Graft Mesenteric Lymph Nodes Research Articles

Recombinant HLA-G as Tolerogenic Immunomodulant in Experimental Small Bowel Transplantation.

The non-classical MHC I paralogue HLA-G is expressed by cytotrophoblast cells and implicated with fetomaternal tolerance by downregulating the maternal adaptive and innate immune response against the fetus. HLA-G expression correlates with favorable graft outcome in humans and recently promising immunosuppressive effects of therapeutic HLA-G in experimental transplantation (skin allograft acceptance) were shown. Consequently, we examined this novel therapeutic approach in solid organ transplantation. In this study, therapeutic recombinant HLA-G5 was evaluated for the first time in a solid organ model of acute rejection (ACR) after orthotopic intestinal transplantation (ITX). Allogenic ITX was performed in rats (Brown Norway to Lewis) with and without HLA-G treatment. It was found that HLA-G treatment significantly reduced histologically proven ACR at both an early and late postoperative timepoint (POD 4/7), concomitant to a functionally preserved graft contractility at POD 7. Interestingly, graft infiltration by myeloperoxidase+ cells was significantly reduced at POD7 by HLA-G treatment. Moreover, HLA-G treatment showed an effect on the allogenic T-cell immune response as assessed by flow cytometry: The influx of recipient-derived CD8+ T-cells into the graft mesenteric lymphnodes at POD7 was significantly reduced while CD4+ populations were not affected. As a potential mechanism of action, an induction of T-reg populations in the mesenteric lymphnodes was postulated, but flow cytometric analysis of classical CD4+/CD25+/FoxP3+Treg-cells showed no significant alteration by HLA-G treatment. The novel therapeutic approach using recombinant HLA-G5 reported herein demonstrates a significant immunosuppressive effect in this model of allogenic experimental intestinal transplantation. This effect may be mediated via inhibition of recipient-derived CD8+ T-cell populations either directly or by induction of non-classical Treg populations.

Tolerance and Long-Lasting Peripheral Chimerism After Allogeneic Intestinal Transplantation in MGH Miniature Swine

Clinical intestinal transplantation (Int-Tx) is limited by high rates of rejection, infection, and graft versus host disease. To improve clinical outcomes and eliminate the comorbidities associated with chronic immunosuppression, the induction of donor-specific tolerance to intestinal grafts is desirable, especially in the pediatric population. This study determined the ability of intestinal grafts to facilitate tolerance induction in major histocompatibility complex (MHC)-inbred miniature swine. Seven MGH-miniature swine received heterotopic intestinal grafts, two across MHC-matched, minor-antigen disparities, three across a class I MHC disparity with 12 days of cyclosporine A, and two across a class I MHC disparity without an immunosuppressant. Chimerism was assessed by FACS analysis and immunohistochemistry. Cell-mediated lympholysis assays were used to assess antidonor responses. Two animals receiving intestinal grafts without an immunosuppressant developed antidonor IgG in 14 days and rejected these completely. All other grafts were accepted with 12 days of cyclosporine A across both MHC-matched and MHC class I barriers. Cell-mediated lympholysis assays showed donor-specific unresponsiveness by day 30 across MHC class I barriers. Greater than 15% peripheral donor cell chimerism persisted for more than 60 days after MHC-matched Int-Tx. Although less than 1.5% peripheral donor cell chimerism was seen during the maintenance period after class I-mismatched Int-Tx, 5% to 10% myeloid chimerism was found in the peripheral blood 14 to 90 days after Int-Tx. FACS analysis demonstrated that 1% to 2% of lymphocytes in the graft mesenteric lymph nodes were CD4/CD25(HIGH+)/Foxp3(+) cells. To our knowledge, this is the first demonstration of tolerance induction and persistence of chimerism in a large animal intestinal transplant model.

