Recipients Of Spleen Cells Research Articles

Allograft TLR9 is required for endogenous donor-reactive memory CD8 T cell activation to mediate CTLA-4Ig resistant rejection of high-risk cardiac allografts

Abstract Organ cold ischemic storage (CIS) is inherent to deceased-donor solid organ transplantation and CIS time correlates with poorer transplant outcomes. Prolonged CIS enhances ischemia-reperfusion injury (IRI) following graft revascularization, generating an inflammatory environment that promotes endogenous donor-reactive memory T cell activation leading to CTLA-4Ig costimulatory blockade-resistant rejection. Using a murine heterotopic heart transplant model, we tested the impact of prolonged (8hr) versus minimal (0.5hr) CIS on cell-free mitochondrial DNA (cf-mtDNA) release and activation of the cf-mtDNA sensor TLR9 to understand their contributions to early post-transplant donor-reactive endogenous memory T cell activation and CTLA-4Ig resistant rejection. Plasma cf-mtDNA was significantly elevated in 8hr versus 0.5hr CIS allograft recipients as early as 12hr post-transplant. Flow cytometry analysis of graft infiltrating leukocytes showed reduced memory CD4 and CD8 T cell proliferation on D2 following 8hr CIS in TLR9KO compared to WT allografts. In contrast, graft TLR9KO did not impact the de novo donor-reactive T cell response. 8hr CIS TLR9KO and WT allografts had similar survival in untreated recipients (MST 7.5 vs 7 days) and there was no difference in IFN-γ producing donor-reactive T cells in recipient spleens on D7. When recipients of allografts subjected to 8hr CIS were conditioned with CTLA-4Ig to inhibit de novo T cell priming, graft TLR9KO significantly prolonged graft survival compared to WT (MST 59 vs 28 days). These findings indicate that IRI-induced cf-mtDNA release and TLR9 activation are novel targets for abrogating CTLA-4Ig resistant donor-reactive memory T cell activation within high-risk allografts. This work was supported by NIH grants R01AI40459, T32AI089474, and T32GM007250.

CLECL1+ CLL Cells: A Key Subset That Drives CLL Survival and Proliferation and Shapes a Th2-Biased Tumor Microenvironment

Chronic lymphocytic leukemia (CLL) cells depend on elements in the tissue microenvironment (TME) that produce factors such as IL-4 to promote leukemic B cell survival and growth. CLECL1, a C-type lectin-like protein, is a T cell co-stimulatory molecule originally identified on dendritic cells, but also expressed on normal B and myeloid cells. Interactions of CLECL1 with naïve T cells (Tn) lead to Th2 cell development and production of IL-4. While IL-4 can have pro- and anti- tumor activities, a role for CLECL1 in tumorigenesis has not been tested. Here we studied the role of CLECL1 in CLL. Flow cytometry analyses of CD19+CD5+ cells from 7 normal people and 77 patients with CLL revealed significantly higher CLECL1 levels on CLL cells (CLL vs Normal = 10.1% vs 3.9%, P = 0.01). CLECL1 was found most in the fraction of leukemic clones enriched in recently divided cells (PF; CXCR4DimCD5Bright) and least in the resting fraction (RF; CXCR4BrightCD5Dim) (PF vs RF = 20% vs 0.4%, P < 0.001). Importantly, high levels of CLECL1 in the PF correlated with shorter time to first treatment (P = 0.01). Notably, effective treatment with ibrutinib significantly reduced CLECL1+ cells (pre vs post treatment: 9.1% vs 1.8%, P = 0.01), and these levels increased when patients developed ibrutinib resistance (sensitive vs resistance = 1.8% vs 7.5%, P < 0.05). Flow cytometry analyses also indicated that, compared to CLECL1- cells, CLECL1+ CLL cells expressed higher levels of other cell interaction molecules such as CD40 and CD80, as well as activation markers, CD23, CD27, CD69, CD71 and CD72. Consistent with this, CLECL1+ PF cells were mainly in the G2/M phase of the cell cycle and express AID. In contrast, CLECL1- PF cells were AID- and chiefly in G1/S phases or undergoing apoptosis. Single cell RNAseq analyses of CLL PF versus RF cells corroborated that the expression of CLECL1 is [1] restricted to the PF, [2] associated with genes expressed in G2/M, and [3] correlated with IL-4-related gene expression. Together, these data suggest CLECL1+ CLL cells are activated cells generated from CLECL1- cells. Indeed, stimulation of CLECL1- cells in vitro revealed that anti-IgM+IL-4 converted CLECL1- to CLECL1+ cells. In CLL patient lymph nodes, CLECL1+ cells expressed Ki67 and co-localized with T cells. Thus, we questioned the abilities of CLECL1+ and CLECL1- cells to interact with T cells and to affect T cell growth and cytokine production. Co-culture of unseparated autologous T cells with CLECL1+, but not CLECL1- CLL cells significantly enhanced proliferation of T cells stimulated through T-cell receptors (CLECL1+ vs CLECL1-: 12.9% vs 2.8%, P = 0.02) and induced increased production of IL-4 (CLECL1+ vs CLECL1-: 15% vs 7%, P < 0.05). Contrary to published results that recombinant CLECL1 (rCLECL1) only binds to healthy Tn, we identified direct binding of rCLECL1 to both Tn and T memory (Tmem) cells in CLL patients. Moreover, when Tn and Tmem cells were incubated with rCLECL1, both T cell subsets responded with enhanced proliferation and IL-4 production. Subsequently, we tried to recapitulate the above in vitro findings in vivo, using patient derived xenografts. Injection of NSG mice with autologous CD3+ T cells plus CLECL1+ or CLECL1- PF cells led to significantly higher numbers of CLL B and T cells in the spleens of recipient mice (CLECL1+ vs CLECL1- for B cells: 508,800 vs 37,000; P = 0.03; for T cells: 273,700 vs 47,000; P = 0.03). Additionally, Th2 cells outgrew Th1 T cells (CLECL1+ vs CLECL1- for Th2 cells: 60.1% vs 12%; P < 0.05; for Th1 cells: 20.1% vs 1.8%; N.S). The same results were seen when we injected CLECL1+ or CLECL1- CLL PF cells plus autologous Tn or Tmem cells into NSG mice, as only CLECL1+ PF cells stimulated Tn or Tmem cells to expand and produce IL-4. In turn, these CLECL1 stimulated T cells facilitated the growth of CLL cells in recipient spleen and bone marrow. Overall, these ex vivo, in vitro, and in vivo results indicate that CLL cells that express CLECL1, specifically those in the recently-divided fraction, engage both Tn and Tmem cells to promote T cell growth and IL-4 production. Consequently, the CLECL1-shaped Th2 TME facilitates the survival and proliferation of tumor cells. Therefore, despite the relatively small numbers, CLECL1+ cells in the PF of CLL clones are potential key targets for therapy. Eliminating CLECL1+ CLL cells or blocking their promotion of IL-4 production could improve treatment, especially for those patients with CLL resistant to ibrutinib.

