cdd donor CD8+ T cells were significantly reduced compared to the other groups. Persistence of host antigen presenting cells did not account for the unrestrained expansion of cdd donor T cells because host dendritic cells were not detected in either the spleen, BM or gut of recipients of cdd CD8+ T cells on day 6 after BMT. In addition, alloantigen expression on epithelial target cells did not enhance GVHD because B6 donor cdd T cells induced equivalently lethal GVHD in [bm1 → B6] and [bm1 → bm1] chimeras (MST of 30 days and 27 days, respectively). We conclude that both perforin and Fas ligand pathways are required for alloreactive CD8+ T cell populations to contract after their initial expansion during a GVH reaction and that the absence of both these pathways results in donor CD8+ T cell unrestricted expansion and more severe GVHD. Table 1 GVHD score (gut) IFN-g (pg/ml) TNF-a (pg/ml) CD8+T cell (x10e6) Annexin+CD8+(%) cdd vs. wt, *P

Beschreibung: Early life exposure to noninherited maternal antigens (NIMAs) may occur via transplacental transfer and/or breast milk. There are indications that early life exposure to NIMAs may lead to lifelong tolerance. However, there is mounting evidence that exposure to NIMAs may also lead to immunologic priming. Understanding how these different responses arise could be critical in transplantation with donor cells expressing NIMAs. We recently reported that murine neonates that received a transplant of low doses of NIMA-like alloantigens develop vigorous memory cytotoxic responses, as assessed by in vitro assays. Here, we demonstrate that robust allospecific cytotoxicity is also manifest in vivo. Importantly, at low doses, NIMA-expressing cells induced the development of in vivo cytotoxicity during the neonatal period. NIMA-exposed neonates also developed vigorous primary and memory allospecific Th1/Th2 responses that exceeded the responses of adults. Overall, we conclude that exposure to low doses of NIMA-like alloantigens induces robust in vivo cytotoxic and Th1/Th2 responses in neonates. These findings suggest that early exposure to low levels of NIMA may lead to long-term immunologic priming of all arms of T-cell adaptive immunity, rather than tolerance.

Beschreibung: Clinical reports suggest significant sex differences in risk for thrombosis-related diseases such as myocardial infarction, stroke, and venous thromboembolism. However, little is known about mechanism for such differences. There is a well-described sexual dimorphism in liver protein synthesis that is growth hormone (GH) dependent. GH secretion from the pituitary is itself highly sexually dimorphic with males (M) secreting in a pulsatile (P) and females (F) a continuous (C) fashion. These patterns induce M- and F-specific signatures of liver gene expression. In the past, we and others have observed significant sex differences in murine thrombosis models. Given that most coagulation proteases and inhibitors are synthesized or modified in the liver, we aimed to test whether sex-specific GH secretion patterns contribute to the observed sex differences in thrombosis. We measured whole blood clotting times (WCT), thrombosis susceptibility in the thromboplastin-mediated pulmonary embolism (PE) model, and hemostasis in the tail bleeding time (BT) model in M and F control (WT) and GH-deficient “little” (LIT) mice. We observed that WT Fs had longer WCTs (mean time 61.38 vs. 56.72 sec) and were significantly protected in the PE model (median survival 232.5 vs 165 sec) as compared to M. There were no differences in the BT model across all experiments. Interestingly, F and M LIT animals both had significantly prolonged WCTs (67.56 and 67.30 sec, respectively) and were substantially protected in the PE model (median survival 900 and 1200 sec) as compared to WT. Next, LIT animals were injected twice daily with GH to simulate the P pattern of GH secretion (LIT+). This resulted in a significant shortening of the F and M WCTs back to WT M levels (53.16 and 50.97 sec). A group of F WT animals were also injected with M pattern GH (WT+). This too resulted in significant shortening of the F WCTs (54.10 sec). To explore for possible mechanisms underlying these differences, we measured activity of coagulation factors II, V, VII, VIII, IX, X, and XI. The average of all factor activity levels was significantly higher in WT M vs F (100 vs. 81.99%), significantly lower and in both M and F LIT (60.85 and 57.97%), and increased to WT M levels in M and F LIT+ animals (106.6 and 99%). To determine whether these changes were mediated by changes in liver gene expression, we measured a panel of 30 coagulation protease and inhibitor genes in liver and vascular tissue by Taqman®. Surprisingly, we found no significant differences in coagulation factor expression, but found that expression of TFPI was significantly increased in F vs M WT vasculature (9431 vs. 7678 gene copy number (GCN)). Expression was increased in M and F LIT animals (10350 and 11710 GCN) and fell to below WT levels in M and F LIT+ animals (4534 and 4194 GCN). These results indicate that sex differences in thrombosis in mice are at least in part mediated by sex differences in GH secretion with F mice relatively protected as compared to M. M and F GH-deficient LIT mice are similarly protected as compared to WT M. Repletion of GH in a P pattern reverts M and F LIT and F WT mice to WT M levels. Finally, P GH secretion may promote increased thrombosis through inhibition of TFPI in the vasculature. This represents a novel mechanism underlying these sex-differences in thrombosis mediated by sexually dimorphic GH secretion and its effect on regulation of TFPI in the vasculature.

