Recipient Spleen Research Articles

Rapamycin Combined with Immature Dendritic Cells Attenuates Obliterative Bronchiolitis in Trachea Allograft Rats by Regulating the Balance of Regulatory and Effector T Cells

Background: Obliterative bronchiolitis (OB) ranks as the major obstacle for long-term survival of lung transplantation patients. Rapamycin (Rapa) has recently been confirmed as an immunosuppressant for antirejection due to its suppressive role in T cell activation. Here, we explore the effect of Rapa combined with immature dendritic cells (imDCs) on OB in trachea allograft rats. Methods: The effect of bone marrow-derived imDCs or Rapa-imDCs on lymphocyte cells and CD4⁺ T cells were evaluated by methyl thiazolyl tetrazolium and flow cytometry. Tracheal transplantation was performed from Lewis rats to Wistar recipients. Recipient rats received Rapa+imDCs for 10 consecutive days after implantation. Allograft rejection was assessed by micro-CT image, hematoxylin/eosinHE staining and flow cytometry. The underlying mechanism was also investigated. Results: Rapa-imDCs inhibited lymphocyte and CD4⁺ T cell growth. Furthermore, Rapa-imDC treatment induced T cell hyporesponsiveness by attenuating T cell differentiation into IFN-γ-producing T cells (Th1), but increased CD4⁺CD25⁺Foxp3⁺ T cell (Treg) contents. Importantly, Rapa-imDC administration ameliorated airway obliteration symptoms and CD4⁺ and CD8⁺ T cell infiltration. Furthermore, the proinflammatory factor levels of IL-6, TNF-α, IFN-γ and IL-17 were decreased, concomitant with the upregulation of immunosuppressive cytokines IL-10 and TGF-β1. Further analysis confirmed that Rapa-imDC treatment attenuated the amounts of infiltrated IL-17⁺CD4⁺ T cells (Th17 cells) and Th1 cells, but increased Treg contents in the spleens of recipients. Conclusions: This research may corroborate a protective role of Rapa-imDCs in OB by regulating the balance between effector T cells and Tregs, suggesting a potential applicable strategy to treat OB after lung transplantation.

MyD88-silenced dendritic cells induce T-cell hyporesponsiveness and promote Th2 polarization in vivo

Background aimsPrevious studies have determined that the absence of MyD88 enhances the tolerogenicity of dendritic cells (DCs), suggesting that inhibiting innate immunity may be a potential strategy to facilitate the induction of transplant tolerance by DCs. However, the underlying mechanism remains unclear. MethodsRecipient rats were preconditioned with MyD88 gene–silenced DCs. In vivo distribution of infused MyD88 gene–silenced DCs in lymphatic organs was also analyzed. The response ability of recipient spleen T cells was determined by cell proliferation assay. The concentrations of interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-5 and IL-10 in cell culture supernates were measured with sandwich enzyme-linked immunosorbent assay. Flow cytometry was used to detect the transfection efficiency and CD4+CD25+FoxP3+ T cell assay. ResultsAfter being infused into allogenic recipient rats, both MyD88-or control-silenced DCs were efficiently trafficked to the lymphatic organs and liver. The ex vivo analysis of proliferative responses revealed the donor-specific inhibition of alloimmune reactivity by MyD88-silenced DCs. This effect was associated with the marked inhibition of Th1-type cytokine production (IFN-γ and IL-2) but with significant promotion of Th2 type cytokine secretion (IL-4 and IL-5). ConclusionsIt was demonstrated that T cells from recipients pretreated with MyD88-silenced DCs exhibited significantly reduced secretion of IFN-γ and IL-2 but markedly enhanced production of IL-4 and IL-5.

Abstract 2945: Nilotinib abrogates DNMT1-depenent DNA methylation: A novel mechanism for induction of AML leukemia regression

