MHC-mismatched Mouse Research Articles

L. amazonensis infection differentially affects migration of phagocyte populations from inflammatory site to the draining lymph node (102.27)

Abstract Leishmania infection interferes with integrin function. In this work we use a model of chronic peritonitis to study the effect of L. amazonensis infection upon the ability of phagocytes populations to migrate from an inflammatory site to the draining lymph node (dLN). First we showed that macrophage recruitment into the peritoneum and migration to the dLN were both maximal 4 days after thioglycollate injection into the peritoneum. At this time point, cell tracking assays using cell transfer between MHC-mismatched mice showed that 1% of the leukocytes injected into the peritoneum migrated to the dLN after 24h. In the mice injected with cells cultivated with medium alone adoptive cells represented 14% of the peritoneal cells. Among these leukocytes 57% were macrophages, 2% were neutrophils and 35% were myeloid dendritic cells. In the LN, 22% of the migrating cells were macrophages, 4% were neutrophils and 25% were myeloid dendritic cells. In the mice injected with leukocytes co-cultivated with Leishmania adoptive cells represented 12% of the peritoneal cells. Among these cells 44% were macrophages, 1% were neutrophils and 46% were dendritic cells. In the LN 19% of the migrating cells were macrophages, 2% were neutrophils and 12% were myeloid dendritic cells. Our data show that: (1) A variety of phagocytes are able to migrate from the inflammatory site to the dLN. (2) Populations of Leishmania-infected dendritic cells appear to be retained in the peritoneum.

Adoptive transfer of DNT cells induces long-term cardiac allograft survival and augments recipient CD4+Foxp3+ Treg cell accumulation

Regulatory T (Treg) cells play an important role in the regulation of immune responses but whether Treg will induce tolerance in transplant recipients in the clinic remains unknown. Our previous studies have shown that TCRαβ+CD3+CD4−CD8−NK1.1− (double negative, DN) T cells suppress T cell responses and prolong allograft survival in a single locus MHC-mismatched mouse model. In this study, we investigated the role of DNT cells in a more robust, fully MHC-mismatched BALB/c to C57BL/6 transplantation model, which may be more clinically relevant. Adoptive transfer of DNT cells in combination with short-term rapamycin treatment (days 1–9) induced long-term heart allograft survival (101±31 vs. 39±13days rapamycin alone, p<0.01). Furthermore adoptive transfer DNT cells augmented CD4+Foxp3+ Treg cells accumulation in transplant recipients while depletion of CD4+ Treg cells by anti-CD25 inhibited the effect of DNT cells on long-term graft survival (48±12days vs. 101±31days, p<0.001). In conclusion, DNT cells combined with short-term immunosuppression can prolong allograft survival, which may be through the accumulation of CD4+Foxp3+ Treg cells in the recipient. Our result suggests that allograft tolerance may require the co-existence of different type Treg cell phenotypes which are affected by current immunosuppression.

DN-Treg cells induce long-term cardiac allograft survival in fully MHC mismatched recipients but do not eliminate T memory cells (145.13)

Abstract Establishment of immune tolerance is a major goal in transplantation. Regulatory T (Treg) cells play an important role in the regulation of immune responses. Our previous studies have shown that TCRαβ+CD3+CD4-CD8-NK1.1- (double negative, DN) Treg cells suppress anti-donor T cell responses and prolong allograft survival in a single MHC-mismatched mouse model. In this study, we tested whether DN-Treg cells can induce long-term graft survival in the stringent BALB/c to C57BL/6 heart transplant model. Adoptive transfer of 107 DN-Treg cells in combination with rapamycin (RAPA) treatment (day 1- 9) induced long-term graft survival (101 vs. 39 days RAPA alone, p&amp;lt;0.01). DN-Treg treated recipients had few graft-infiltrating CD4+ and CD8+ T cells on day 40 compared to massive infiltration in RAPA treated controls. Memory T (Tm) cells (CD3+CD44+) were found to be resistant to DN-Treg cell-mediated suppression in vitro. The effect of DN-Treg on Tm in vivo was tested in B6-Rag1-/- mice. DN-Treg cells along with CD44+ Tm cells modestly prolonged BALB/c skin graft survival in contrast to DN-Treg with CD44- T cells (26 vs 43 days, P&amp;lt;0.001). In conclusion, DN-Treg cells combined with short term immunosuppression, can prolong allograft survival in the absence of tolerance. Furthermore, this may be related to the resistance of Tm cells to DN-Treg cell suppression, suggesting alternative strategies will be needed to control Tm cells and establish tolerance after transplantation.