Beschreibung: Despite the potential to cure both acquired and inherited disorders involving the hematopoietic compartment, application of allogeneic bone marrow transplantation (BMT) is limited by the frequent and severe outcome of Graft vs. Host Disease (GVHD). Unfortunately, efforts to reduce GVHD by purging the donor graft of T cells have resulted in poor engraftment and elevated disease recurrence. Alternative cell populations capable of supporting allogeneic engraftment without inducing GVHD could increase the potential for donor-recipient matching and decrease treatment associated risks. We have observed that GVHD-suppressive donor CD4+CD25+ T cells are capable of supporting allogeneic hematopoietic engraftment, as demonstrated by initial donor progenitor activity and long-term chimerism and tolerance. Using a murine MHC mismatched model transplanting 0.5–2x106 GFP+ C57BL/6 (B6) T cell-depleted bone marrow cells into 7.0 Gy sublethally irradiated BALB/c recipients, splenic CFU assessment demonstrated that co-transplantation of 1x106 B6 CD4+CD25+ T cells lead to increased donor lineage-committed GM (p

Beschreibung: Abstract 4286 In patients with hematologic cancers, such as lymphoma, aggressive radiation and chemotherapy followed by autologous hematopoietic stem cell transplantation (auto-HSCT) can achieve a minimal residual disease state. Nonetheless, relapse remains the major cause of morbidity and mortality in such patients, and there remains a critical need for more effective anti-tumor therapy. Vaccination with tumor cells secreting the heat shock fusion protein gp96-Ig can induce robust CTL expansion in non-transplanted mice, however its efficacy in HSCT recipients where the opportunity to manipulate the immune response during hematolymphoid reconstitution has not been studied. We examined strategies to expand antigen specific CD8 T cells using lymphoma cells secreting gp96-Ig heat shock fusion protein in the early post auto-HSCT setting. B6 mice (CD45.2+) received ablative conditioning (9.5 Gy TBI) followed by the infusion of 5×106 congenic (CD45.1+) T cell depleted BMC. To investigate vaccine timing, E.G7-OVA lymphoma cells secreting gp96-Ig (E.G7-gp96-Ig: 107/120 Gy) were introduced either 2 or 7 days following auto-HSCT. Two days prior to vaccination, recipients received 106 CD8 OT-I T cells. Following single intraperitoneal E.G7-gp96-Ig vaccination, OT-I expansion occurred at the site of vaccination, reaching maximal levels 5 days post-vaccination. Expansion was clearly superior following vaccination on day 7. Multiple vaccinations (day 7 and 13) improved this response including enhancement of OT-I accumulation in the spleen and LN. Greater than 80% of the OT-I CD8 T cells displayed an antigen-experienced effector memory phenotype (CD44hiCD62Llo) after vaccination with E.G7-gp96-Ig. Notably, little OT-I expansion occurred following vaccination with parental E.G7-OVA (Fig 1), and the OT-I present displayed a central memory phenotype (CD44hiCD62Lhi). Since gp96 chaperoned peptides are cross-presented by APC, we determined the source of B7.1/2 costimulation with regard to donor and recipient APC. Interestingly, following day 7 vaccination CD8 OT-I expansion was markedly diminished when donor BMC were transplanted from B7.1/2 KO mice into WT or B7.1/2 KO recipients (Fig 1). In contrast, following day 2 vaccination, recipient B7.1/2+ APC clearly contributed to OT-I expansion. These observations are consistent with the transition to predominant donor APC presence early following auto-HSCT. To attempt to enhance vaccine induced CD8 expansion, immune complexes comprised of IL-2 and anti-IL-2 mAb clone S4B6 (IL-2/S4B6) were administered following day 7 vaccination. This specific IL-2 immune complex has been shown to interact with CD8 T cells expressing the intermediate affinity IL-2 receptor (IL-2Rβγ), which is expressed on memory CD8 T (concomitantly expressing CD44) and NK cells. Combination treatment with E.G7-gp96-Ig and