Abstract Nilotinib has been approved to treat newly diagnosed, imatinib-resistant or -intolerant leukemia, but the mechanisms underlying its antileukemia actions remain elusive. Here, we report that nilotinib blocks AML growth through its DNA hypomethylating activity. We showed that, in addition to inducing a reduction of KIT and FLT3 protein expression and phosphorylation, nilotinib downregulated DNMT1, 3a and 3b in Kasumi-1, MV4-11 and C1498 cells. Since DNMT1 is maintenance and de novo DNMT, we focused on DNMT1 and proved that nilotinib impaired DNMT1 expression via Sp1 dysfunction, since the luciferase activity driven by DNMT1 promoter containing Sp1 binding sites was decreased by 2.6 fold by nilotinib, while increased by 10.6 fold after Sp1 overexpression. Electrophoretic mobility shift assay disclosed that nilotinib abolished Sp1 binding on DNMT1 promoter. Dotblot using 5mC antibody revealed global DNA hypomethylation and qPCR showed p15INK4B upregulation in Kasumi-1 (3.4 fold) or MV4-11 (5.2 fold). Bisulfite sequencing found a decrease of DNA methylation on p15INK4B promoter from 100% to 89% in Kasumi-1 or 13% to 0% in MV4-11. The functional assays showed a decrease of colony formation in Kasumi-1 (60%) or MV4-11 (58%), and an increase of cell apoptosis in Kasumi-1 (6.9 fold) or MV4-11 (3.2 fold). We verified these findings in AML patient blasts (n = 3) by showing that nilotinib treatment led to the dephosphorylation of KIT, the downregulation of DNMT1, 3a, 3b and Sp1, the reduction of global DNA methylation and the re-expression of p15INK4B (4.4 fold) via promoter demethylation (from 14% to 6%), which was followed by suppressed colony formation (77%), promoted cell apoptosis (1.84 fold) and increased caspase-3 and caspase-8. To test if nilotinib induces AML regression via DNA hypomethylation in vivo, 0.1×106 C1498 cells were injected into C57BL/6 mice (n = 3 mice/group) via the tail vein. The leukemic mice were treated with 5, 10 or 15 mg/kg of nilotinib in PEG400 plus saline via intraperitoneal injection twice a week for a total of 6 doses. No toxicity was observed for the tested drug dosage and schedule, because of no evident change of mouse body weight. Compared to the control group, nilotinib-treated mice had a decrease in spleen size. H&E staining showed that the control mice exhibited increased infiltration of AML cells into the spleen, lung and liver of recipients. The bone marrow (BM) histopathology identified a higher degree of mature post-mitotic cells containing metamyelocytes, bands and segmented neutrophils in nilotinib-treated mice than control. DNMT1 was downregulated (39%), p15INK4B was upregulated and global DNA methylation was reduced (60%) in BM cells from nilotinib-treated mice. Thus, our findings unveil DNA methylation machinery as an innovative target behind nilotinib's anticancer activities, and provide a new therapeutic avenue for nilotinib in overcoming aberrant epigenetics in AML. Note: This abstract was not presented at the meeting. Citation Format: Na Shen, Fei Yan, Jiuxia Pang, Naseema Gangat, Mark R. Litzow, Aref Al-Kali, Shujun Liu. Nilotinib abrogates DNMT1-depenent DNA methylation: A novel mechanism for induction of AML leukemia regression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2945. doi:10.1158/1538-7445.AM2015-2945

Rat model of heterotopic toe allotransplantation

BackgroundFinger allotransplantation is a promising treatment for severe finger destruction. However, more research is required to decrease the risks of this procedure to a level at which the clinical use of this non–life-saving procedure is justified. A proper animal model is essential for the required experiments. MethodsIn this article, we established a toe transplantation model based on anatomic studies. A tapered dose of Cyclosporine A (CsA) was used as an immunosuppressive therapy in the Brown Norway-to-Lewis allotransplantation experimental group, whereas isotransplantation or allotransplantation without treatment or with ligated pedicles was performed on the control groups. Recipients were assessed daily after operation for any signs of rejection and complications. On postoperative day 90, skin graft test was used to test the level of donor-specific tolerance in the recipients. On postoperative day 120, x-rays and micro-computed tomographies were performed for bone morphology evaluation. The chimerism in the recipient peripheral blood, lymph node, spleen, and thymus was tested by flow cytometry and immunohistochemical staining. And histologic study of the toe grafts and skin grafts were carried out. ResultsThe blood supply of the toe graft was confirmed, and accordingly, transplantations were performed. The isografts survived indefinitely. The allografts with ligated pedicles experienced necrosis within 5 d. The allografts without treatment exhibited necrosis within 14 d. Forty percent, 20%, and 40% of the allografts associated with the CsA treatment experienced severe rejection, mild rejection, and nonrejection, according to gross graft appearance, respectively. Skin grafting tests showed three different types of results. X-rays and micro-computed tomographies reveal nearly normal bone morphologies in the bone structures of all surviving animals with grafts. Low levels of donor-specific chimerism were detected in the peripheral blood samples. Spleen, lymph node, and thymus chimerism were also confirmed in the long-term surviving animals with allografts. Histologic evaluation of the long-term survivals revealed similar graft morphologies in the isografts and the nonrejected allografts, inflammatory cell infiltration in the mildly rejected allografts, and degraded cutaneous appendages in the severely rejected allografts. ConclusionsWe established a toe allotransplantation model. Moreover, the low rate of chimerism did not introduce specific tolerance, which might explain the high rejection rate. This model might facilitate future research in finger allotransplantation.