CD11b− Donor Dendritic Cells Enhance Donor T-Cells' Th1 Polarization and Graft Versus Leukemia Activity in Allogeneic Hematopoietic Stem Cell Transplantation

Background: Based on a clinical association of donor plasmacytoid dendritic cell (DC) content with leukemia relapses after allogeneic BMT (Waller, Blood 2001), we have previously reported that CD11b− donor DC added to a graft containing FACS-purified hematopoietic stem cells (HSC) and T-cells enhanced interferon-γ (IFN-γ) production and GvL activity in MHC-mismatched allogeneic transplant mouse models (Li, Blood 2007).Objective: In this study, we studied the mechanisms whereby donor DC in the graft modulate donor T-cell activity and the graft-versus-leukemia (GvL) effect in MiHA (C3H.SW → C57BL/6J)- and MHC (C57BL/6J → B10.BR)- mismatched models of allogeneic hematopoietic stem cell transplantation (HSCT).Methods: Mice irradiated to 11 Gy received 5 × 104 log-phase viable MMB3.19 myeloid lymphoma cells via intraperitoneal injection or intravenous injection of 1 x 105 LBRM T-cell lymphoma cells one day before transplant. Allografts consisted of 5 × 104 FACS-purified donor BM CD11b− DC or CD11b+ DC plus 3 × 103 FACS-purified c-kit+ sca-1+ lineage− hematopoietic stem cells (HSC) in combination with either 3 × 105 T-cells, 3 × 105 CD8+ T-cells or no additional T-cells transplanted via tail vein. Graft-versus-host disease (GvHD) clinical scores (based on body weight loss, posture, skin, fur texture, activity) were recorded twice weekly in non-tumor bearing recipients. In vitro proliferation and cytotoxic activity of donor-derived T-cells against tumor targets was assessed by CFSE staining and a caspase flow cytometry assay (CyToxiLux PLUS) using donor T-cells harvested from recipients on day 34 and day 82 post transplant. Serum and intracellular Th1 cytokines (IFN-γ, IL-2, and TNF-α) and Th2 cytokines (IL-4, IL-5, and IL-10) from recipients' peripheral blood and spleens day 3 and day 10 post-transplant was measured by ELISA and flow cytometry. IFN-γ direct killing of leukemia cells was tested by in vitro IFN-γ exposure.Results: In non-tumor bearing mice, recipients of all combinations of donor DC subsets, with and without donor T-cells had equivalent survival (75% – 85%) at 3 months post transplant without significant clinical signs of GvHD. Transplantation of tumor cells to recipients of HSC alone, HSC plus donor T-cells, or HSC plus T-cells and CD11b+ DC in the MiHA- and the MHC-mismatched transplant models led to 0% or 5% 3 month survival, respectively. Strikingly, tumor-bearing mice transplanted with CD11b− DC had significantly enhanced 3 month survival (35% in the MiHA-mismatched model and 45% in the MHCmismatched model) without increased GvHD (p<0.001). There was no significant difference in survival between mice that received HSC plus CD11b− DC and a mixture of CD4+ and CD8+ donor T-cells versus mice that received HSC plus CD11b− DC and only CD8+ donor T-cells. Donor T-cells harvested from recipients of CD11b− DC 34 days after transplant in the MiHA-mismatched model as well as 82 days after transplant in the MHC-mismatched model displayed increased cell proliferation following co-culture with irradiated hosttype splenocytes as a source of alloantigen compared with donor T-cells harvested from recipients of CD11b+ DC or recipients of HSC plus T-cells without donor DC. Leukemia cell killing was greater following incubation of purified donor T-cells recovered from recipients of CD11b− DC with tumor targets compared to T-cells recovered from other treatment groups. Recipients of CD11b− DC had higher serum levels of Th1 cytokines IFN-γ and IL-2 and higher number of Th1 positive donor T-cells compared with recipients of other treatment groups. In contrast, recipients of CD11b+ DC had higher serum levels of Th2 cytokines IL-4, IL-5, and IL-10 and higher number of Th2 positive donor T-cells. IFN-γ added to in vitro cultures with MMB3.19, and LBRM, had no direct cell killing effect.Conclusion: CD11b− donor DC enhanced Th1 polarization of donor T-cells and GvL without increasing GvHD. Donor CD8+ T-cells mediated tumor killing effect. CD11b+ donor DC enhanced Th2 polarization of donor CD4+ T-cells and led to limited GvHD and GvL.