IL-2/S4B6 immune complex resulted in 4x greater expansion of OT-I T cell numbers at the site of vaccination and in the spleen versus E.G7-gp96-Ig vaccination alone. Thus, 1 week following vaccination, 〉6×106 OT-I were identified in the compartments analyzed. Interestingly, residual host CD8 T cells were also expanded following complex administration. In summary, we hypothesize that gp96-Ig vaccination post-HSCT results in cross presentation of tumor peptides to CD8 T cells by mature or newly derived B7.1/2 expressing APC of donor origin. Addition of IL-2 via specific mAb/cytokine immune complex provides a strategy to augment gp96-Ig induced CD8 expansion. Studies are underway to address the efficacy of this approach in pre-clinical lymphoma models involving T cell replete auto-HSCT. Disclosures: Podack: Heat Biologics, Inc.: Consultancy, Equity Ownership.

Beschreibung: Abstract 3751 Following myeloablative as well as reduced intensity conditioning regimens, recipients of allogeneic T cell replete (TCR) HLA matched sibling)or unrelated hematopoietic stem cell transplants (HSCT) experience significant acute as well as chronic graft vs. host disease (GVHD). Recent studies have reported that post-transplant cyclophosphamide (PTC) can function as an effective single agent for prophylaxis of acute and chronic GVHD (Biol. Blood Marrow Transpl. 16:482, 2010; Blood. 116:3224, 2010). To investigate the fate of non-host alloreactive donor cells, which are critical to providing immune reconstitution post-transplant, T cell replete HSCT were performed between MHC-matched allogeneic donor–recipient pairs. C3H.SW (H2b) mice were lethally conditioned 4 hrs prior to receipt of B6-CD45.1 (H2b) T cells and bone marrow. Recipients developed severe GVHD as assessed by weight loss (Fig. 1A) and clinical analyses during the first 4–5 wks post-transplant. Recipients had inverted CD4/CD8 ratios as well as an activated phenotype (CD44hiCD62Llo), severe B and T cell lymphopenia, and virtually undetectable thymic tissue. A single dose of post-transplant cyclophosphamide at day 3 (66mg/kg/body weight) blocked onset of weight loss and clinical signs of GVHD as well as lethality in C3H.SW recipients, consistent with the reduction of alloreactive anti-recipient T cells. In contrast to non-cyclophosphamide treated animals, treated recipients expressed typical CD4/CD8 ratios, significant numbers of spleen and lymph node cells (Fig. 1B) and readily detectable thymi 7–8 weeks post-HSCT. Notably, the majority of T as well as B cells in the periphery of these recipients exhibited a non-activated phenotype and were derived from donor bone marrow. Together with the observation that approximately 50% of T cells exhibited a CD44hiCD62Llo phenotype characteristic of TN, the findings are consistent with de novo derived lymphopoiesis in the thymus and marrow of post-transplant cyclophosphamide treated recipients. To assess the impact of D.3 cyclophosphamide treatment on a non-host reactive T cell population, B6 OT-I CD8 T cells (Vα2/Vβ5 TcR) were added (1-2×106) to the donor inoculum. In non-PTC treated recipients, low numbers of OT-I T cells (below input levels) were detected by 3–4 weeks post-HSCT. In contrast, in PTC-treated recipients, significant numbers of OT-I CD8 T cells were present in the recipient spleen and lymph nodes (Fig. 1C), consistent with the finding of CFSE labeled OT-I cells exhibiting division following cyclophosphamide treatment. Notably, the overall numbers of OT-I CD8 T cells in cyclophosphamide treated recipients was greater than the input levels, indicating that these cells a) survived post-cyclophosphamide treatment and b) underwent lymphopenic expansion. Consistent with these findings, CFSE studies at 72 hrs post-HSCT illustrated OT-I T cells exhibited low proliferation vs. a population of presumed B6 anti-C3H.SW alloreactive T cells. We therefore conclude that non-host reactive donor T cells undergo sufficient repair following cyclophosphamide