Protection of Mouse Small Bowel Allografts by FTY720 and Costimulation Blockade

The clinical application of small bowel transplantation (SBTx) is hampered by its pronounced immunogenicity. We aimed to test the hypothesis that prolonged sequestration of lymphocytes in secondary lymphoid organs may enhance the alloprotective effect of costimulation blockade. For this purpose, recipients of intestinal allografts were treated with MR1, FTY720, combined FTY720 plus MR1, or were left untreated. Grafts were examined 6 and 14 days after transplantation by applying a histologic rejection score, multiparameter-immunofluorescent staining, and flow cytometry. FTY720 or MR1 monotherapy did not prevent the rejection of mouse intestinal allografts, whereas combined therapy with FTY720 plus MR1 profoundly inhibited rejection at day 6 and day 14 after transplantation. In FTY720-treated mice infiltration of host lymphocytes in graft mesenteric lymph nodes, Peyer's patches, intraepithelial lymphocytes, and lamina propria lymphocytes (LPLs) was reduced on day 6. Anti-CD40L antibody improved the rejection score at day 14 but had no effect at day 6. Importantly, host CD8 T-cell infiltration in graft LPLs was significantly reduced compared with all other groups. FTY720 plus MR1 effectively inhibited intestinal allograft rejection in mice, possibly by enhancing the alloprotective effects of costimulation blockade by prolonged sequestration of lymphocytes in secondary lymphoid organs.

Control of intestinal allograft rejection by FTY720 and costimulation blockade

Introduction The clinical application of small bowel transplantation (SBTx) is hampered byits pronounced immunogenicity. In this study we examined the effects of the novelimmunosuppressant FTY720 and costimulation blockade by an anti-CD40L mAb (MR-1) ina stringent mouse model of SBTx. Methods SBTx was performed in mice with a full MHC mismatch (donors: C3H=H-2 k;recipients: C57BL/6=H-2 b). Recipients were divided into four groups: 1, untreated group; 2,MR1 monotherapy (500 μg IV on days 0, 2, 4, and 7); 3, FTY720 monotherapy (1 mg/kg bodyweight PO for 14 consecutive days after transplantation); 4, FTY720 plus MR1–treated group.Graft rejection grades were assessed by H&E staining. Graft mesenteric lymph nodes (MLNs),Peyer's patches (PPs), as well as intraepithelial lymphocytes (IELs) and lamina proprialymphocytes (LPLs) were analyzed by flow cytometry and three-color immunofluorescencestaining. Results Neither FTY720 nor MR1 monotherapy was efficient in preventing the rejection ofmouse intestinal allografts, whereas FTY720 plus MR1 profoundly inhibited the rejectionresponse at the 14th posttransplant day. The infiltration of host lymphocytes was reduced ingraft MLNs, PPs, IELs, and LPLs by FTY720 therapy. FTY720 plus MR1 inhibited hostCD8 + T-cell infiltration in graft LPLs when compared with grafts treated with FTY720 only.Additionally, two subpopulations, CD11b +high Gr1 − and CD11b +intermediate Gr1 + cells, weredecreased in FTY720-treated grafts. Conclusions FTY720 plus MR1 efficiently delayed intestinal allograft rejection in a mousemodel by preventing the infiltration of host lymphocytes, particularly of CD8 + cells.

