Tail Skin Grafts Research Articles

Platelet-Targeted Gene Therapy Induces Robust Immune Tolerance Even in a Primed Model Via Peripheral Clonal Deletion of CD4 and CD8 T Cells and Expansion of Treg Cells

Our previous studies have demonstrated that platelet-specific gene transfer under control of the αIIb promoter (2b) results in effective immune tolerance induction to FVIII and FIX as well as the non-coagulant protein ovalbumin (OVA) in the unprimed model. Here we explored how the reactivity of immune responses affects the efficacy of transgene expression and the potential immune tolerance mechanisms in platelet-specific gene transfer in the primed model. We used OVA as a surrogate protein for this study. Recipient wild-type (WT) CD45.1 mice were primed with OVA protein. Platelet-OVA expression was introduced by 2bOVA lentivirus transduction of Sca-1+ cells from either WT/CD45.2 or OTII/CD45.2 donors followed by transplantation into OVA-primed WT/CD45.1 recipients preconditioned with 6.6Gy irradiation. We found that pre-existing high-reactive immune responses devastate platelet-OVA expression in recipients in both the WT/WT and OTII/WT models. Using the WT/WT mouse model, 6 of 7 2bOVA-transduced recipients had sustained platelet-OVA expression with levels ranging from 4.53-8.84 ng/108 platelets. Anti-OVA total IgG titers declined with time in 2bOVA-transduced OVA-primed recipients even when rechallenged with OVA. In contrast, recall memory immune responses were elicited in 2bGFP and untransduced transplanted controls. Furthermore, full-thickness tail skin grafts from CAG-OVATg mice were successfully engrafted onto 2bOVA-transduced recipients and survived throughout the remaining lifetimes of the animals. In contrast, skin grafts were completely rejected in control 2bGFP-transduced and untransduced transplanted recipients. To ensure that the immune system was not inactive in 2bOVA-transduced recipients, animals were immunized with unrelated antigen recombinant human FVIII (rhF8) using a protocol known to induce anti-F8 immune responses even in WT mice. All 2bOVA-transduced recipients developed anti-F8 inhibitory antibodies after rhF8 immunization with no differences between the 2bOVA group and the 2bGFP and untransduced control groups. Using the OVA-specific CD4 TCR transgenic model (OTII/WT), we showed that sustained platelet-OVA expression was achieved in 90% of 2bOVA-transduced recipients. OVA-specific CD4 T cells were deleted and Treg cells were expanded in peripheral lymphoid organs in 2bOVA-transduced OVA-primed recipients. The levels of platelet-OVA expression negatively correlates with the percentages of OVA-specific CD4 T cells, but positively correlates with Treg cells. Immune tolerance was also achieved in the OTII/WT model even though all donor-derived CD4 T cells are OVA-specific. However, our studies showed that high-reactive immune responses could influence the neoprotein expression in platelet-targeted gene therapy. Using the OVA-specific CD8 TCR transgenic model (OTI/WT), in which all donor-derived CD8 T cells are OVA-specific, we found that low levels of platelet-OVA expression were obtained at week 5 after transplantation (0.72±0.65 ng/108 platelets) and then dropped to barely detectable at subsequent time points. Flow cytometry analysis showed that there were 33.9±12.9% platelets positive with GFP in 2bGFP LV-transduced recipients, demonstrating that high transduction efficiency and successful transplantation were achieved. Of note, OVA-specific CD8 T cells in the 2bOVA group were 48% lower than in the 2bGFP control group, demonstrating that antigen-specific CD8 T cells were partially deleted in the OTI/WT model after platelet-targeted OVA gene transfer. Interestingly, we found that the percentage of the antigen-specific CD8+Foxp3+ cells in the 2bOVA group was significantly higher than in the 2bGFP group (0.19±0.14% vs. 0.05±0.01%, respectively). These results suggest that there is a counterpace process between immune reaction and immune tolerance after platelet-targeted gene therapy. In conclusion, our studies demonstrate that platelet-targeted gene therapy can induce antigen-specific immune tolerance even when the immune response has been mounted via peripheral clonal deletion of antigen-specific CD4 and CD8 T cells and expansion of Tregs, suggesting that platelet-targeted gene therapy is a promising approach to induce immune tolerance for treatment of diseases with undesired immune responses or even with pre-existing immune responses, such as hemophilia A with inhibitors or autoimmune diseases. Disclosures No relevant conflicts of interest to declare.