Sphingosine-1-phosphate receptor 1 agonist SEW2871 prolongs heterotopic heart allograft survival in mice.

Sphingosine-1-phosphate (S1P) is a biologically active metabolite of plasma-membrane sphingolipids that is essential for immune cell trafficking. Recent studies have revealed immunomodulatory functions of S1P and its receptors (S1PR1-S1PR5) in many inflammatory conditions, such as asthma and autoimmunity. Here, we explore the efficacy of SEW2871, a selective S1PR1 agonist, in the prevention of acute allograft rejection in a murine cardiac transplantation model. Treatment of recipient mice with SEW2871 significantly prolongs cardiac allograft survival as compared to those recipients treated with control vehicle. The enhanced graft survival is associated with reduced circulating lymphocytes and allograft inflammatory cell infiltration. The cytokine analysis showed decreased allograft expression of TNF-α, IFN-γ and IL-2 in the SEW2871-treated mice. Moreover, administration of SEW2871 increases the percentage of CD4(+) T regulatory cells and FoxP3 expression in spleen of allograft recipients. Therefore, SEW2871 plays a critical role in regulation of lymphocyte trafficking and development, which directly contributes to prolongation of the allograft survival.

Flow cytometric analysis of the graft-versus-Leukemia-effect after hematopoietic stem cell transplantation in mice.

Acute Graft-versus-Host-Disease (aGvHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Although rather helpful, the use of conventional immunosuppressive drugs leads to general immunosuppression and is toxic. The effects of CD4(+) T-cells, in respect to the development of aGvHD, can be altered by administration of antihuman CD4 monoclonal antibodies, here MAX.16H5 IgG1 . This approach must be tested for possible interference with the Graft-versus-Leukemia-Effect (GvL). Thus, in vitro experiments were conducted, exposing P815 leukemic cells to bone marrow and splenocytes from cd4(-/-) -C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice, [TTG]) as well as previously irradiated splenocytes from Balb/c(wt) mice. Using flow cytometry, the vitality of the various malignant and graft cells was analyzed over the course of 4 days. The survival rate of P815 cells did not change significantly when exposed to MAX.16H5 IgG1 , neither did the viability of the graft cells. This provides evidence that MAX.16H5 IgG1 does not impair the GvL effect in vitro. Additionally, P815-Balb/c(wt) leukemic mice were transplanted with P815(GFP) cells, bone marrow, and splenocytes from TTG mice with and without MAX.16H5 IgG1 . Without transplantation, P815(GFP) leukemic cells could be detected by flow cytometry in the liver, the bone marrow, and the spleen of recipients. The antibodies prevented aGvHD while leaving the GvL effect intact. These findings indicate no negative effect of MAX.16H5 IgG1 on the GvL effect in vitro and in vivo after HSCT in a murine model.

Identification of CD34’ and CD34− leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia

AbstractTranslocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34’CD38’CD19’ and CD34−CD19’ cells initiated leukemia, and in MLL-AF9 patients, CD34−CD19’ cells were LICs. In MLL-ENL patients, either CD34’ or CD34− cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34’CD38−CD19−CD33− cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34’CD38−CD19−CD33− cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4’ single positive (SP), CD8’ SP, and CD4’CD8’ double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34’CD38’ and CD34− LICs but not in CD34’CD38−CD19−CD33− HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia.

Permanent acceptance of mouse cardiac allografts with CD40 siRNA to induce regulatory myeloid cells by use of a novel polysaccharide siRNA delivery system.