CD8+ but not CD4+ T cells require cognate interactions with target tissues to mediate GVHD across only minor H antigens, whereas both CD4+ and CD8+ T cells require direct leukemic contact to mediate GVL

AbstractWhether T-cell antigen receptors (TCR) on donor T cells require direct interactions with major histocompatibility complex class I or class II (MHCI/MHCII) molecules on target cells to mediate graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) is a fundamental question in allogeneic stem-cell transplantation (alloSCT). In MHC-mismatched mouse models, these contacts were not required for GVHD. However, this conclusion may not apply to MHC-matched, multiple minor histocompatibility antigen-mismatched alloSCT, the most common type performed clinically. To address this, we used wild-type (wt)→MHCI−/− or wt→MHCII−/− bone marrow chimeras as recipients in GVHD experiments. For GVL experiments, we used MHCI−/− or MHCII−/− chronic-phase CML cells created by expressing the BCR-ABL cDNA in bone marrow from MHCI−/− or MHCII−/− mice. TCR/MHCI contact was obligatory for both CD8-mediated GVHD and GVL. In contrast, CD4 cells induced GVHD in wt→MHCII−/− chimeras, whereas MHCII−/− mCP-CML was GVL-resistant. Donor CD4 cells infiltrated affected skin and bowel in wt→MHCII−/− recipients, indicating that they mediated GVHD by acting locally. Thus, CD4 cells use distinct effector mechanisms in GVHD and GVL: direct cytolytic action is required for GVL but not for GVHD. If these noncytolytic pathways can be inhibited, then GVHD might be ameliorated while preserving GVL.

Naive and Memory T Cells Induce Different Types of Graft-versus-Host Disease

The goal of this study was to compare the ability of donor naive and alloantigen-primed effector memory T cells to induce graft-vs-host disease after bone marrow transplantation in MHC-mismatched irradiated host mice. Purified CD4(+) naive (CD62L(high)CD44(low)) T cells and CD4(+) effector memory (CD62L(low)CD44(high)) T cells obtained from unprimed donors and donors primed to host alloantigens, respectively, were injected into host mice, and the rapidity, severity, and pattern of tissue injury of graft-vs-host disease was assessed. Unexpectedly, the naive T cells induced a more acute and severe colitis than the primed memory cells. Whereas the naive T cells expressing CD62L and CCR7 lymph node homing receptors vigorously expanded in mesenteric lymph nodes and colon by day 6 after transplantation, the primed memory T cells without these receptors had 20- to 100-fold lower accumulation at this early time point. These differences were reflected in the significantly more rapid decline in survival and weight loss induced by naive T cells. The primed memory T cells had a greater capacity to induce chronic colitis and liver injury and secrete IL-2 and IFN-gamma in response to alloantigenic stimulation compared with memory T cells from unprimed donors. Nevertheless, the expected increase in potency as compared with naive T cells was not observed due to differences in the pattern and kinetics of tissue injury.

Suppression of Ewing's Sarcoma Tumor Growth, Tumor Vessel Formation, and Vasculogenesis Following Anti–Vascular Endothelial Growth Factor Receptor-2 Therapy

We previously showed that bone marrow cells participate in new tumor vessel formation in Ewing's sarcoma, and that vascular endothelial growth factor 165 (VEGF(165)) is critical to this process. The purpose of this study was to determine whether blocking VEGF receptor 2 (VEGFR-2) with DC101 antibody suppresses tumor growth, reduces tumor vessel formation, and inhibits the migration of bone marrow cells into the tumor. An H-2 MHC-mismatched bone marrow transplant Ewing's sarcoma mouse model was used. Bone marrow cells from CB6F1 (MHC H-2(b/d)) mice were injected into irradiated BALB/cAnN mice (MHC H-2(d)). TC71 Ewing's sarcoma cells were s.c. injected 4 weeks after the bone marrow transplantation. Mice were then treated i.p. with DC101 antibody or immunoglobulin G (control) twice a week for 3 weeks starting 3 days after tumor cell injection. DC101 antibody therapy significantly reduced tumor growth and tumor mean vessel density (P < 0.05) and increased tumor cell apoptosis. Decreased bone marrow cell migration into the tumor was also shown after DC101 therapy as assessed by the colocalization of H-2K(b) and CD31 using immunohistochemistry. DC101 inhibited the migration of both human and mouse vessel endothelial cells in vitro. These results indicated that blocking VEGFR-2 with DC101 antibodies may be a useful therapeutic approach for treating patients with Ewing's sarcoma.

Peripheral deletion of mature alloreactive B cells induced by costimulation blockade

Alloreactive B cells can contribute to graft rejection. Anti-CD154 treatment together with donor-specific transfusion (DST) results in the long-term survival of MHC-mismatched mouse heart grafts and inhibition of alloantibody production. To characterize the mechanism of B cell tolerance induced by the anti-CD154 and DST, we used 3-83Igi mice, on BALB/c (H-2K(d)) background, that express a B cell receptor that reacts with MHC class I antigens H-2K(b). Transplanting C57BL/6 (H-2K(b)) hearts into 3-83Igi mice, followed by tolerance induction, resulted in the peripheral deletion of mature but not immature 3-83 B cells. The sustained deletion of mature alloreactive B cells required the presence of the allograft and can be explained by the absence of T cell help.