B lymphocytes contribute to indirect pathway T cell sensitization via acquisition of extracellular vesicles.

B cells have been implicated in transplant rejection via antibody-mediated mechanisms and more recently by presenting donor antigens to T cells. We have shown in patients with chronic antibody-mediated rejection that B cells control the indirect T cell alloresponses. To understand more about the role of B cells as antigen-presenting cells for CD4+ T cell with indirect allospecificity, B cells were depleted in C57BL/6 mice, using an anti-CD20 antibody, prior to receiving MHC class I-mismatched (Kd ) skin. The absence of B cells at the time of transplantation prolonged skin graft survival. To study the mechanisms behind this observation, T cells with indirect allospecificity were transferred in mice receiving a Kd skin transplant. T cell proliferation was markedly inhibited in the absence of recipient B cells, suggesting that B cells contribute to indirect pathway sensitization. Furthermore, we have shown that a possible way in which B cells present alloantigens is via acquisition of MHC-peptide complexes. Finally, we demonstrate that the addition of B cell depletion to the transfer of regulatory T cells (Tregs) with indirect alloresponse further prolonged skin graft survival. This study supports an important role for B cells in indirect T cell priming and further emphasizes the advantage of combination therapies in prolonging transplant survival.

Skin transplantation in mice as an animal model for functional analysis of T regulatory cells.

The role of T regulatory cells (Tregs) in transplantology has been widely studied over the years. The mouse model of skin transplantation is used as a quick and easy way to monitor the immune response of Tregs, vital for creation and maintenance of homeostasis and immunological tolerance. This model is extremely useful in the assessment of allograft rejection and immune tolerance induction dependent on Tregs. The procedure of skin transplantation is widely used not only in transplantology, but also in studies on the effectiveness of various immunosuppressive compounds and studies on autoimmune diseases, including diabetes, multiple sclerosis and albinism. The improved protocol of mouse tail skin grafting is easy to master, and its wide application gives many opportunities for new solutions in biomedical research..

An Inhibitor of Thyroid Hormone Synthesis Protects Tail Skin Grafts Transplanted to Syngenic Adult Frogs.

Tail regression in amphibian tadpoles during metamorphosis is one of the most dynamic morphological changes in animal development and is induced by thyroid hormone (TH). It has been proposed that tail resorption is driven by immunological rejection in Xenopus laevis, based on experimental evidence showing that larval skin grafts become atrophic on syngenic recipient adult frogs. This led to the hypothesis that tail regression is induced by an immunological rejection against larval skin-specific antigens called Ouro proteins. However, our group has demonstrated that ouro-knockout tadpoles undergo normal metamorphosis, including tail resorption in Xenopus tropicalis, which indicates that the expression of ouro genes is not necessary for tail regression. In the present study, we showed that an inhibitor of TH synthesis promotes the survival of larval tail skin grafts on syngenic adult Xenopus tropicalis frogs. The levels of endogenous THs in adult frogs were also comparable to those in metamorphosing tadpoles of Xenopus laevis with a regressing tail, and TH induced the regression of tadpole tail tips of Xenopus tropicalis in organ culture. Taken together, these results strongly suggest that endogenous THs in the recipient adult frog induce the degeneration of syngenic tail skin grafts.