The CD40/CD154 co-stimulatory pathway is crucial in alloimmune response. We developed a novel small interfering RNA (siRNA) delivery system with a poly-dA extension at the 5'-end of the siRNA sense strand that was stably incorporated into 1,3-β-glucan (schizophyllan, SPG). This was captured and incorporated into dendritic cells (DCs) through its receptor, Dectin-1, specifically silencing CD40 genes (siCD40) to exert immunoregulatory activity. siCD40/SPG-treated CBA mice permanently accepted B10 fully mismatched cardiac allografts. Consistent with graft survival, the infiltration of CD4(+), CD8(+) T cells into the graft was lower, and that the numbers of CD40(low)CD11c(+) DCs cells and CD4(+)Foxp3(+)cells were increased in both the graft and in the recipient spleen. In addition, naive CBA recipients given an adoptive transfer of splenocytes from the primary recipients with siCD40/SPG accepted a heart graft from donor-type B10, but not third-party Balb/c mice. In conclusion, the treatment with siCD40/SPG targeting DCs could generate antigen-specific Tregs, resulting in the permanent acceptance of mouse cardiac allografts. These findings have important implications for clarifying the mechanism underlying the induction of tolerance in DCs, and also highlight the potential of immunomodulation and the feasibility of siRNA-based clinical therapy in the transplantation field.

Upregulation of Immune Checkpoint Blockade Molecules Post Allogeneic Stem Cell Transplantation Is Necessary but Insufficient to Prevent GvHD

Donor T-lymphocytes are effective adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT), but life threatening complications related to GVHD limits its clinical application. Recent advancement in the field of immunotherapy has directed our interest to enhancing the anti-tumor response of donor T cells by modulating expression of checkpoint blockade molecules including programmed death-1 (PD-1), cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and foxp3, the transcription factor associated with regulatory T cells. The two ligands of PD-1, PD-L1 or PD-L2 are highly expressed in the presence of inflammatory signal induced by infection or cancer and PD-1/PD-L1 interaction negatively regulates T-cell antigen receptor (TCR) signaling and dampen T cell cytotoxic activity. Herein, we studied the role of PD-1, CTLA-4 and transcription factor foxp3 expressing donor CD4+ and CD8+ T cells in the development of GVHD.Methods: We have used two established allo-HSCT murine GvHD models. Lethally irradiated wild type (WT) B6, PD-L1 knock out (KO) B6 and PD-L2 KO B6 mice were transplanted with 2 x 106 splenic T cells and 2 x 106 T cell depleted bone marrow (TCD BM) cells from H-2Kdonors. Lethally irradiated CB6F1 recipients were similarly transplanted with splenocytes and TCD BM cells from B6 donors. Acute GvHD scores were determined by combining scores obtained from histological tissue sections and weight-loss, posture, activity, fur texture and skin integrity following standard published procedures. The activation status of donor T-cells and BM and host-derived non-T cells in GvHD target organs was analyzed by flow cytometry. Data from allo-HSCT recipients were compared with the respective data obtained from B6 à B6 syngenic HSCT (syn-HSCT) recipients. Serum cytokines were determined by Luminex assay.Results: PD-L1 KO B6 allo-HSCT recipients had significantly increased acute GvHD scores compared with WT B6 allo-HSCT recipients (p<0.0005) and B6 PD-L2 KO allo-HSCT recipients (p<0.0005) measured on day 8 after transplant. All PD-L1 KO allo-HSCT recipients died within 10 days post transplant while WT B6 and PD-L2 KO allo-HSCT recipients had 20% mortality until 36 days post transplant. Increased acute GvHD was associated with increased amount of serum inflammatory cytokines and increased numbers of activated PD-1+CD69+CD4+ donor T cells. Interestingly, PD-1 expression on donor CD4+ T cells significantly increased in the spleen of transplant recipients but not in BM, while PD-1 expression was significantly increased on donor CD8+ T cells in both spleen and BM compartments of allo-HSCT recipients compared with the syn-HSCT recipients. CTLA-4 expression on CD4+ and CD8+ donor T cells were significantly increased in spleen in the first two weeks post transplant but decreased at later time points compared with syn-HSCT. Again, CTLA-4 expression on CD4+ donor T cells in the BM remained significantly higher measured on 100+ days post transplant in allo-HSCT recipients compared with the syn-HSCT but similar levels of CTLA-4 expression on CD8+ T cells were measured in BM between these two HSCT recipients. Foxp3 expression on donor T cells and the numbers of CD4+CD25+foxp3+ regulatory T (Tregs) were markedly suppressed in donor T cells on day 4 post HSCT of allo-HSCT recipients compared with the syn-HSCT recipients. Although total numbers of donor T cells in the spleen of allo-HSCT recipients remained low over time, the percentage of PD-L1-expressing donor T cells in spleen were significantly higher (p<0.005) at early time points (day 4) in allo-HSCT recipients compared with the syn-HSCT. While total numbers of host-derived cells in spleen decreased over time in mice that developed GvHD, host-derived PD-L1 expressing CD3+ T cells persisted at higher levels through day 36 post transplant. Additionally, PD-L1 expression was also increased in donor BM-derived T cells and non-T cells populations over time.Collectively, these data indicate that severe GvHD occurs in allo-HSCT recipients in spite of increased numbers of PD-1, CTLA-4 and PD-L1 expressing donor and host cells. The occurrence of severe GvHD in these allo-HSCT models systems was associated with markedly reduced levels of CTLA-4 and foxp3 transcription factor expressing Tregs indicating that these pathways may be more relevant to controlling GvHD than PD-1:PD-L1 expression. DisclosuresNo relevant conflicts of interest to declare.