A limited course of soluble CD83 delays acute cellular rejection of MHC-mismatched mouse skin allografts

CD83 is a surface marker expressed on matured dendritic cells (DCs). It plays a pivotal role in the mediation of DC/T cell interaction and induction of T-cell activation. Previous studies have suggested that a soluble form of CD83 could suppress DC maturation and inhibit T-cell activation and, as a result, it can prevent paralysis associated with experimental autoimmune encephalomyelitis. Here, we explored its potential effect on allograft rejection in a fully major histocompatibility complex-mismatched murine skin transplantation model. A form of mouse soluble CD83 (CD83-Ig) fused the extracellular domain of murine CD83 with human IgG1alpha Fc tail was purified from transfected COS-7 cell. It was found that the treatment of recipient mice with CD83-Ig significantly delayed allograft rejection. Especially, when T cells originated from recipients treated with CD83-Ig re-stimulated with donor-specific splenocytes, they showed a significant reduced responding capability as compared with that of originated from control recipients. In line with these results, a reduction for serum IFN-gamma and IL-2 and a decreased mRNA expression of IFN-gamma and IL-2 in allograft infiltrated immune cells were also observed. Our results suggest that CD83-Ig could be useful for the treatment of allograft rejection in combination with other therapeutic strategies.

Therapeutic Potential of Ovarian Monocyte Progenitor Cells (MPCs) in SLE-Murine Model.

We have previously shown that the progeny of resident ovarian stromal progenitor cells is restricted to the production of cells of the monocyte-macrophage lineage. These ovarian monocyte progenitor cells (MPCs) formed hematopoietic colonies on methyl cellulose, and were CD45+, CD11b+, CD11c+, and Ly6-Gr-1. A distinct CD45low population within these cells reacted with CD117 (C-kit) surface marker, suggestive of a primitive hematopoietic progenitor. To determine the therapeutic potential of MPCs, we transplanted 50,000 cells into normal and autoimmune BXSB autoimmune mouse model for SLE. MPCs did not rescue lethally irradiated mice from the consequences of irradiation, and were not engrafted in MHC-mismatched mouse strains. However, murine SLE-like symptoms were improved, and substantial reduction in inflammatory infiltrate was observed in tissue sections from transplanted mice including kidneys, lungs, and spleen. Restructuring of renal glomerural sclerosis was also observed in the transplant recipients. To determine whether glomerular vascular regeneration was directly mediated by donor MPCs, we tested the hypothesis that MPCs co-express markers for endothelial progenitor cells and monocytes. After culture in conditions permissible to endothelial cell differentiation, large percentage of MPCs (73%) expressed endothelial cell marker CD106. When their culture supernatant was applied to a rat aortic ring in matrigel tube forming assay, robust tube formation was observed in the experimental group. However, similar tube formation was also observed in the control well containing 20% FBS, and cultured MPCs failed to produce tube formation in matrigel assay. Interestingly, phenotypic analysis showed that only 52% of MPCs express MHC class I suggesting that these cells may induce less rejection when transplanted in mismatched hosts. These data suggest that vascular tissue repair initiated by MPCs is mediated by anti-inflammatory factors, and not by direct contribution to the reconstruction of glomeular vasculature.

A Radiation-Free Immune and Epigenetics Based Conditioning Regimen for Allogeneic HCT: Combination of Anti-CD3 and Romidepsin.

Hematopoietic cell transplantation (HCT) is a curative therapy for hematological malignancies as well as refractory autoimmune diseases. However, graft versus host disease (GVHD) remains a major obstacle in classical HCT, where recipients are usually conditioned with total body irradiation or high dose chemotherapy. We recently reported that donor CD8+ T cells facilitated engraftment and mediated graft versus leukemia (GVL) without causing graft versus host disease (GVHD) in young (6–8 weeks old) MHC-mismatched mouse recipients conditioned with anti-CD3 mAb (Blood 2005). Thereafter, we observed that anti-CD3 conditioning alone was not sufficient for induction of chimerism in old (>12 weeks) recipients, due to the higher percentage of residual host CD8+ T cells in the old recipients. Romidepsin (Desipeptide), a histone deacetylase inhibitor, was reported to induce apoptosis of human T cell lymphoma lines. We hypothesize that depsipeptide will induce the apoptosis of anti-CD3 activated proliferating T cells, and that conditioning with a combination of anti-CD3 and depsipeptide will markedly reduce the residual host T cells and allow donor stem cell engraftment.To test our hypothesis, we first added Romidepsin (1.25–10 ng/ml) to cultures of T cells with or without anti-CD3 stimulation. We found that, although Romidepsin showed no effect on un-stimulated T cells, it augmented apoptosis of anti-CD3 activated T cells in a dose dependent manner, and the maximum augmentation was 5 fold. In addition, when Romidepsin (1.25–10 ng/ml) was added to a culture of mixed lymphocyte reaction (MLR), we found that it suppressed MLR in a dose dependent manner also, and the maximum suppression was greater than 98%. Second, old (> 12 weeks) BALB/c recipients were conditioned with one I.V. injection of anti-CD3 (20μg/g) and three I.P. injections (every other day) of Romidepsin at a dose of 0.4 μg/g. 7 days after anti-CD3 injection, recipients were injected with donor bone marrow cells (100×106) and CD4+- T depleted spleen (CD4−-SPL) cells (100×106). CD4−-SPL cells were injected again 7 days later. We found that, 4 weeks after HCT, 7/8 of the recipients conditioned with a combination of anti-CD3 and Romidepsin but only 1/8 of the recipients conditioned with anti-CD3 alone became chimeric. The recipients showed healthy appearance without signs of GVHD. The results are combined from two replicate experiments. This radiation free and GVHD preventive conditioning regimen may provide a novel approach for clinical HCT.