The Synergistic Immunoregulatory Effects of Culture-Expanded Mesenchymal Stromal Cells and CD4+25+Foxp3+ Regulatory T Cells on Skin Allograft Rejection

Mesenchymal stromal cells (MSCs) are seen as an ideal source of cells to induce graft acceptance; however, some reports have shown that MSCs can be immunogenic rather than immunosuppressive. We speculate that the immunomodulatory effects of regulatory T cells (Tregs) can aid the maintenance of immunoregulatory functions of MSCs, and that a combinatorial approach to cell therapy can have synergistic immunomodulatory effects on allograft rejection. After preconditioning with Fludarabine, followed by total body irradiation and anti-asialo-GM-1(ASGM-1), tail skin grafts from C57BL/6 (H-2kb) mice were grafted onto the lateral thoracic wall of BALB/c (H-2kd) mice. Group A mice (control group, n = 9) did not receive any further treatment after preconditioning, whereas groups B and C (n = 9) received cell therapy with MSCs or Tregs, respectively, on days −1, +6 and +13 relative to the skin transplantation. Group D (n = 10) received cell therapy with MSCs and Tregs on days −1, +6 and +13. Cell suspensions were obtained from the spleens of five randomly chosen mice from each group on day +7, and the immunomodulatory effects of the cell therapy were evaluated by flow cytometry and real-time PCR. Our results show that allograft survival was significantly longer in group D compared to the control group (group A). Flow cytometric analysis and real-time PCR for splenocytes revealed that the Th2 subpopulation in group D increased significantly compared to the group B. Also, the expression of Foxp3 and STAT 5 increased significantly in group D compared to the conventional cell therapy groups (B and C). Taken together, these data suggest that a combined cell therapy approach with MSCs and Tregs has a synergistic effect on immunoregulatory function in vivo, and might provide a novel strategy for improving survival in allograft transplantation.

Skin allograft rejection.

Skin allograft rejection is a test of the competence of T lymphocytes to mediate in vivo tissue destruction, which in turn reflects their role in critical functions such as anti-viral and tumor immunity. The tail-skin graft procedure described in this unit is useful predominantly because of the ease of preparation and resistance to ischemic (nonspecific) necrosis. Additionally, it is not necessary to sacrifice the donor mouse. However, rejection of tail-skin grafts should not be used to test for genetic homogeneity in breeding experiments or to detect minor histocompatibility (minor-H) antigens because tail skin is less sensitive than trunk skin in detecting such differences.

Prolonged Survival of Vascularized Skin Grafts Across a Full MHC Mismatch Using Donor Antigen-Pulsed Tolerogenic GM+Rapa Dendritic Cells (102.22)

Abstract We compared the tolerogeneic properties of BM-derived GM+IL-4 and GM+Rapa host DCs and their potential to induce long-term acceptance of vascularized skin allograft in rats across a full MHC mismatch with transient immunosuppression. Results show that both DCs were CD11b+ and expressed low levels of CD86 and produced low levels of IL-12p70 following TLR activation. GM+Rapa DC produced lower levels of anti- and pro-inflammatory cytokines in response to LPS. Both DCs had low T cell stimulatory capacity in vitro. Interestingly, donor Ag-pulsed GM+Rapa DC (GM+Rapa DCp) induced long-term survival of the vascularized skin grafts and this was significant compare to GM+IL-4 DC and control groups. PBMC from long-term graft survivors that were previously treated with donor Ag-pulsed GM+Rapa DC were hyporesponsive to donor Ag, but proliferated in response to the third-party Ag and produced IL-10 in addition to IFN-gamma. Recipients of the long-term surviving allografts were challenged with full-thickness tail skin grafts. While the third-party grafts were rejected fast the donor graft had delayed rejection, suggesting that this treatment induced a regulatory mechanism that prevented the acute rejection of donor skin grafts. Further experiments demonstrated presence of CD4+ Foxp3+ T cells in the vascularized skin and secondary lymphoid tissues. Taken together, these results demonstrate that the donor Ag-pulsed tolerogenic GM+Rapa DC have ability to induce regulatory T cells and prevent vascularized skin graft rejection across a full MHC mismatch.