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.

Oral Administration of Ibrutinib Is Ineffective at Preventing Scleroderma in Chronic Gvhd in Two Preclinical Mouse Models

We have established two mouse models of chronic graft versus host disease (cGVHD): one is an MHC-matched DBA/2 donor to BALB/c recipient, and this model amplifies the role of CD4+ T and B cells in transplants; the other model is an MHC-mismatched C57BL/6 donor to BALB/c recipient, and this model amplifies the role of de novo-developed CD4+ T and B cells. BALB/c recipients in both models started to develop scleroderma ~35 days after transplantation of donor spleen and bone marrow cells and this peaked ~50 days after transplantation. BALB/c recipients given DBA/2 transplants also developed proteinuria due to IgG anti-dsDNA autoantibody deposition in the glomeruli. We have reported that donor CD4+ T and B cells play critical roles in the pathogenesis of chronic GVHD (J. Immunol 2012 and 2013). Administration by I.V. injection of depleting anti-CD20 prevented induction of chronic GVHD in both models (Biol. Blood and Marrow Transplant 2014). In the current studies, using these two models, we tested whether oral administration of Ibrutinib could prevent induction of cGVHD. Ibrutinib was gavaged at 12.5 mg/kg/day from day 0 to day 30 or given in drinking water at 35 mg/kg/day (0.16 mg/ml) from day 0 to day 50. We found that administration of Ibrutinib significantly delayed the onset of proteinuria in the model of DBA/2 donor to BALB/c recipient, but accelerated the onset and increased the severity of scleroderma in 20/20 recipients given DBA/2 transplants and 9/9 recipients given C57BL/6 transplants as measured by a clinical score system. Administration of Ibrutinib significantly reduced CD138+B220lo plasma cells but not CD138-B220+ B cells in the spleen and peripheral lymph nodes of both recipients. Administration of Ibrutinib slightly reduced CD4+ T cell numbers in the spleen but significantly increased CD4+ T cell numbers in the peripheral lymph nodes, although there was no significant impact on CD4+ T cell expression of IFN-g and TNF-α in both recipients. These results indicate that, although oral administration of Ibrutinib is able to reduce B cell activation and differentiation into plasma cells, it is ineffective at inhibiting the CD4+ T cell expansion that mediates scleroderma in the models of DBA/2 donor to MHC-matched BALB/c recipient and C57BL/6 donor to MHC-mismatched BALB/c recipient. Our result is in contrast to a previous report showing that oral administration of Ibrutinib prevented induction of chronic GVHD with bronchiolitis obliterans in a mouse model and reversed scleroderma in another mouse model (Poster 2591, AACR 2014, San Diego). Further discussion about the differences is warranted. (This work was supported by Nesvig Lymphoma Foundation). DisclosuresNo relevant conflicts of interest to declare.

On discovering thymus-marrow synergism.