Evaluation of interferon-gamma producing-cells using enzyme linked immunospot assay in mice model of acute graft versus host disease

To investigate IFN-gamma producing-cells (IFN-gamma PCs) in allogeneic mixed lymphocyte reaction (MLR) and acute graft versus host disease (aGVHD) model of mice. Enzyme linked immunospot assay (ELISPOT) was applied to study IFN-gamma PCs in MHC mismatched mice spleen cell MLR and aGVHD model of mice. IFN-gamma PCs increased significantly in MLR after allogeneic mice spleen cell stimulation. In the experimental mice aGVHD model, IFN-gamma PCs were significantly higher in the severe aGVHD group than those in the moderate aGVHD. In the moderate aGVHD group, mice with GVHD prophylaxis regimen demonstrated significantly lower level of IFN-gamma PCs, compared with those without prophylaxis. IFN-gamma PCs were significantly correlated with the GVHD clinical scores in the group with moderate aGVHD and prophylaxis regimen. ELISPOT is a fast, sensitive and specific approach to evaluate alloresponse in allogeneic mice MLR and IFN-gamma PCs are correlated closely with the severity of aGVHD and prophylaxis regimen in the MHC-mismatched mice model.

Suppression of (5R)-5-hydroxytriptolide (LLDT-8) on Allograft Rejection in Full MHC-Mismatched Mouse Cardiac Transplantation

(5R)-5-hydroxytriptolide (LLDT-8) is a new compound derived from triptolide, which is the major immunosuppressive fraction of Tripterygium wilfordii Hook. F (TWHF). Studies in vitro and in vivo have demonstrated that LLDT-8 had potent immunosuppressive activities. Here we tested LLDT-8 in major histocompatibility complex (MHC)-mismatched cardiac transplantation and investigated the mechanisms underlying the prevention of transplant rejection. LLDT-8 was administered orally to recipients in Balb/c to C57BL/6 murine cardiac transplantation model. Allograft survival after transplantation was recorded in recipients. The T cell immunity and cytokine production were observed. Histological analysis was performed. The chemokine and its receptor were analyzed by reverse transcriptase-polymerase chain reaction on cardiac graft RNA. LLDT-8 administered orally significantly induced the survival prolongation of allogeneic cardiac graft. Histological results showed that LLDT-8 well preserved myocardium and significantly reduced infiltration of the graft with inflammatory cells. LLDT-8 decreased IL-2 production in recipient splenocytes stimulated by concanavalin A (ConA) ex vivo. LLDT-8 significantly inhibited the immunoreactivity of recipient to specific donor alloantigens, but preserved immunity to third-party alloantigens and mitogen. However, the flow cytometry analysis of the proportion of CD4+, CD8+ T cell subgroup in recipient spleens showed LLDT-8 had a normalizing effect on the splenic lymphocytes population. LLDT-8 decreased CC chemokine receptor 5 (CCR5) and their ligands macrophage inflammatory protein 1 alpha (MIP-1alpha) and beta (MIP-1beta) mRNA expressions in allografts. The results outline the great potential of LLDT-8 as a therapeutic tool in transplant rejection.

An Antagonist Mutant IL-15/Fc Promotes Transplant Tolerance

IL-15 is a proinflammatory and antiapoptotic T-cell growth factor that plays an important role in a variety of autoimmune disorders and transplant rejection. To inhibit IL-15 function and to target IL-15 receptor (IL-15R) bearing cells, we have generated a unique lytic antagonistic mutant IL-15/Fc fusion protein (mIL-15/Fc). In this study, we further examined the efficacy of mIL-15/Fc in preventing allograft rejection cross minor and major histocompatibility barriers. A short-course treatment with mIL-15/Fc fusion protein is sufficient to prevent cardiac allograft rejection and induce antigen-specific tolerance in minor histocompatibility complex-mismatched recipients, and permit prolonged cardiac allograft survival in fully MHC mismatched recipients. In addition, mIL-15/Fc treatment, in combination with a suboptimal dose of anti-CD154 antibody, confers permanent cardiac allograft engraftment in a fully MHC-mismatched mouse strain combination. In a murine islet allograft model, mIL-15/Fc monotherapy is capable to permit permanent allograft survival in 50% fully MHC-mismatched recipients. Immunochemistry studies demonstrated that prolonged graft survival was accompanied by reduced intragraft mononuclear cell infiltration and pro-inflammatory cytokine gene expression in the mIL-15/Fc treated recipients. Moreover, parallel experiments employing a mutated nonlytic IgG2a Fc demonstrate that the Fc portion of mIL-15/Fc contributes to the overall efficacy of the molecule in vivo.