Wasabi Component 6-(Methylsulfinyl)hexly Isothiocyanate and Derivatives Improve the Survival of Skin Allografts.

We tested the effect of 6-(Methylsulfinyl)hexyl Isothiocyanate (6-MITC) and derivatives (I7447 and I7557) on the differentiation and maturation of human myeloid dendritic cells (DCs) in vitro, and skin transplantation in vivo. Triggering of CD14+ myeloid monocyte development toward myeloid DCs with and without 6-MITC and derivatives to examine the morphology, viability, surface marker expression, and cytokine production. Stimulatory activity on allogeneic naive T cells was measured by proliferation and interferon-γ production. The skin allograft survival area model was used to translate the 6-MITC and derivatives’ antirejection effect. All of the compounds had no significant effects on DC viability and reduced the formation of dendrites at concentrations higher than 10 μM. At this concentration, 6-MITC and I7557, but not I7447, inhibited the expression of CD1a and CD83. Both 6-MITC and I7557 exhibited T-cells and interferon-γ augmentation at lower concentrations and suppression at higher concentration. The 6-MITC and I7557 prolonged skin graft survival. Both the 6-MITC and I7557 treatment resulted in the accumulation of regulatory T cells in recipient rat spleens. No toxicity was evident in 6-MITC and I7557 treatment. The 6-MITC and I7557 induced human DC differentiation toward a tolerogenic phenotype and prolonged rat skin allograft survival. These compounds may be effective as immunosuppressants against transplant rejection.

Activation of the Aryl Hydrocarbon Receptor Ameliorates Acute Rejection of Rat Liver Transplantation by Regulating Treg Proliferation and PD-1 Expression.

Aryl hydrocarbon receptor (AhR) plays important roles in modulating immune responses. However, the role of AhR in rat liver transplantation (LT) has not been explored. Safety and side effects of N -(3,4-dimethoxycinnamonyl) anthranilic acid (3,4-DAA) and 2-methyl-2H-pyrazole-3-carboxylic acid amide (CH223191) were evaluated. We used optimal doses of 2 drugs, 3,4-DAA, a drug used for mediating AhR activation, and CH223191, antagonist of AhR (3,4-DAA, CH223191, and 3,4-DAA + CH223191), intraperitoneally administered to recipients daily to investigate the role of AhR in the rat LT model. The recipient livers were used to observe the pathological changes, the cells infiltrating the graft, and changes of AhR and programmed death-1 (PD-1) by Western blot, real-time polymerase chain reaction, and immunofluorescence assays. The contents of Foxp3 + and PD-1 + T cells in the recipient spleen and peripheral blood mononuclear cells were evaluated by flow cytometry. In vitro, after isolating CD4 + T cells, they were treated with different AhR ligands to observe the differentiation direction and PD-1 expression level. The activation of AhR by 3,4-DAA prolonged survival time and ameliorated graft rejection, which were associated with increased expression of AhR and PD-1 in the livers and increased Foxp3 + T cells and PD-1 + T cells in recipient spleens, livers, and peripheral blood mononuclear cells. In vitro, primary T cells incubated with 3,4-DAA mediated increased proportion of Treg and PD-1 + T cells. However, the suppression of AhR with CH223191 reverses these effects, both in the LT model and in vitro. Our results indicated that AhR activation might reduce the occurrence of rat acute rejection by increasing the proportion of Treg and the expression of PD-1.

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.

IL-Y Aggravates Murine Chronic Graft-Versus-Host Disease by Enhancing T and B Cell Responses.

IL-Y, a synthetic member of IL-12 cytokine family, was found to exert potent immunosuppressive effects by inhibiting the differentiation and activation of Th1 and Th17 cells. However, the role of IL-Y in the development of chronic graft-versus-host disease (cGVHD) remains unknown. Here, using murine models of scleroderma-like and lupus-like cGVHD, we examined the function of IL-Y in the pathogenesis of cGVHD by hydrodynamically injecting minicircle-IL-Y expressing plasmids (MC IL-Y). In contrast with the reported immune suppressive function of IL-Y, administration of MC IL-Y enhanced cGVHD severity reflected by deteriorated multi-organ pathologic damages. In lupus-like cGVHD model, urine protein and the serum anti-dsDNA antibody (IgG) were significantly upregulated by IL-Y treatment. Further study demonstrated that IL-Y impacts both donor T and B cell response. In T cells, IL-Y inhibited the generation of CD4+Foxp3+ regulator T (Treg) cells during the development of cGVHD. IL-Y may also increase the infiltration of pathogenic TNF-α producing CD4+ and CD8+ T cells through IL-27Rα in recipient spleens, as this effect was diminished in IL-27Rα deficient T cells. Moreover, IL-Y enhanced the differentiation of ICOS+ T follicular helper (Tfh) cells. In B cells, the percentage of germinal center (GC) B cells in recipient spleens was significantly upregulated by MC IL-Y plasmid administration. The levels of co-stimulatory molecules, MHC-II and CD86, on B cells were also enhanced by IL-Y expression. Taken together, our data indicated that IL-Y promoted the process of cGVHD by activating pathogenic T and B cells.