In the 1960s, the thymus was an organ of mystery. Although it was full of lymphocytes, they made no antibodies. Furthermore, thymectomy (in mature animals) failed to produce immunological inadequacy. This situation changed when J.F.A.P. Miller did neonatal thymectomy, which was indeed followed by a syndrome including crippling of the immune response (1–3). The possible role of the thymus was the focus of a several-day symposium organized by Robert A. Good in November, 1962 in Minneapolis. Its proceedings, The Thymus in Immunobiology, summarized the clinical and experimental data (4). This mystery organ intrigued me. Systemically, immunized animals did not make antibody in the thymus as they did in lymph nodes or spleen. Maybe, we thought, there was some kind of blood–thymus barrier, which prevented systemic antigen from interacting with thymocytes in the thymic parenchyma. We used adoptive transfer of syngeneic cells into irradiated recipients. In this model, spleen cell suspensions responded to sheep erythrocyte (sheep red blood cells, SRBC) antigens by making hemolytic antibody in the recipient spleens and serum. Would thymus cell suspensions similarly prepared (so as to break any blood–thymus barrier) do the same? I was a young investigator working in the late David W. Talmage’s lab at the University of Colorado Medical School in Denver. It was a stimulating environment. Edward A. Chaperon and R. Faser Triplett were post-doctoral fellows. On day 0, the mice were irradiated and then injected i.v. with spleen cells or thymus cells. On day 1, we injected the SRBC antigen IV. On day 5, we sacrificed the mice and looked for anti-SRBC-producing cells in the recipient spleens. The results were clear-cut. Recipients of donor spleen cells made many antibody-producing cells while recipients of donor thymus cells did not. Perhaps, we thought, 4-days of exposure to antigen after transfer might have been sufficient to get the mature spleen cells to make antibody but insufficient for the (putatively) immature thymocytes to do the same. We needed to lengthen the protocol. The next experiments were identical on days 0 and 1, but on day-4 recipients got a booster injection of SRBC antigen, and we planned to sacrifice on day 8. This worked well in the group that received spleen cells but the recipients of thymus cells (the test group) were all dead by day 8. We figured that this represented radiation death in the thymus recipients, whereas the spleen recipients survived because of the hematopoietic stem cells in the inoculum. We knew of the radio-protective effects of bone marrow cells, so it made sense to add an aliquot of such cells to the thymus inoculum. Indeed, the recipients of thymus-plus-bone marrow survived until day 8, but to our surprise, these recipients produced almost as much antibody as did spleen recipients. (Later we added a new group as another control, i.e., bone marrow cells only. They caused no significant antibody production.) We called this phenomenon “thymus–marrow synergism,” and it was the first demonstration that two (presumably lymphoid) cell populations were needed for significant antibody production. We speculated that one sort of cell (the “effector”) made the antibody while another variety of cell from the other inoculum performed in an “auxiliary” mode. On the basis of indirect evidence, we postulated that the bone marrow provided the effector cells and the thymus cells were “auxiliary.” Support for this view had to await the definitive experiments by others. Our experiments were published in 1966 in Proc. Soc. Exptl. Biol. Med. (5). The paper was widely acknowledged to have demonstrated cell–cell interaction in the antibody response. Additional findings by the three of us were considered important enough many years later when they were chosen as the first article to be mentioned in the Journal of Immunology’s new historical series, Pillars of Immunology. This discovery was unexpected – almost representing serendipity. Not everyone was convinced. However, others used the paradigm to provide further elucidation of the mechanism of thymus–marrow synergism. Mitchell and Miller made great progress by identifying the antibody-forming cell as originating in the bone marrow (6). Additionally, Avrion Mitchison added the brilliant insight that the carrier effect was an example of T–B collaboration where the anti-hapten antibody was made by bone marrow-derived cells while the thymus-derived cells provided “help” (7–9).

5-Aminolevulinic acid with ferrous iron induces permanent cardiac allograft acceptance in mice via induction of regulatory cells

5-Aminolevulinic acid (5-ALA), a precursor of heme biosynthesis, plays a fundamentally important role in aerobic energy metabolism. Heme oxygenase (HO)-1 cleaves heme to form biliverdin, carbon monoxide (CO) and iron (Fe(2+)). The anti-inflammatory properties of biliverdin and CO help to alleviate ischemia/reperfusion injury as well as acute and/or chronic allograft rejection. We investigated whether 5-ALA and Fe(2+) exerts salutary effects in the setting of organ transplantation. An in vitro mixed-lymphocyte reaction (MLR) assay and cardiac allotransplantation model (CBA to C57BL/10) were used to evaluate the effects of 5-ALA and Fe(2+) on transplantation tolerance. Treatment with 5-ALA and sodium ferrous citrate (SFC) resulted in permanent acceptance in the murine cardiac allografts in a dose-, SFC- and HO-1-dependent manner. The number of graft-infiltrating CD8 T cells was lower and the survival response of recipient spleen T cells to donor-type alloantigens was less compared with control recipients; however, numbers of both regulatory T cells and dendritic cells were significantly increased in 5-ALA/SFC-treated recipients. Our findings show that 5-ALA/SFC inhibits T-cell proliferation in response to alloantigens and an increased number of regulatory cells, resulting in permanent cardiac allograft acceptance in mice. These findings highlight the major roles of CO and/or HO-1 in inducing tolerance and suggest that 5-ALA/SFC may be a clinically effective treatment for allograft rejection.