The Effects of Mixed Chimerism Conducted by Natural Killer Cell Depletion with Non Myeloablation on Islet Allograft Rejection

Background: Because of the shortage of human pancreas and immunorejection, very small fraction of patients with type 1 diabetes can be treated with islet transplantation. The immune tolerance induction for overcoming the immume rejectin of trausplamted islets could be conducted by hematopoietic mixed chimerims with various invasive methods. The purpose of this study is to investigate the effect of mixed chimerims conducted by newly developed minimally invasive methods on islet allografts rejection in streptozotocin induced diabetic mice. Methods: Recipient, Balb/c (H-) mice were injected intraperitoneally with antiasialoGM1 antibody at one day before bone marrow transplantation. There were received total body irradiation at a dose of 500 cGy and followed by tail vein injection of the 2 T-cell depleted bone marrow cells from C57BL/6 (H-). Mixed chimerism mice were determined by gDNA PCR of lymphocyte MHC class I gene (H-2K) on 21st day. Streptozotocin induced diabetic mixed chimera mice were received islet transplantation from bone marrow donors. Grafts, spleen and peripheral blood were obtained from the mixed chimera mice, and there were used by Immunohistochimeical staining, flow cytometric analysis and gDNA PCR on 21st day. Results: The blood glucose levels of streptozotocin induced diabetic mice were normalized by transplantation of bone marrow donor islets and maintained during 30 days. After removal of first islet allografts, hyperglycemia was re-established. We could re-confirmed donor specific tolerance of transplanted islets by second transplantation of bone marrow donor islets. Normoglycemia was maintained during 21 days after second islet transplantation. Furthermore islet grafts from MHC-mismatched third party mice were immediately rejected. Flow cytometric analysis results suggest that the mixed chimerism mice were maintain during the whole study period. Conclusion: The mixed chimerism model conducted by newly developed and minimally invasive method effectively prevents the islet allo grafts rejection in STZ-induced mixed chimerism mice.

Anti-CD3/Anti-CD28 Bead-Activated T Cells Facilitate Engraftment of Murine Histoincompatible Allogeneic Transplants after Low-Dose Radiation.

Donor T cells are known to facilitate hematolymphoid engraftment following allogeneic stem cell transplantation. The recent development of the Xcellerate Technology has made it feasible to activate and expand T cells to large numbers for therapeutic applications. In the present study, we tested whether large numbers of donor T cells can enable engraftment with less intensive conditioning regimens thereby improving outcome and survival in allogeneic transplants. To test this hypothesis, we conducted transplant experiments in congenic MHC-mismatched mice in which we varied the infusional dose of both T cell-depleted bone marrow (TCDBM) and splenic T cells after conditioning animals with different amounts of total body radiation.For these experiments, we utilized a fully mismatched murine model in which BALB/C (H-2d) recipients were transplanted with C57/BL6 (H-2b) TCDBM. Previous studies by our group have demonstrated that a minimum radiation dose of 950 rads (single fraction) is required to achieve complete donor chimerism in this model. Recipient mice given a similar dose of radiation and donor BM, but supplemented with 2 x 106 naïve T cells, demonstrate evidence of donor engraftment, but all animals suffer from severe GVHD and are dead by day 30 post-transplant (n=45).In the present study, we studied the effects of administering donor T cells activated ex vivo in conjunction with lower radiation doses on engraftment, GVHD and survival in this histoincompatible murine model. Mice were treated with a single fraction of whole body irradiation at doses ranging from 100 to 400 rads. For each radiation dose, animals received either 2 x 106 naïve T cells or increasing doses of activated and expanded T cells (range from 2 to 50 x 106). Freshly isolated splenic T cells were activated and expanded using the Xcellerate Technology in which T cells are cultured ex vivo using microbeads coated with monoclonal antibodies directed against mouse CD3 and CD28 molecules. Six to eight mice were assigned to each treatment group.Control mice given TCDBM alone without T cells displayed no evidence of donor chimerism at any radiation dose level. At 100 rads radiation, none of the animals showed evidence of donor chimerism, whether treated with naïve or activated T cells. At 200 rads, complete donor chimerism was achieved in some animals given the highest dose of activated T cells (50 x 106). At doses of 300 and 400 rads, all animals given activated T cells at doses of 50 x 106 activated T cells showed complete donor chimerism. Animals, who were treated with 200–400 rads and given naive T cells showed no evidence of engraftment. Severity of GVHD was positively correlated with radiation dose and chimerism.These data provide evidence of the graft-promoting effect of high doses of activated T cells generated using the Xcellerate Technology. Further studies are ongoing to optimize the schedule and dose of donor stem cells and T cells in this murine histoincompatible transplant model. Additional studies are ongoing to document the durability of engraftment and characterize immune reconstitution, which may be facilitated by the early administration of large numbers of donor T cells. If the results reported here can be confirmed, Xcellerated T Cells may provide another approach to making histoincompatible transplants safer by enabling the use of less intensive conditioning regimens.