C5aR1 regulates migration of suppressive myeloid cells required for costimulatory blockade-induced murine allograft survival.

Costimulatory blockade-induced murine cardiac allograft survival requires intragraft accumulation of CD11b+ Ly6Clo Ly6G- regulatory myeloid cells (Mregs) that expand regulatory T cells (Tregs) and suppress effector T cells (Teffs). We previously showed that C5a receptor (C5aR1) signaling on T cells activates Teffs and inhibits Tregs, but whether and/or how C5aR1 affects Mregs required for transplant survival is unknown. Although BALB/c hearts survived >60days in anti-CD154 (MR1)-treated or cytotoxic T-lymphocyte associated protein 4 (CTLA4)-Ig-treated wild-type (WT) recipients, they were rejected at ~30days in MR1-treated or CTLA4-Ig-treated recipients selectively deficient in C5aR1 restricted to myeloid cells (C5ar1fl/fl xLysM-Cre). This accelerated rejection was associated with ~2-fold more donor-reactive T cells and ~40% less expansion of donor-reactive Tregs. Analysis of graft-infiltrating mononuclear cells on posttransplant day 6 revealed fewer Ly6Clo monocytes in C5ar1fl/fl xLysM-Cre recipients. Expression profiling of intragraft Ly6Clo monocytes showed that C5aR1 deficiency downregulated genes related to migration/locomotion without changes in genes associated with suppressive function. Cotransfer of C5ar1fl/fl and C5ar1fl/fl xLysM-Cre myeloid cells into MR1-treated allograft recipients resulted in less accumulation of C5ar1-/- cells within the allografts, and in vitro assays confirmed that Ly6Chi myeloid cells migrate to C5a/C5aR1-initiated signals. Together, our results newly link myeloid cell-expressed C5aR1 to intragraft accumulation of myeloid cells required for prolongation of heart transplant survival induced by costimulatory blockade.

Establishment of Acute GVHD Mouse Model of Haplo identical Hematopoietic Stem Cell Transplantation and Its Pathogenesis

To establish the acute graft-versus-host disease(GVHD) mouse model based on haplo identical hematopoietic stem cell transplantation (HSCT) and to further investigate the pathogenesis of GVHD. Recipient mice ([C57BL/6×CBA/Ca]F1(H-2b×k)) received lethal irradiation (11 Gy) of γ ray (137Cs), followed by transplantation with donor-derived [C57BL/6(H-2b)] T cell-depleted bone marrow cells (TD-BM) with or without donor-derived T cells. Recipients were randomly divided into 4 groups, including irradiation group, TD-BM group, TD-BM+T cell group 1 and TD-BM+T cell group 2. Survival rate and weight change were detected after HSCT. Pathological scoring were performed on the collected organs from recipients on day 14. Moreover, bone marrow chimerism was detected on days 14 and 18 after HSCT. Additionally, differentiation of donor-derived T cells towards Th1 cells in vivo was analyzed by flow cytometry. Furthermore, proliferation of donor-derived T cells in vivo was tested using CFSE. The average survival in TD-BM, irradiation, TD-BM+T cell group 1 and TD-BM+T cell groups 2 were 60, 13.29±5.50, 33±2.3 and 29.14±1.77 days, respectively. On day 14 after transplantation, recipients of TD-BM + T cell group 1 displayed significantly more severe pathology in liver, colon, lung and skin. On days 14 and 28 after HSCT, bone marrow chimerism in recipients of both TD-BM group and TD-BM+ T cell groups 1 was over 94%. On day 14 after HSCT, serum cytokines IFN-γ, TNF-α and IL-1 levels in TD-BM+T cell group 1 were significantly higher than those in TD-BM group. Percentage of H-2b+H-2k-CD4+IFN-γ+ in spleen cells of recipients in TD-BM+T cell group 1 was higher than that of TD-BM group, with (0.5240±0.08447)% vs (7.912±0.6087)% (P<0.05). On days 14 after HSCT, donor-derived T cells displayed obvious proliferation in vivo. C57BL/6(H-2b)→［C57BL/6×CBA/Ca］F1(H-2b×k) HSCT model can be used as a aGVHD model of haploidentical HSCT. Additionally, Th1 may play an important role in the pathogenesis of aGVHD resulting from haplo-identical HSCT.