Heart, but Not Skin, Allografts from Donors Lacking Flt3 Ligand Exhibit Markedly Prolonged Survival Time

Fms-like tyrosine kinase 3 ligand (Flt3L) administration leads to dramatic increases in dendritic cells (DC) in lymphoid and nonlymphoid tissues. Conversely, mice lacking Flt3L (Flt3L(-)/(-)) show severe reductions in both myeloid (CD11c(+)CD8alpha(-)) and lymphoid-related DC (CD11c(+)CD8alpha(+)) in the thymus and secondary lymphoid organs. In this study marked reductions in CD11c(+) interstitial cardiac DC and in dermal, but not epidermal, DC (Langerhans cells) were also observed. CD11c(+) cells that migrated from Flt3L(-/-) skin explants expressed lower surface MHC class II and costimulatory molecules and naive T cell allostimulatory activity than migratory wild-type (wt) C57BL/6 (B6) CD11c(+) cells. We examined the survival of Flt3L(-)/(-) heart or tail skin grafts (H2(b)) in allogeneic wt (BALB/c; H2(d)) recipients. The outcome of transplantation of BALB/c organs into Flt3L(-)/(-) recipients was also determined. Flt3L(-)/(-) mice rejected BALB/c heart or skin grafts with similar kinetics as B6 wt recipients. Trafficking of donor DC into host spleens or draining lymph nodes was markedly reduced after transplantation of Flt3L(-)/(-) heart, but not skin grafts, respectively. Compared with wt hearts, survival of Flt3L(-)/(-) hearts was markedly prolonged in BALB/c recipients (median survival time, 37 and 15 days, respectively; p < 0.001). Skin graft survival was unaffected. Rejection of Flt3L(-/-) hearts was precipitated by infusion of wt donor DC at the time of transplant. Thus, severe depletion of interstitial heart DC resulting from targeted gene disruption prolongs, but does not indefinitely extend, heart survival. Acute rejection of wt grafts in Flt3L(-/-) recipients reflects presumably an intact role of the direct pathway of allorecognition.

Rejection of grafts with no H-2 disparity in TAP1 mutant mice: CD4 T cells are important effector cells and self H-2 b class I molecules are target

Our previous results showed that TAP1 mutant mice rejected heart and skin grafts from donors with no H-2 disparity that express normal density of MHC class I molecules at the cell surface. During rejection, CD4 cells were predominant and essentially, no CD8 cells were found infiltrating the grafts. We hypothesized that TAP1 mutant mice, which developed and matured in an MHC class I-deficient environment, may have selected a repertoire of T cells with distinct reactivity to self class I molecules. The rejection of grafts with no H-2 disparity could be mediated by CD4 + T cells reactive to wild type H-2 b class I molecules, or derived peptides, in the context of self-APC. Accordingly, we observed that transplanted TAP1 mutant mice presented a significant amplification of the proliferative T cell response to H-2K b peptides, indicating that the stimulus with the graft was sufficient to induce peripheral expansion of these T cell repertoires. Therefore, the response to H-2K b molecules could be a relevant pathway of activating T cells and triggering rejection of grafts expressing normal levels of these class I molecules. To test our hypothesis, we investigate the effect of pre-transplantation H-2K b peptide-immunization on TAP1 mutant, which were then transplanted with C57BL/6 skin grafts (H-2 b). Mice were immunized with a pool of five peptides derived from the polymorphic region of K b α chain, before tail skin grafting. To study the role of CD4 + T cells in the rejection of C57BL/6 skin grafts, mice were in vivo depleted with an anti-CD4 monoclonal antibody GK1.5, and transplant evolution was observed. Sensitization of TAP1 mutant mice with H-2K b peptides accelerated the rejection of skin grafts. Immunized mice rejected grafts with a MST of 13 days, compared to 16 days for the non-immunized mice ( P=0.0089). The significant acceleration of graft rejection, induced by immunization with H-2K b peptides, indicates that these peptides are capable of mobilizing effector T-cells that participate in rejection. These results support our hypothesis that class I molecules may be a target in the rejection of grafts with no MHC disparity. Depletion of CD4 T-cells resulted in a significant delay in rejection compared with the untreated control group. The MST of skin grafts in the controls was 16 days, whereas CD4-depleted recipients rejected skin grafts with a MST of 41 days ( P=0.025). Moreover, some animals did not show macroscopic signs of rejection up to >100 days posttransplantation. The contribution of CD4 + T cells to skin graft rejection, in our model, may reflect the occurrence of the presentation of H-2 b peptides during graft rejection, in the context of self-APC. In conclusion, our results demonstrate an important role for H-2 b molecules and CD4 T cells in the rejection of C57BL/6 grafts by TAP1 mutant mice. The low expression of MHC-I molecules on TAP1 −/− mice may be determinant in the selection of a T cell repertoire strongly reactive to self MHC class I molecules which probably escapes the control of peripheral regulatory mechanisms.