Adipocyte IL-15 regulates local and systemic NK cell development.

NK cell development and homeostasis require IL-15 produced by both hematopoietic and parenchymal cells. Certain hematopoietic IL-15 sources, such as macrophages and dendritic cells, are known, whereas the source of parenchymal IL-15 remains elusive. Using two types of adipocyte-specific Il15(-/-) mice, we identified adipocytes as a parenchymal IL-15 source that supported NK cell development nonredundantly. Both adipocyte-specific Il15(-/-) mice showed reduced IL-15 production specifically in the adipose tissue but impaired NK cell development in the spleen and liver in addition to the adipose tissue. We also found that the adipose tissue harbored NK progenitors as other niches (e.g. spleen) for NK cell development, and that NK cells derived from transplanted adipose tissue populated the recipient's spleen and liver. These findings suggest that adipocyte IL-15 contributes to systemic NK cell development by supporting NK cell development in the adipose tissue, which serves as a source of NK cells for other organs.

HMGB1 Is Involved in Chronic Rejection of Cardiac Allograft via Promoting Inflammatory-Like mDCs

Chronic rejection that leads to diffuse narrowing and occlusion of graft vessels is the most important cause of morbidity and mortality following cardiac transplantation. The role and underlying mechanism of high-mobility group box 1 (HMGB1), as an established inflammatory mediator in acute rejection, remains poorly understood in chronic rejection. Here, we assessed the effects and mechanisms of HMGB1 on the chronic rejection using single MHC Class II-mismatched mouse cardiac transplantation model. It was found that HMGB1 was increased accompanying with the development of chronic rejection, while blockade of HMGB1 with specific neutralizing mAb substantially ameliorated chronic rejection-mediated vasculopathy and fibrosis of allograft, as well as markedly decreased T cell infiltration and production of IL-17A and interferon-gamma in allograft and recipient's spleen. Further, anti-HMGB1 antibody treatment significantly declined the number and frequency of mature dendritic cells (DCs) in allograft and recipient's spleen, especially CD11b(+) Ly6C(high) matured DCs that share the phenotypes with inflammatory-DCs. These findings indicate that HMGB1 contributes to chronic rejection, and HMGB1 blockade may be a novel mean to disrupt the proinflammatory loop after heart transplantation.

Role of Graft-Derived CCL2/MCP-1 in Antibody-Mediated Rejection of Cardiac Allografts.

The role of monocytes/macrophages in antibody-mediated rejection (AMR) remains unclear. We have reported that murine CCR5-/-/CD8-/- recipients produce high titers of donor-specific antibody and reject MHC-mismatched heart allografts. The current studies tested the magnitude of injury and alloimmune responses to grafts deficient in expression of the monocyte chemoattractant MCP-1/CCL2 during AMR. B6.CCR5-/-/CD8-/- recipients rejected MHC-mismatched wild-type A/J allografts with high donor-reactive antibody titers and diffuse C4d deposition in the large vessels and myocardial capillaries, features consistent with AMR. Donor-specific CD4 T cells producing IFN-γ in the recipient spleen were significantly decreased in response to A/J.CCL2-/- vs. A/J allografts and this resulted in decreased donor-reactive antibody production and C4d deposition in allografts in B6. CCR5-/-/CD8-/- recipients at day 7 post-transplant. Consequently, A/J.CCL2-/- allograft survival was slightly but significantly longer than that of A/J allografts (MST: wild-type A/J, day 8 vs. A/J.CCL2-/-, day 10, P < 0.01). Allograft CCL2 deficiency reduced classically activated (M1) macrophage and other inflammatory leukocyte infiltration into the allografts. Strikingly, A/J.CCL2-/- allograft survival was much longer than wild-type A/J allografts in recipients treated with 0.25 mg anti-CD40L mAb at the time of transplant (MST: wild-type A/J, day 21.5 vs. A/J.CCL2-/-, day 58, P < 0.01) with lower titers of donor-reactive antibody and decreased leukocyte infiltration. CCL2 deficiency also reduced pro-fibrotic gene expression and delayed the development of interstitial fibrosis when compared to wild-type A/J allografts. The results indicate that allograft-derived CCL2/MCP-1 plays an important role in directing M1 macrophages and other inflammatory cell infiltration into allografts and in the development of donor-reactive antibody and vascular rejection and suggest CCL2/MCP-1 as a therapeutic target for antibody-mediated vascular rejection and chronic graft injury.