Effect of Infusion of Recipient Spleen Cells at Different Time after Murine Haploidentical Hematopoietic Stem Cell Transplantation on Graft Versus Host Disease

To explore the effect of infusing G-CSF mobilized recipient spleen cells at different time after haploidentical stem cell transplantation(HSCT) on graft-versus-host disease (GVHD) in mice and its possible mechanism. Forty mice after HSCT were randomly divided into 4 groups (n=10): GVHD positive control group (control group), 1st d recipient cell infusion group after transplantation (+1 d group), 4th d recipient cell infusion group after transplantation(+4 d group), 7th d recipient cell infusion group after transplantation(+7 d group). The mice in control group were injected the normal saline of same equivalent with experimental group which were given the same amount of G-CSF-mobilized recipient spleen cells. The general manifestation and pathological change of GVHD were observed. The expression changes of CD3+CD4+, CD3+CD8+ cell subsets and FasL in peripheral blood were detected by flow cytometry. The incidence of GVHD was significantly decreased in +4 d group and the median survival time was longer than 60 days, which was significantly higher than that of control group (24 d), +1 d group (21 d), +7 d group (28 d). (P<0.01, P<0.01, P<0.01). The Fasl expression of peripheral blood T lymphocytes in +4 d group were significantly lower than that in the other 3 groups(P<0.05). The +4 d infusion of G-CSF mobilized recipient spleen cells on 4th day after haploidentical HSC transplantation can inhibit the expression of FasL in donor T lymphocytes, and significantly reduce the incidence of GVHD.

Investigation for the effect of donor endothelium bcl-xL gene transfection on transplant immunoreaction in rat aorta transplantation

Objective To investigate the influence of bcl-xL overexpression in donor endothelia on recipient immunoreaction after rat aorta transplantation. Methods Enhanced green fluorescent protein (EGFP)-marked lentivirus express vector with Tie2 promoter of anti-apoptotic gene bcl-xL was constructed and transfected into donor SD rat by tail vein injection. The isogeneic and allogeneic aorta transplantation was performed between SD and SD rats as well as SD and Wistar rats. After one week of transplantation, the overexpression of bcl-xL gene in transplant aorta was detected by fluorescence microscope and real-time quantitative polymerase chain reaction (Real-time PCR). The serum from recipients was collected and transplant aortic endothelial cells, spleen cells as well as cytotoxic T lymphocytes (CTL) from donor and recipient were separated and cultured. The mixed lymphocyte reaction, CTL activity and complement-dependent antibody-mediated cytotoxicity were then performed to assess the influence of bcl-xL overexpression in donor endothelia on recipient immunoreaction after rat aorta transplantation. Results Bcl-xL gene was significantly overexpressed (8.5±1.1 vs. 1.0±0.2, t=17.050, P=0.002) in transplant aorta after transfection of lentivirus express vector with Tie2 promoter. The proliferation rate of recipient spleen cells stimulated by allogeneic donor spleen cells was significantly increased in contrast to stimulated by isogeneic donor spleen cells (14.2±1.8 vs. 1.0±0.1, t=23.599, P=0.001; 14.7±2.2 vs. 0.8±0.1, t=25.037, P=0.001). The survival rate of transplant aortic endothelial cells incubated by allogeneic recipient CTL and serum was significantly reduced in contrast to incubated by isogeneic recipient CTL and serum (14.5% vs. 92.7%, χ2=244.558, P=0.000; 8.9% vs. 95.8%, χ2=290.494, P=0.000; 15.1% vs. 96.7%, χ2=269.251, P=0.000; 10.1% vs. 94.7%, χ2=286.190, P=0.000). However, there were no significant differences of the proliferation rate (1.0±0.1 vs. 0.8±0.1, t=2.108, P=0.068; 14.2±1.8 vs. 14.7±2.2, t=-1.012, P=0.341) and survival rate (92.7% vs. 96.7%, χ2=3.078, P=0.079; 95.8% vs. 94.7%, χ2=0.497, P=0.481; 14.5% vs. 8.9%, χ2=2.917, P=0.088; 15.1% vs. 10.1%, χ2=2.286, P=0.131; and 96.6% vs. 97.3%, χ2=0.344, P=0.558; 91.5% vs. 92.1%, χ2=0.033, P=0.856; 19.8% vs. 15.4%, χ2=0.387, P=0.239; 10.2% vs. 13.8%, χ2=1.515, P=0.218) between transfected groups and nontransfected groups in both isogeneic and allogeneic transplantation. Conclusion No significant effect of bcl-xL overexpression in donor endothelia on recipient immunoreaction after rat aorta transplantation. Key words: Bcl-xL; Aorta; Transplantation; Immune

A specific immune tolerance toward offspring cells is to exist after the mother lymphocyte infusion

PurposeTo examine immune tolerance between maternal lymphocytes and offspring tissue after a donor lymphocyte infusion. MethodsMouse models were established by mating female BALB/c mice with male C57BL mice. Splenic lymphocytes from donors of different genetic backgrounds were labeled with carboxyfluorescein succinimidyl ester (CFSE), and 1×107 of the labeled cells were intravenously injected into a recipient. At 6h, 24h, 72h and 120h after the infusion, mononuclear cells in recipient spleen, liver, thymus, lymph nodes, and peripheral blood were collected. CFSE+, CFSE-, CD3+, CD8+, CD4+, CD19+, NK1.1+, CD25+, and CD127+ lymphocytes in those samples were analyzed by flow cytometry. The distribution of donor T cells, B cells, NK cells, helper T cells, cytotoxic T cells, and recipient regulatory T cells in the tissues were then analyzed. ResultsMaternal lymphocytes were more likely to survive in offspring. At 120h after infusion, the percentages of maternal cells in the offspring were 0.52±0.11% in lymph nodes, 0.97±0.04% in peripheral blood, and 0.97±0.11% in the spleen. Few donor cells, if any, were detected in these tissues at 120h after aunt to child, father to child, and unrelated allogeneic infusions were performed. The subtype proportion of donor lymphocytes changed significantly in the recipient tissues. Recipient Treg cells increased in the mother to child group, but not in the aunt to child, father to child, and unrelated allogeneic groups, suggesting a decreased cellular immune response to allogeneic cells in the mother to child group. At 120h after the infusion, no donor cells were detected in the recipient livers and thymuses of all groups, implying that donor cells were barely able to colonize in the liver and thymus. ConclusionSpecific immune tolerance to maternal lymphocytes exists in offspring. An infusion of maternal donor lymphocytes may produce a relatively persistent effect of adoptive immunotherapy with reduced side-effects.