Prolonged Hya-disparate skin graft survival in ethanol-consuming mice: correlation with impaired delayed hypersensitivity.

Ethanol consumption impairs cell-mediated immunity and enhances humoral immunity. Among cell-mediated immune reactions, little is known of the effect of ethanol on chronic graft rejection. Allograft responses against the male-specific minor histocompatibility antigen, Hya, are widely used to study chronic graft rejection. Female C57BL/6 (B6) mice were fed ethanol-containing liquid diets, were pair-fed an isocaloric liquid control diet, or were fed solid diet and water ad libitum. One week after diet initiation, the mice were grafted with split thickness, orthotopic male tail skin grafts, and the integrity of the grafts was monitored as the diet continued. Delayed hypersensitivity (DTH) was also determined in these same mice. In addition, Hya-cytolytic T-cell-deficient syngeneic major histocompatibility complex mutant B6.C-H2bm13 (bm13) and B6.C-H2bm14 (bm14) mice were assessed for skin graft rejection, DTH, and cytotoxic T-lymphocyte (CTL) activity. Ethanol-consuming female B6 mice are impaired in their ability to reject syngeneic male skin grafts and to develop Hya-specific DTH responses. To address the underlying mechanism, we show that Hya graft rejection correlates with DTH and not with CTL activity. Female B6 mice clearly differ from female bm13 and bm14 mice in their ability to generate CTLs against Hya antigen. Despite their inability to make Hya-specific CTL responses, bm13 and bm14 female mice, nevertheless, make Hya-specific DTH responses and ultimately reject Hya-disparate skin grafts, indicating that Hya-specific graft rejection results from DTH. Ethanol, by impairing Hya-specific DTH, inhibits Hya-specific skin graft rejection. We demonstrate that ethanol consumption impairs Hya-specific graft rejection. In addition, experiments with mice unable to generate anti-Hya CTLs support previous observations suggesting that DTH responses are sufficient to cause rejection of Hya-incompatible grafts.

Prolonged Hya‐Disparate Skin Graft Survival in Ethanol‐Consuming Mice: Correlation With Impaired Delayed Hypersensitivity

Background: Ethanol consumption impairs cell‐mediated immunity and enhances humoral immunity. Among cell‐mediated immune reactions, little is known of the effect of ethanol on chronic graft rejection. Allograft responses against the male‐specific minor histocompatibility antigen, Hya, are widely used to study chronic graft rejection.Methods: Female C57BL/6 (B6) mice were fed ethanol‐containing liquid diets, were pair‐fed an isocaloric liquid control diet, or were fed solid diet and water ad libitum. One week after diet initiation, the mice were grafted with split thickness, orthotopic male tail skin grafts, and the integrity of the grafts was monitored as the diet continued. Delayed hypersensitivity (DTH) was also determined in these same mice. In addition, Hya‐cytolytic T‐cell–deficient syngeneic major histocompatibility complex mutant B6.C‐H2bm13 (bm13) and B6.C‐H2bm14 (bm14) mice were assessed for skin graft rejection, DTH, and cytotoxic T‐lymphocyte (CTL) activity.Results: Ethanol‐consuming female B6 mice are impaired in their ability to reject syngeneic male skin grafts and to develop Hya‐specific DTH responses. To address the underlying mechanism, we show that Hya graft rejection correlates with DTH and not with CTL activity. Female B6 mice clearly differ from female bm13 and bm14 mice in their ability to generate CTLs against Hya antigen. Despite their inability to make Hya‐specific CTL responses, bm13 and bm14 female mice, nevertheless, make Hya‐specific DTH responses and ultimately reject Hya‐disparate skin grafts, indicating that Hya‐specific graft rejection results from DTH. Ethanol, by impairing Hya‐specific DTH, inhibits Hya‐specific skin graft rejection.Conclusions: We demonstrate that ethanol consumption impairs Hya‐specific graft rejection. In addition, experiments with mice unable to generate anti‐Hya CTLs support previous observations suggesting that DTH responses are sufficient to cause rejection of Hya‐incompatible grafts.