Specific Elimination of Alloreactive T Lymphocytes Using Photodynamic Therapy Prevents GVHD and Enables Rapid Immune Reconstitution.

Graft-versus-host disease (GVHD) and impaired immune reconstitution are the primary obstacles limiting the efficacy of allogeneic stem cell transplantation (SCT).Graft-versus-host disease (GVHD) and impaired immune reconstitution are the primary obstacles limiting the efficacy of allogeneic stem cell transplantation (SCT). The purpose of this study was to determine whether selective depletion of donor alloantigen-specific T lymphocytes using photodynamic therapy (PDT) would prevent GVHD and enable immune reconstitution in the context of MHC-mismatched SCT. This question was addressed in an MHC-incompatible mouse model of GVHD. The donor (C57BL/6; H-2b) derived spleen cells were first activated against C3H/HeJ (H-2k) host spleen cells in a one-way mixed lymphocyte culture and then exposed to photodynamic treatment, using dibromorhodamine methyl ester (TH9402) as a photosensitizer. Activated T cells showed preferential retention of this photosensitizer compared to resting lymphocytes. In addition, in vitro experiments revealed that PDT eradicated a significantly higher proportion of activated than resting T cells. When lethally irradiated H-2k mice (C3H/HeJ and B10BR) were transplanted with C57BL/6 derived T cell-depleted bone marrow cells supplemented with C57BL/6 derived spleen cells activated with C3H/HeJ targets, they rapidly succumbed to acute GVHD (within 10–47 days). In contrast, both mouse strains receiving histoincompatible C57BL/6 T cells previously exposed to PDT after activation against C3H/HeJ survived until the end of the observation period (>100 days)(p<0.0001). Additionally, transplantation of treated T cells induced lethal GVHD in C57BL/6 histoincompatible strains of mice (third party), suggesting PDT specifically eradicated activated T cells while sparing most resting T lymphocytes. Analysis of immune recovery, evaluating T and B cell populations in thymus and spleen, activated T cells components (CD44, CD62L, CD69), proliferative responses (anti-CD3 and conA), and Vβ repertoire indicated that T and B cell reconstitution in MHC-mismatched mice transplanted with treated primed cells was similar to that of mice transplanted with treated or control autologous cells indicating that immune cells were preserved and functional. These results demonstrate that PDT can selectively eliminate alloreactive T cells and prevent the development of GVHD, while sparing T cells reactive against non-host antigens, thus offering protection against infection and disease relapse.