induced alkylation to enable greater survival in contrast to rapidly dividing anti-host (GVHD) reactive populations. Vaccination studies in cyclophosphamide treated and non-treated HSCT recipients are being performed using heat shock fusion protein-transfected tumor cells to investigate the capacity to elicit anti-tumor (i.e., GVL) responses in the presence and absence of GVHD. Fig. 1: Post-transplant cyclophosphamide treated recipients demonstrate little severe weight loss vs. non-ptc recipients (panel A). Enhanced lymph node cell numbers and non-host reactive donor CD8 OT-I cell levels in ptc recipients 1 month post MHC-matched MiHA-mismatched HSCT (panels B and C). Fig. 1:. Post-transplant cyclophosphamide treated recipients demonstrate little severe weight loss vs. non-ptc recipients (panel A). Enhanced lymph node cell numbers and non-host reactive donor CD8 OT-I cell levels in ptc recipients 1 month post MHC-matched MiHA-mismatched HSCT (panels B and C). Disclosures: No relevant conflicts of interest to declare.

Beschreibung: The role CD4+CD25+FoxP3+ (Treg) cells play in the establishment and maintenance of immune tolerance makes them attractive candidates for therapeutic prevention and treatment of autoimmune and transplant rejection responses. Autoimmune diseases have diverse pathologies/underlying effector mechanisms and how Treg cells control each disorder remains unclear. Granzyme B in murine and perforin in human Treg cells have been reported to be involved with suppression of proliferation and lysis of activated T cells, respectively. Perforin/fas-ligand double cytotoxic deficient (B6-cdd) mice develop a spontaneous lymphoproliferative disorder with selective tissue infiltration, principally in the pancreas and ovaries, together with weight loss and death by ~15 weeks of age (i.e. cdd syndrome). Uncontrolled expansion of the effector T cell pool is proposed to result as a consequence of cytotoxically impaired, activated T cells unable to lyse Ag-presenting monocytes and macrophages. Since CD4+CD25+FoxP3+ cells have the capacity to regulate lymphocyte proliferation, this compartment was examined in B6-cdd (H2b) mice. At 6–7 weeks of age-prior to complete onset of the syndrome-splenic CD4+CD25+ T cells are present and functionally suppressive in vitro. We were therefore interested in investigating their capacity to regulate auto and allo-reactive T cell responses in situ in the context of defined spontaneous autoimmune disease (IL2Rβ−/−)and GVHD (MHC Class I/II disparate) models. Purified CD4+CD25+ T cells isolated from B6-cdd mice were injected into B6-IL2Rβ−/− (CD122−/−) newborns and effectively prevented autoimmune disease assessed by hematocrit, absence of lymphoproliferation and clinical signs. B6-cdd Tregs were identified and collected several (~3) months post-injection from the rescued animals and found to exhibit functional suppression in vitro. Interestingly, identical results were obtained in allogeneic BALB/c (H2d) IL2Rβ−/− recipients. Purified CD4+CD25+ T cells isolated from B6-cdd or B6-wt mice were transplanted together with T cell replete B6 BMC into lethally irradiated, MHC-mismatched BALB/c recipients. B6-cdd Tregs were found to be comparable to B6-wt Tregs in their ability to prevent development of ac ute GVHD as assessed by weight loss, clinical signs and survival post-BMT. In total, these observations demonstrate that CD4+CD25+FoxP3+ Treg cells do not require the cytolytic effector molecules perforin and fas-ligand for in vitro suppression, in vivo regulation of a spontaneous (CD122−/−) autoimmune disease as well as for acute GVHD. Their inability to control autochthonous B6-cdd lymphoproliferative syndrome could ultimately be a result of death or dysregulation of Treg cells in these mice. Alternatively, distinct Treg effector molecules may be required to control different T cell responses associated with various autoimmune/non-self reactivity and ongoing experiments are investigating these possibilities.