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.

Sensitization to MHC Antigen Is Characterized By Heightened Early Proinflammatory Cytokine Release Rather Than Lymphocyte Proliferation: Implications for Sensitization After Allograft Rejection.

Purpose: This study aims to evaluate lymphocyte proliferation and cytokine production in response to mismatched MHC antigen in a mouse model of sensitization. Methods: Two consecutive skin grafts at 14-day interval were performed on C57BL/6 (H2kb) recipients using allogeneic (H2kd) or syngeneic (H2kb) donors. 14 days after the second graft, recipient spleens were harvested for mixed lymphocyte culture (MLC) with T-cell-depleted H2kd splenocytes, for 6h, 24h, 72h and 5 days. At each time-point, intracytoplasmic cytokine expression in recipient splenocytes and their proliferation by CFSE dilution were assessed using flow cytometry. Cytokine release was measured in the supernatant using Luminex. Results: Allogeneic, but not syngeneic, recipients became sensitized, as evidenced by antibody binding to H2kd splenocytes. Following MLC, proliferation did not significantly differ between sensitized (S) and non-sensitized (NS) groups over 5 days (Figure 1, p>0.05). Although percentages of CD4+IL-6+ (S:1.14%, NS:1.66%; p=0.13), CD4+IFN-γ+ (S:5.29%, NS:6.41%; p=0.51) and CD4+IL-4+ (S:1.63%, NS:2.65%; p=0.13) cells were not different at 24h, sensitized cells released higher concentrations of IL-6, IL-4 and IFN-γ than the non-sensitized (Figure 2). Sensitized cells also released IL-17A sharply at 72h, unlike the non-sensitized, even though the percentages of CD4+IL-17A+ cells were not significantly different at 72h (S:3.90%, NS:3.32%; p=0.28). Conclusion: These results suggest that sensitization to MHC antigen is not marked by a difference in lymphocyte proliferation or their expression of pro-inflammatory cytokines, but by the individual cell release of pro-inflammatory cytokines, particularly those involved in the IFN-γ and IL-6/IL-17 pathways.Figure: No Caption available.Figure: No Caption available.DISCLOSURES:Sahakian, S.: Grant/Research Support, Astellas Pharma Canada, Inc. Tchervenkov, J.: Grant/Research Support, Astellas Pharma Canada Inc.

Trafficking of Intra-Graft Donor T Cells After Skin Transplantation in Mice.

It is firmly established that the skin contains T lymphocytes including significant numbers of FoxP3+ regulatory T cells (Tregs). However, their role in skin transplantation is still unknown. Recent evidence has been provided by W. Burlingham's laboratory suggesting that intra-graft T cells may influence the alloresponse and rejection process (bi-directional alloimmunity). In this study, we investigated the trafficking of intra-graft donor T cells to the recipient's lymphoid organs after skin transplantation in mice. To test this C57BL/6 (B6) mice received a skin transplant from a double transgenic B6 donor whose T cells expressed DSred while FoxP3 expression was associated with GFP (Tregs being double positive). At different time points after transplantation, cell suspensions of recipients' lymph nodes, spleen and thymus were prepared. The presence and characterization of fluorescent donor cells was investigated using Image Stream (Amnis) technology. First, the presence of T cells (DSred+ FoxP3-) was detected only at d4-d7 posttransplantation i.e. after skin graft revascularization and essentially in the host's thymus. On the other hand, double positive (DSred+ FoxP3+) Tregs were found in all recipient's lymphoid organs. Surprisingly, high frequencies of single positive GFP+ cells were found in both ipsilateral lymph nodes and thymus. The nature of these cells is being investigated. In summary, our results show that most T cells leaving the graft are Tregs spreading through all recipient's lymphoid organs while intra-graft effector T cells migrate primarily to the host's thymus.