Combined FTY720/cyclosporine A treatment promotes graft survival and lowers the peripheral lymphocyte count in DA to Lewis heart and skin transplantation models

Objective: The immunomodulator, FTY720, lowers the peripheral lymphocyte count (PLC) by inducing migration of circulating lymphocytes to secondary lymphoid organs. We investigated the efficacy of mono- vs. combined-FTY720/CsA therapy on graft survival (GS) and on lowering the PLC in a solid organ and a skin graft model, using strains with strong MHC disparity. Methods: Heterotopic cardiac or tail skin grafting was performed using the DA (RT1 a) to Lewis (RT1 1) rat strain combination. FTY720 was administered as a single daily dose by gavage alone or in combination with subcutaneously delivered CsA. PLC, body weight and drug concentrations were determined on day 7, 28, or the day of rejection. Main findings: In placebo-treated animals the heart and skin allografts rejected after 6 and 8 days. FTY720 delayed rejection of both the solid organ and skin grafts. The maximal effect was achieved at 1 mg kg −1 day −1 FTY720, resulting in a median survival time (MST) of 14 days for both allotransplants comparable to the effect achieved by 1 mg kg −1 day −1 CsA in both models. In the cardiac graft experiment with CsA co-administration, doses of 0.3 and 1 mg/kg were used. Under these conditions very small doses of FTY720 were effective in maintaining grafts throughout the treatment period. Adding higher FTY720 doses to the 1 mg kg −1 day −1 CsA was needed to effectively extend the skin GS, e.g. 0.3 mg kg −1 day −1 FTY720 prolonged GS from 13 to 47.5 days MST, i.e. well beyond the 28 day-treatment period. CsA did not influence the PLC at clinically relevant doses. FTY720 lowered the PLC significantly and dose-dependently, at doses lower than those needed for the prolongation of both cardiac and skin GS with FTY720 monotherapy. In rats with skin grafts the PLC was markedly lowered up to 1 mg kg −1 day −1 FTY720, whereas, in the heart model, it was lowered up to 0.1 mg kg −1 day −1. Independently of the graft type, within the combination regimens 0.3 mg kg −1 day −1 FTY720 achieved a maximal PLC depletion. Conclusions: Combining FTY720 and CsA was very well tolerated with respect to weight gain and lack of any clinically detectable infections. In the strain combination used FTY720 monotherapy was less effective than previously reported in maintaining grafts. The two-drug regimens extended strikingly the GS for both models. However, the prolongation of the heart GS was smoothly dose-related with FTY720 doses ranging from 0.01 to 1 mg kg −1 day −1, whereas, the skin graft prolongation was modest at doses up to 0.1 mg kg −1 day −1 and remarkably enhanced at 0.3 and 1 mg kg −1 day −1 FTY720.

MODULATION OF CD95 OR BCL-2 EXPRESSION INFLUENCE GRAFT REJECTION INDUCED BY DENDRITIC CELLS.