MiR-17-92 Is Required for the Pathogenicity of T and B Cells in Chronic Gvhd

Chronic graft-versus-host disease (cGVHD) remains to be a major cause of mortality and morbidity after allogeneic hematopoietic cell transplantation (allo-HCT). cGVHD is characterized as autoimmune-like fibrosis and antibody production, mediated by pathogenic T and B cells. Through producing pro-inflammatory cytokines, CD4 T cells are the driving force of cGVHD. Donor B cells augment the pathogenesis of cGVHD not only by acting as antigen-presenting cells (APCs) and promoting CD4 T-cell expansion and survival, but also by producing autoantibodies. microRNA (miR)-17-92 has been shown to regulate T-cell immunity including allogeneic, anti-viral, and anti-tumor responses. Recently, miR-17-92 was found to act together with Bcl-6 to promote the differentiation of Follicular help T (Tfh) cells. Furthermore, B-cell deficiency of miR-17-92 impairs IgG2c production. Since Tfh differentiation and antibody production are required for the development of cGVHD, we hypothesize that miR-17-92 contributes to the pathogenesis of cGVHD by promoting pathogenic T- and B-cell responses.By using Cre-loxp system, we generated B6 mice with conditional deficiency of miR-17-92 in T cells (CD4cre), B cells (CD19cre), or both (CD4CD19cre). aGVHD to cGVHD transition model (B6 to BALB/c) was utilized to test the effects of individual and combinational deficiency of miR-17-92 in T and/or B cells in the development of cGVHD. BALB/c mice were lethally irradiated and transferred with splenocytes plus BM cells derived from CD4cre, CD19cre or CD4CD19cre miR-17-92flox/flox B6 mice. WT B6 (Cre- miR-17-92flox/flox) mice were used as control donors. A significantly reduction of GVHD mortality was observed only in the recipients with CD4CD19cre grafts, but not with CD4cre or CD19cre grafts. Deficiency of miR-17-92 in donor T or B cells indeed improved the clinical manifestation of cGVHD, but the deficiency in both T and B cells showed further improvement, indicating the additive role of miR-17-92 in T and B cells in the pathogenesis of cGVHD. Mechanistically, deficiency of miR-17-92 in T cells resulted in the reduction of Tfh generation (Fig. A), germinal center (GC) B-cell and plasma cell differentiation, and the expression of MHC-II and CD86 on donor B cells in recipient spleens. Furthermore, deficiency of miR-17-92 in B cells significantly reduced the levels of total IgG and IgG2c in recipient serum (Fig. A). These data suggest that miR-17-92 contributes to both T- and B-cell differentiation and function, which is required for the development of cGVHD.To extend our findings, we used a bronchiolitis obliterans cGVHD model (B6 to B10.BR). Recipient mice were pre-conditioned and received either BM alone from WT or CD19cre B6 mice, or BM plus purified T cells from WT or CD4cre B6 mice. Deficiency of miR-17-92 in T cells or BM-derived B cells resulted in significant improvement in pulmonary functions in recipient mice, as demonstrated by a decrease in resistance and elastance and an increase in compliance (Fig. B). Consistently, we found that miR-17-92 promoted Tfh and GC B-cell differentiation (Fig. B), while inhibiting differentiation of T follicular regulatory cells in recipient spleens 60 days after allo-HCT.For translational purpose, we tested whether inhibition of miR-17-92 could ameliorate cGVHD using locked nucleic acid (LNA) antagomirs specific for miR-17 or miR-19, key members in this microRNA cluster. In a SLE cGVHD model (DBA2 to BALB/c), administration of anti-miR-17, but not anti-miR-19, significantly suppressed the incidence of proteinuria and the severity of clinical manifestation by inhibiting donor splenocyte expansion, expression of costimulatory molecules on donor B cells, and differentiation of GC B cells and plasma cells (Fig. C). In addition, systemic delivery of anti-miR-17 significantly improved skin cGVHD by restraining IL-17 producing CD4 T-cell infiltration in skin-draining lymph nodes in a scleroderma-cGVHD model (B10.D2 to BALB/c).Taken together, the current work reveals that miR-17-92 is required for T- and B-cell differentiation and function, and thus for the development of cGVHD. Furthermore, pharmacological inhibition of miR-17 represents a potential therapeutic strategy for the control of cGVHD after allo-HCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting the IL-2/CD25 and TL1A/TNFRSF25 Pathways: A New Approach to Markedly Expand Donor Tregs in Multiple Compartments Leads to in Situ Immune Regulation