911. MHC Class I and II Mismatched Marrow Stromal Cells from C57Bl/6 Mice Are Immune Rejected by Recipient Balb/c Mice

Bone marrow stromal cells (MSCs) can be easily harvested, culture expanded, and genetically engineered, and thus can be utilized therapeutically in regenerative medicine and in transgenic cell therapy strategies for delivery of therapeutic proteins. Recent studies have suggested that MSCs possess immunosuppressive properties and can be “tolerated” in allogeneic transplant setting without immunosuppression of the host. The objective of our investigation was to determine if the implantation of primary murine MSCs genetically engineered to release a soluble protein, erythropoietin (Epo), would be feasible in MHC-mismatched allogeneic mice without immunosuppression. Firstly, primary MSCs from donor C57Bl/6 (H-2Kb, H-2Db, I-Ab) mice were retrovirally engineered to release murine Epo (Epo+MSCs). These cells were admixed in a collagen-based matrix at 20 million cells/ml. Balb/c (H-2Kd, H-2Dd, I-Ad, I-Ed) mice are class I and II MHC-mismatched relative to C57Bl/6 donors and 5 recipient Balb/c mice were injected subcutaneously with 0.5 ml of Epo+MSCs/matrix. In parallel, 5 recipient syngeneic C57Bl/6 mice were likewise implanted with Epo+MSCs. In these latter syngeneic recipients, the hematocrit (Hct) rapidly rose from a baseline 54 ± 0.6 % (mean ± SEM) to 86 ± 0.3 % within 4 weeks post-implantation and remained >88 % for >200 days. However, in MHC-mismatched recipient Balb/c mice, the Hct rose from a basal 56 ± 0.6 % to a peak 79 ± 2.0 % at 4 weeks post-implantation and then rapidly declined to baseline values by day 52. Moreover, when these allogeneic mice received a 2nd implant of the same Epo+MSCs, the Hct increase was significantly more modest and of shorter duration. A 3rd identical implant in these same Balb/c mice led to no significant effect on Hct. Repeat experiments performed in the absence of collagen matrix led to similar results. To elucidate the host-derived immune response to the MHC-mismatched Epo+MSC implant, allogeneic Balb/c and syngeneic C57Bl/6 mice were identically implanted with C57Bl/6 derived EpoEpo+MSCs, sacrificed at day 15 and implants recovered. Implants were collagenase dissociated and flow cytometric analysis conducted on host-derived lymphoid cells revealed that allogeneic implants compared to syngeneic comprised significantly greater proportions of CD8+, NK T, CD11c, and NK cells. These results are consistent with a robust host cellular immune response to donor allogeneic EpoEpo+MSCs. To determine whether Epo production was causative in the observed immune response, “null” C57Bl/6 MSCs, lacking Epo production, were implanted in Balb/c mice and splenocytes from recipients were isolated 15 days later and tested for their interferon gamma (IFNγ) activation by C57Bl/6 MSCs in vitro. We found that splenocytes from these test Balb/c mice displayed a robust, specific, and significant IFNγ response to C57Bl/6 MSCs when compared to controls. Therefore, our results reveal that class I and II MHC-mismatched MSCs will elicit a cellular immune response by allogeneic hosts with normal immune systems, and that rejection is amplified by repeated challenge. These results strongly suggest that MSCs, at least in the murine system, cannot serve as a “universal donor” in cell therapy applications.

Embryonic stem cells as an alternate marrow donor source: engraftment without graft-versus-host disease.

A single embryonic stem cell (ESC) line can be repetitively cryopreserved, thawed, expanded, and differentiated into various cellular components serving as a potentially renewable and well-characterized stem cell source. Therefore, we determined whether ESCs could be used to reconstitute marrow and blood in major histocompatibility complex (MHC)-mismatched mice. To induce differentiation toward hematopoietic stem cells (HSCs) in vitro, ESCs were cultured in methylcellulose with stem cell factor, interleukin (IL)-3, and IL-6. ESC-derived, cytokine-induced HSCs (c-kit+/CD45+) were isolated by flow cytometry and injected either intra bone marrow or intravenously into lethally irradiated MHC-mismatched recipient mice. From 2 wk to 6 mo after injection, the peripheral blood demonstrated increasing ESC-derived mononuclear cells that included donor-derived T and B lymphocytes, monocytes, and granulocytes without clinical or histologic evidence of graft-versus-host disease (GVHD). Mixed lymphocyte culture assays demonstrated T cell tolerance to both recipient and donor but intact third party proliferative responses and interferon γ production. ESCs might be used as a renewable alternate marrow donor source that reconstitutes hematopoiesis with intact immune responsiveness without GVHD despite crossing MHC barriers.

Allogeneic versus syngeneic killer splenocytes as effector cells for the induction of graft-versus-tumor effect

The effect of allogeneic versus syngeneic killer cells derived from normal or severe combined immunodeficiency disease (SCID) mice was evaluated for induction of antitumor reaction in a murine model of mammary carcinoma. Tumor cells of H-2 d origin were injected intravenously into H-2 d/b mice 24 hours after total body irradiation (4 Gy). On the following day, lymphokine-activated killer (LAK) splenocytes, derived from either minor (H-2 d) or major (H-2 b) histocompatibility complex (MHC)-mismatched parental normal mice or MHC (H-2 b)-mismatched SCID mice, were given intravenously. LAK cells of H-2 d normal or SCID mice, syngeneic to the tumor, were inoculated in parallel. The results show that LAK cells derived from minor histocompatibility complex-mismatched or MHC-mismatched parental normal mice improved the probability of tumor-free survival as compared with LAK cells syngeneic to the tumor cells, but they aggravated the severity of graft-versus-host disease. SCID splenocytes serving as a source of natural killer (NK) cells were expanded and activated in vitro by rIL-2 to obtain a sufficient number of DX5 + CD3 − CD8 − NK cells (SCID-LAK). H-2 b SCID-LAK cells did not cause graft-versus-host disease and significantly delayed tumor growth compared with syngeneic H-2 d SCID-LAK cells, as indicated by tumor colony assays in vitro and adoptive transfer experiments. However, the graft-versus-tumor effect was not long lasting, and treated mice finally died of tumor. Our results show an advantage of allogeneic over syngeneic cell therapy for achieving a graft-versus-tumor effect by rIL-2-activated T cells and NK cells. Periodic repetition of NK treatments may be required to achieve more durable antitumor effects.