CONTROL OF INTESTINAL ALLOGRAFT REJECTION BY FTY720 AND COSTIMULATION BLOCKADE

P1038 Aims: The clinical application of small bowel transplantation (SBTx) is hampered by its pronounced immunogenicity. To date no immunosuppressive protocol has proven to prevent intestinal allograft rejection in mouse models. Therefore, in this study we examined the effects of the novel immunosuppressant FTY-720 and costimulation blockade by an anti-CD40L mAb (MR-1) in a heterotopic model of small bowel transplantation. Methods: SBTx was performed in mice with a full MHC haplotype mismatch (donors: C3H=H-2k; recipients: C57BL/6=H-2b). Recipients were divided into 4 groups: 1, non-treated group; 2, MR1 monotherapy (500 μg iv on day 0, and 500 μg i.p. on days 2, 4 and 7); 3, FTY720 monotherapy (1 mg/kg body weight po for 14 consecutive days after transplantation); 4, FTY720 plus MR1 treated group. Each group contained 6 mice for histological assessment at day 14, and another 6 mice for FACS analysis and histology at day 6 after transplantation. Analyses were performed on donor and recipient mesenteric lymph nodes, Peyer’s Patches, as well as Intraepithelial Lymphocytes (IEL) and Lamina Propria Lymphocytes (LPL) that were purified by differential collagenase digestion. Tissue sections were analyzed by three colour immunofluorescence staining on an Axiovert M200 microscope. Lymphocytes were stained with monoclonal antibodies, and were studied by three-colour flow cytometry on a FACSCalibur (Becton Dickinson). Results: Both FTY720 and MR1 monotherapy were not efficient in alleviating the rejection of mouse intestinal allografts, whereas FTY720 plus MR1 profoundly improved the rejection response both at early (day 6) and later (day 14) time points. In the latter, the infiltration of host lymphocytes was profoundly reduced in graft mesenteric lymph nodes (MLNs), Peyer’s patches (PPs), intraepithelial lymphocytes (IELs), and lamina propria lymphocytes (LPLs) by FTY720 therapy. FTY720 plus MR1 inhibited host CD8+ T cell infiltration in graft LPLs when compared with grafts treated with FTY720 only. Infiltration by CD44+ T cells was inhibited in most of the FTY720 plus MR1 treated grafts (4/6). Additionally, two sub-populations, CD11b+high Gr1+ and CD11b+high Gr1+ cells were decreased in FTY720 treated grafts. Conclusions: FTY720 plus MR1 efficiently delayed intestinal allograft rejection in a mouse model by preventing the infiltration of host lymphocytes, particularly of CD8+ cells.

Immune responses of graft mesenteric lymph node in small bowel transplantation

Background The high proportions of lymphoid tissues are thought to be one of the underlying factors inducing severe allograft rejection following small bowel transplantation. Mesenteric lymph nodes (MLN) contained in the intestinal graft are not only a source of donor-derived professional antigen-presenting cells, but also offer a field for immune interaction between donor and host cells. We investigated immune responses in graft MLNs with or without FK506 to develop a novel strategy to control small bowel allograft rejection. Materials and methods Heterotopic small bowel transplantations were performed from Brown Norway donors to Lewis recipients. Changes in population of lymphocytes, expressions of costimulatory molecules, apoptosis, and cytokine profiles in graft MLNs were evaluated. Results The increase in apoptotic cells and cytokine responses relating to rejection in the graft MLNs developed prior to those in graft jejunum. While donor lymphocytes in graft MLNs were rapidly replaced to host-derived lymphocytes independent of FK treatment, increase in CD8 + T cells in host population was seen only in recipients without FK506 treatment. The expressions of B7 molecules on donor cells in graft MLNs were significantly lower in the recipients with FK treatment. Conclusions Immune responses in graft MLNs have significant impact on the outcome of the small bowel allograft. Apoptosis of graft MLN cells was well correlated with and ahead of progression of acute rejection. Modulation of costimulatory molecules on donor-derived MLN cells in the allograft and specific suppression of host CD8 + T cells are possible ways to control severe rejection after allogeneic small bowel transplantation.

Long-lasting donor passenger leukocytes after hepatic and intestinal transplantation in rats