Regulatory T cells (Treg) are critical for the maintenance of self-tolerance and Treg expansion is being investigated as a promising therapy for graft-versus-host disease (GVHD) after allogeneic stem cell transplantation. Because the numbers of donor graft Tregs necessary for maximal efficacy are significant, ex vivo expansion presents both practical and scientific challenges. We and others have demonstrated that in situ expansion of CD4+ FoxP3+ Tregs using IL-2 may be used to inhibit alloreactive T cells that mediate GVHD. Recently, we developed a novel combination approach to expand donor Tregs, wherein we administered a fusion protein (TL1A-Ig) and IL-2, which target, respectively, TNF receptor super family 25 (TNFRSF25) and CD25, the high affinity IL-2 receptor. Following a 6-day sequence of Tl1A-Ig and IL-2, we observed markedly greater peripheral Treg expansion (5-8 fold) vs. either reagent alone (2-3 fold). The resulting Treg expansion in healthy animals is transient, peaking by 7-8 days, and results in no long-term complications, as assessed by changes in blood counts, immune phenotype / function and tissue pathology 6 months following cessation of treatment. Analyses of animals 1 week following sequential therapy revealed Treg expansion in blood, spleen, lymph nodes, and the colon but not bone marrow. Expanded Tregs were highly activated (Ki67+) with an elevated KLRG1+ fraction. High functional activity was also reflected in their hypomethylated FoxP3 promoter region, elevated pSTAT5 expression and the ability of sorted Tregs to mediate functional suppression of lymphocyte proliferation. Notably, assessment of the splenic vs. LN Treg compartment indicated that there were consistently higher Treg frequencies an d greater levels of Treg effector proteins (Gzmb/TNFa/IFNg/IL-10) in the splenic Treg compartment. To assess the functional significance of this difference, we stimulated splenocytes and lymph node cells from TL1A-Ig/IL-2 treated animals with anti-CD3 mAb and LPS, and observed significantly greater suppression in the spleen vs LN cultures. Importantly, even when cultures of LN cells contained the same or greater numbers of Tregs compared with the spleen, greater suppression was detected in the splenic cultures. Based on the heightened suppressive environment in spleens post-Treg expansion, we performed HSCT across a complete MHC mismatch using the B6àBALB/c donor/recipient combination. Groups of 8.5 Gy TBI-conditioned mice received T cell depleted B6 bone marrow and either spleen or LN cells from TL1A-Ig/IL-2 treated or untreated B6 mice. While GVHD was reduced in recipients of either spleen or lymph node T cells, relative to recipients of control (non-expanded) donors, suppression of GVHD was significantly greater in recipients of spleen cells (by weight loss, clinical score and survival). In summary, we have identified a novel strategy to rapidly and transiently expand donor Treg cell numbers in situ. The splenic compartment contained increased numbers and more functionally active Tregs compared to lymph nodes. Overall, TL1A-Ig/IL-2 expanded Tregs demonstrated excellent suppressive function both ex vivo and in vivo, as assessed by GVHD incidence and severity following allogeneic transplantation. This approach may be a promising strategy for GVHD prevention or therapy in the clinical setting. [Display omitted] DisclosuresPodack:Heat Biologics: Equity Ownership, Patents & Royalties: Dr. Podack is an inventor of patents used in the study and stands to gain royalties from future commercialization of "immunomodulating tumor necrosis factor receptor 25 (TNFR25) agonists, antagonists and immunotoxins, Research Funding. Levy:Allergan: Consultancy.

A Specific Immune Tolerance Toward Maternal Cells Is Found in the Donor Lymphocyte Infusion

PurposeTo observe the immunological responses of recipients to infused lymphocytes from donors of different genetic backgrounds.MethodsMouse models were set up by mating female BALB/c mice with male C57BL mice. Splenic lymphocytes from donors of different genetic backgrounds were labeled with CFSE, and 1∙107 of the cells were intravenously injected into a recipient. At 6, 24, 72 and 120 h after the infusion, mononuclear cells in recipient spleen, liver, thymus, lymph node and peripheral blood were collected, and CFSE+, CFSE-, CD3+, CD8+, CD4+, CD19+, NK1.1+, CD25+, CD127+ lymphocytes in these samples were analyzed by flow cytometry. The distribution of donor T cells, B cells, NK cells, helper T cells, cytotoxic T cells, and recipient regulatory T cells in these tissues were then obtained.ResultsMaternal lymphocytes were more likely to survive in offspring. At 120 h after the infusion, the ratio of maternal cells was 0.52¡À0.11% in lymph node, 0.97¡À0.04% in peripheral blood, and 0.97¡À0.11% in spleen of the offspring. In aunt to child, father to child, and unrelated allogeneic groups at 120 h after the infusion, few donor cells, if any, were detected in these tissues. The subtype proportion of donor lymphocytes changed significantly in recipient tissues. Recipient Treg cells increased in mother to child group but not in aunt to child, father to child, and unrelated allogeneic groups, suggesting the decrease of cellular immune responses to allogeneic cells in mother to child group. At 120 h after the infusion, no donor cells were detected in recipient liver and thymus in all groups, implying that donor cells colonized hardly in liver and thymus.ConclusionSpecific immune tolerance to maternal lymphocytes existed in offspring. Donor lymphocyte infusion of maternal origin may obtain a relatively persistent effect of adoptive immunotherapy with less side-effect. [Display omitted] Black is male C57BL£¨H-2b£©mouse; white are female BALB/c mice (H-2d); the gray is offspring produced by mating C57Bland BALB/c. The lymphocyte infusion relationship between donor and recipient: ¢Ùmother to child group; ¢Úfather to child group; ¢Ûaunt to child group; ¢Üunrelated allogeneic group; ¢Ýsyngeneic group. [Display omitted] M1 are CFSE-positive cells; M2 are the total cells. A. spleen£¨0.97¡À0.11%£©; B. liver£¨0.20¡À0.06%£©; C. thymus£¨0.01¡À0.01%£©; D. lymph nodes£¨0.52¡À0.11%£©; E. peripheral blood£¨0.97¡À0.04%£© [Display omitted] Proportion of Treg cells in lymph node, peripheral blood and spleen. Treg cells increased significantly in lymph node, peripheral blood and spleen in mother to child group, as compared with those in aunt to child group, unrelated allogeneic group and control group. DisclosuresNo relevant conflicts of interest to declare.

VLA-4/VCAM-1 Interactions Are Required for Endogenous Memory CD8 T Cell Mediated Cardiac Allograft Rejection.

We recently reported that endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts and are activated to produce IFN-γ in response to donor class I MHC molecules within 24 hours after reperfusion in mice and reject allografts subjected to prolonged ischemic storage in the absence of de novo priming of donor-reactive effector T cells. The current study tested the efficacy of anti-VLA-4 mAb to inhibit this early CD8 T cell allograft infiltration and activation during cardiac allograft rejection. Syngeneic or A/J (H-2a) hearts subjected to 0.5 or 8 hrs of cold ischemic storage were transplanted to C57BL/6 (H-2b) recipients. Anti-VLA-4 mAb (200 μg) was given on days -1 and 0. CD4 and CD8 T cell, macrophage, and neutrophil infiltration into grafts was assessed on day 2 post-transplant by flow cytometry and immunohistochemistry. Levels of mRNA encoding inflammatory cytokines were measured by qPCR. Donor-reactive T cell priming to IFN-γ and IL-2 producing cells in recipient spleens was assessed by ELISPOT assay. Recipient treatment with anti-VLA-4 mAb decreased CD4 and CD8 T cell and macrophage, but not neutrophil, infiltration into allografts by 70% of that observed into allografts from control IgG-treated recipients on day 2 post-transplant and completely inhibited donor-reactive CD4 and CD8 T cell priming to IFN-γ and IL-2 producing cells until day 14. Anti-VLA-4 mAb significantly reduced intragraft mRNA levels of CXCL9, CXCL10, and IFN-γ, but not the acute phase cytokines, TNF-α, IL-1β and IL-6, on day 2 post-transplant. These effects led to substantial prolongation in allograft survival (MST: day 12.5 vs day 7.5 in control-Ig treated recipients, p < 0.01). In recipients given allografts subjected to prolonged cold ischemic storage, anti-VLA-4 mAb treatment inhibited endogenous memory CD8 T cell infiltration into allografts and the inflammatory response and significantly prolonged allograft survival. These data indicate that peri-transplant anti-VLA-4 mAb inhibits endogenous memory CD8 T-cells infiltration into allografts subjected to prolonged cold ischemic storage and the accompanying inflammation. Therapeutic targeting of this pathway will reduce the negative impact of early memory CD8 T cell-dependent inflammatory events on graft outcome that will be particularly important for recipients with high frequencies of donor-reactive memory CD8 T cells.

Absence of Allograft Interleukin (IL)-1 Receptor Signaling Attenuates Early Graft Inflammation and Donor-Reactive CD8 T Cell Priming.

Endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts and produce IFN-γ in response to donor class I MHC within 24 hrs after graft reperfusion in mice. This study tested the role of graft IL-1 receptor signaling in the early post-transplant inflammation and downstream alloimmune response to the allograft. Wild type (WT) C57BL/6 (H-2b) or B6.IL1R-/-hearts were transplanted to A/J (H-2a) mice. CD4 and CD8 T cell, macrophage, and neutrophil infiltration into allografts on day 2 post-transplant was assessed by flow cytometry and immunohistochemistry. Levels of mRNA encoding inflammatory cytokines were measured by qPCR. Donor-reactive T cell priming to IFN-γ producing cells in recipient spleens was assessed by ELISPOT assay. Marked decreases in CD8 T cell and neutrophil, but not CD4 T cell and macrophage, infiltration were observed in B6.IL-1R-/- vs. WT B6 allografts on day 2 post-transplant. The absence of allograft IL-1R signaling also resulted in significant decreases in intragraft mRNA expression of CXCL9, CXCL10, IFN-γ, FasL, granzyme B, perforin and the acute phase cytokines, TNF-α, IL-1β and IL-6. Whereas donor-reactive CD4 T cell priming to IFN-g producing cells in response to B6.IL-1R-/- and WT B6 allografts was similar, donor-reactive CD8 T cell priming was low-absent in response to B6.IL-1R-/- allografts until day 11. These effects resulted in modest but significant prolongation of B6.IL-1R-/- allograft survival (MST: day 11.5 vs day 7.5 for WT B6 allografts, p < 0.05). A/J mice depleted of CD8 T cells rejected WT B6 and B6.IL-1R-/- allografts by day 15. A/J mice depleted of CD4 T cells rejected WT B6 cardiac allografts by day 25, whereas CD4 depletion promoted long term survival of the B6.IL-1R-/- allografts. These data indicate that the absence of IL-1R signaling in allografts inhibits early endogenous memory CD8 T cell infiltration into the allograft and the accompanying inflammation. The results suggest that therapeutic targeting of this pathway will reduce the negative impact of early memory CD8 T cell-dependent inflammatory events that will be particularly important for recipients with high frequencies of donor-reactive memory CD8 T cells.

Generation of Suppressive Blood Cells for Induction of Donor- Specific Tolerance in a Rat Heart Transplant Model: A Clinically Relevant Model