Background: Donor passenger leukocytes (DPLs) that migrate after organ transplantation stimulate the recipient immune system and normally cause rejection and graft vs. host reaction. However, DPLs also contribute to the unresponsiveness to the donor organ. The quantity and quality of these migrating cells are considered dependent on individual transplanted organs. We compared the DPLs of the liver, which might contain somatic stem cells, with those of intestinal grafts that have highly immunogenetic cells. To study DPLs over a long period, we used green fluorescent protein (GFP) transgenic (Tg) rats developed by us as donors. Methods: We performed orthotopic liver transplantation (OLT) and small bowel transplantation (SBT) from GFP Tg rats to wild recipients. A short course of tacrolimus (0.64 mg/kg, intramuscularly) was used to prevent antigenicity of the GFP. The fate of the DPLs in the peripheral blood and the recipient bone marrow was monitored by flow cytometry. Using long-surviving recipients, the GFP + cells in the graft and various host immunologic organs were measured and characterized by immunohistochemical staining. Results: In both groups, the numbers of the GFP + cells in the peripheral blood increased transiently and then gradually decreased to undetectable levels. While no GFP + cells were identified in the long-surviving-recipient bone marrow, there were a few GFP + cells in the graft liver, graft mesenteric lymph nodes and the recipient spleen. These cells showed major histocompatibility complex (MHC) class II antigen. There was no significant difference in the migration patterns of the GFP + cells in the OLT and SBT rats. Conclusions: In both the OLT and SBT groups, the DPLs migrated transiently in the recipient peripheral blood. A small numbers of MHC class II-positive DPLs were present at the graft site and in the host spleen, but not in the bone marrow. There were no significant differences in the migration patterns of the DPLs between the OLT and SBT rats over the long term.

Flow Cytometric Analysis of Graft-and Host-Specific Cell Migration after Allogeneic Small Bowel Transplantation

Background: After small bowel transplantation (SBT), a two-way traffic of graft and host lymphocytes is observed. We compared the composition and distribution of mobile cells under immuno-suppression (FK 506 monotherapy and FK 506 therapy in combi-nation with anti-ICAM-1 monoclonal antibody) with the untreated situation after transplantation and during rejection. Materials and Methods: Heterotopic SBT was performed in the fully allogeneic BN (RT1_n) to LEW (RT1₁) rat model. Monoclonal antibodiesspecific for leukocyte subsets and strain-specific MHC I antigens were used to analyze cell migration with flow cytometry. Results: After transplantation, predominantly graft CD4+ cells were detected in the peripheral blood, spleen, and mesenteric lymph nodes (MLN) of the host. The persistence of these cells was dependent on whether animals were immunosuppressed ornot. After replacement of graft lymphocytes in the graft MLN, the majority of host lymphocytes were also CD4+ cells. In the untreatedgroup a greater number of these cells expressed the IL-2 receptor. Under immunosuppression the expression of IL-2 receptor and the proportion of natural killer cells significantly decreased at the onset of rejection. Conclusions: In the drug-treated as well as in the untreated groups simultaneous exchange migration between graft and host leukocytes was observed. At the onset of rejection, distinct differences between drug-treated and untreated groups were found in graft MLN. Yet no characteristic changes in cell subsets could be found, correlating with the onset of rejection.

Identification of migratory graft and host cell populations after allogeneic rat small bowel transplantation.

This study used flow cytometry to identify graft cells in the recipient peripheral blood and spleen and host cells infiltrating the graft mesenteric lymph node and Peyer's patches after heterotopic rat small bowel transplantation. Transplantation had little effect on the overall cell subset composition of these compartments and no changes appeared characteristic or indicative of developing rejection, suggesting that physiological control of cell migration remained unaltered. A small and transient population of graft cells was detected in the peripheral blood and spleen of the recipient which disappeared after 5 and 3 days respectively. Graft-derived cells in the peripheral blood comprised predominantly CD4+ cells on day 1 with B cells predominating on day 5. Graft cells infiltrating the spleen were predominantly B cells. Host cells infiltrated the graft mesenteric lymph nodes and Peyer's patches to a lesser extent than previously reported using immunohistochemical analysis. For both tissues, infiltrating host-derived cells initially comprised mainly CD4+ cells. On day 4 approximately equal proportions of CD4+ and B cells were present in the mesenteric lymph node, whereas B cells were predominant in the host cell infiltrate of the graft Peyer's patches. In summary, these findings indicate that the cell subset composition of recipient and graft lymphoid compartments does not change after small bowel transplantation, even in the presence of a substantial recipient cell infiltration. The reasons for the apparent discrepancies in the degree of host cell infiltration when assessed using immunohistochemical and flow cytometric techniques are currently uncertain, but may result from the localised release of soluble MHC class I in graft tissues as a consequence of infiltrating host cell activation or localised cell destruction.