Purpose: Our previous studies showed that the chemotherapeutic drug mitomycin c (MMC) renders strongly stimulatory dendritic cells (DCs) suppressive. Donor-derived MMC DCs induced specific tolerance in rat heart allograft recipients. Since in clinical transplantation (Tx) peripheral blood mononuclear cells (PBMCs) are easier available than DCs we tried to replace DCs by PBMCs. Materials and methods: Donor blood (1ml) or PBMCs (108) were incubated for 30 min. with MMC, washed and injected i.v. into recipients 1 week before allogeneic heart Tx (DA to PVG). Blood and spleen cells (SPCs) of tolerant recipients were analyzed by FACS for regulatory cells (Tregs) and adoptively transferred into syngeneic animals transplanted with DA grafts. Grafts were immunohistochemically analyzed for cellular infiltration, vascular lumen narrowing and complement (C4d) deposition. The cytokine profile of recipient blood was determined by multiplex immunoassay. Results: MMC-treated donor blood injected into recipients prolonged allograft survival (34.43 ± 3.95 vs. 8.56 ± 0.27 in untreated and 21 ± 4.16 days in donor-blood transfused recipients). A stronger effect up to tolerance (50% of the animals) was obtained when donor blood was replaced with MMC- PBMCs (64.8 ± 16.8 vs. 8.56 ± 0.27 in untreated and 34.43 ± 3.95 days in PBMC treated recipients). The effect was abrogated by elimination of monocytes from PBMCs. Third-party heart allograft survival showed no prolongation indicating donor-specific tolerance. Tolerated grafts had cellular infiltrates with a significantly increased number of Foxp3+ cells and decreased deposition of CD4d in blood vessels in comparison to rejected grafts. FACS analysis of PBMCs and SPCs of tolerant animals revealed an increased percentage of CD4+CD25+Foxp3+ Tregs when compared to rejecting animals (PBMCs: 6.12 + 0.99 vs. 5.52 + 0.28%, p > 0.05; SPCs: 8.31 + 1.11 vs. 6.74 + 0.13%, p = 0.02). Both, PBMCs and SPCs of tolerant animals prolonged allograft survival up to tolerance by adoptive transfer into syngeneic recipients. Cytokine profile analysis suggests a Th2 deviation early after transplantation. Non-significant reduction of vascular lumen in allografts (as measure for chronic rejection) of tolerant animals in comparison to syngeneic ones was observed. Conclusions: A single pretransplant infusion of MMC-PBMCs is able to induce donor-specific suppression up to tolerance in a heart allotransplant model without concomitant use of immunosuppressants. The cell subpopulation which induces suppression is mainly monocytes. Suppression might be mediated by CD4+CD25+Foxp3+ Tregs, since these cells were found in increased number in blood, spleen and grafts of tolerant animals, and tolerance can be adoptively transferred by PBMCs and SPCs. MMC-PBMCs reduce but do not fully prevent chronic rejection. The described model has clinical relevance.

The impact of Trichinella spiralis excretory–secretory products on dendritic cells

Parasitic nematode Trichinella spiralis exert immunomodulatory effect on the host immune response through excretory-secretory products (ES L1) released from the encysted muscle larvae. Rat bone-marrow derived dendritic cells (DCs) stimulated with ES L1 antigens acquire semi-matured status and induce Th2 and regulatory responses in vitro and in vivo. Priming naïve T cells in vitro with ES L1 pulsed DCs caused strong Th2 polarization, accompanied by elevated production of regulatory cytokines IL-10 and TGF-β and no increase in the proportion of CD4+CD25+Foxp3+ among the effector T cell population. In vivo T cell priming resulted in mixed Th1/Th2 cytokine response, with the dominance of the Th2 type and elevated levels of regulatory cytokines. Significant increase in the proportion of CD4+CD25+Foxp3+ cells was found among recipient's spleen cells. We have achieved to create immune status characteristic for the live infection by in vivo application of DCs educated with ES L1 antigens.

Roles of CD28, CTLA4, and Inducible Costimulator in Acute Graft-versus-Host Disease in Mice

T cells deficient for CD28 have reduced ability to expand and survive, but still cause graft-versus-host disease (GVHD). Inducible costimulator (ICOS), a member of the CD28 family, is expressed on antigen-activated T cells and plays unique roles in T cell activation and effector function. We hypothesized that ICOS contributes to the development of GVHD in the absence of B7:CD28/CTLA4 costimulation. In this study, we evaluated the roles of CD28, CTLA4, and ICOS in the pathogenesis of acute GVHD after myeloablative allogeneic bone marrow transplantation. Unexpectedly, we found that blocking CD28 and CTLA4 signals using the clinically relevant reagent CTLA4-Ig increases the severity of GVHD mediated by CD4(+) T cells, and that such treatment does not add any benefit to the blockade of ICOS. In contrast, selectively blocking CD28 and ICOS, but not CTLA4, prevents GVHD more effectively than blocking either CD28 or ICOS alone. Taken together, these results indicate that CD28 and ICOS are synergistic in promoting GVHD, whereas the CTLA4 signal is required for T cell tolerance regardless of ICOS signaling. Thus, blocking CD28 and ICOS while sparing CTLA4 represents a promising approach for abrogating pathogenic T cell responses after allogeneic bone marrow transplantation.

Interleukin-17 Promotes Early Allograft Inflammation

Acute cellular rejection of organ transplants is executed by donor-reactive T cells, which are dominated by interferon-gamma-producing cells. As interferon-gamma is dispensable for graft destruction, we evaluated the contribution of interleukin-17A (IL-17) to intragraft inflammation in major histocompatibility complex-mismatched heart transplants. A/J (H-2(a)) cardiac allografts placed into wild-type BALB/c (H-2(d)) mice induced intragraft IL-17 production on day 2 after transplant. Allografts placed into BALB/c IL-17(-/-) recipients demonstrated diminished production of the chemokines CXCL1 and CXCL2 and delayed neutrophil and T cell recruitment. However, by day 7 after transplant, allografts from IL-17(-/-) and wild-type recipients had comparable levels of cellular infiltration. The priming of donor-specific T cells was not affected by the absence of IL-17, and the kinetics of cardiac allograft rejection were similar in wild-type and IL-17(-/-) recipients. In contrast, IL-17(-/-) mice depleted of CD8 T cells rejected A/J allografts in a delayed fashion compared with CD8-depleted wild-type recipients. Although donor-reactive CD4 T cells were efficiently activated in both groups, the infiltration of effector T cells into allografts was impaired in IL-17(-/-) recipients. Our data indicate that locally produced IL-17 amplifies intragraft inflammation early after transplantation and promotes tissue injury by facilitating T cell recruitment into the graft. Targeting the IL-17 signaling network in conjunction with other graft-prolonging therapies may decrease this injury and improve the survival of transplanted organs.