817 In this study we investigated how modulation of apoptotic pathways influence graft rejection induced by dendritic cells (DCs). To explore this question we took advantage of a previously described transplant model in which graft rejection is selectively induced by DCs. In this model tail skin grafts from B6Bm7 (Bm7) mice are permanently accepted by B6 (H-2b) mice across a limited MHC Class I difference. However, small numbers of Bm7 DCs readily induce rejection of stable tail grafts after i.p. or i.v. injection, while other antigen presenting cells are ineffective. Thus, this model selectively allows us to explore the role of DCs in graft rejection. Programmed cell death (apoptosis) is thought to be an important mechanism in T cell mediated rejection. Expression of Fas ligand (fasL) promotes apoptosis by ligation with the Fas receptor (CD95). Conversely, overexpression of Bcl-2 inhibits activa-tion of the apoptotic cascade and promotes cell survival. To further examine the importance of fas mediated apoptosis in DC induced graft rejection, we examined the survival of Bm7 tail skin grafts transplanted onto Fas deficient B6 (B6.MRL-lpr, H-2b) mice. In the first set of studies we challenged established Bm7 tail grafts on B6lpr mice with donor-derived DCs. Interestingly, our results showed that challenge with as many as 2×106 donor DCs did not induce rejection in the CD95 deficient mice (1/4), whereas, 4/4 wildtype B6 rejected their transplants. These results suggest that rejection induced by DCs is dependent on Fas mediated apoptotic activity of the recipient. To test this hypothesis we used, as graft recipient, the transgenic bcl-2 mouse (H-2b) which overexpresses Bcl-2 but still express CD95. Bm7 tail skin was grafted onto transgenic mice having the bcl-2 gene expressed from either the human (high expressor) or chicken (low expressor) β-actin promoter. The results showed that without DC challenge, a late rejection ocurred at days 22-27 in 5/5 high expressing bcl-2 mice and at days 31-40 in 4/5 low expressing bcl-2 mice. In comparison, only 1/4 of the wildtype B6 mice with Bm7 tail skin rejected within this observation time (p<0.05). In conclusion, these results suggest that an abrogation of the CD95 apoptosis pathway blocks DC mediated allograft rejection in our mouse model. Furthermore, we see enhanced rejection with conditions that may stimulate recipient T cell survival, thus enhancing interactions between recipient T-cells and donor DCs. Because injection of recipient DCs can not induce a rejection we hypothesize that direct stimulation by donor DCs determine the graft survival.

The Influence of Tissue Culture on Corneal Immunogenicity

In this study we examined whether immunization with heterotopic corneal graft can be suppressed by usage of cultured corneal tissue. Starting from the hypothesis that the corneal antigenicity might change during long-time storage, we compared, in a mouse model, the immunization obtained with fresh and>stored corneas. Heterotopic (chest wall) mice corneal allografts were exchanged between donors and hosts: (1) mismatched at multiple minor H loci and (2) only H-Y mismatched animals. Median survival time (MST) of primary and secondary skin grafts exchanged between mentioned donors and hosts was recorded. Recipient mice were immunized with either: (a) tail-skin graft, (b) fresh cornea graft or (c) corneal graft stored for three weeks in tissue culture. Three weeks later, recipients were challenged with skin graft placed at the opposite side of the chest wall and MST of these skin grafts was recorded. MST of secondary skin grafts in animals that had been immunized by skin served as a control. In case of multiple minor H disparity, MST of a first-set skin graft was 12 days, as compared to 9 days in case of secondary skin graft (P<0·05). MST of secondary skin graft following immunization by both fresh and stored corneas was 10 days. These data suggest that stored corneas don't loose ability to sensitize the multiple minor H disparate host. It also show that both cultured and fresh corneas, when placed in non-privileged site, have same immunizing capacity as skin (MST of 10 and 9 days, respectively;P>0·1). When only H-Y disparate animals were used, MST of a first-set skin grafts was 26 days and of secondary skin graft 11 days (P<0·01). In case of H-Y disparity, MST obtained after immunization with fresh and stored corneal tissue (19 and 18 days, respectively) was significantly longer as compared to skin (P<0·05). However, no significant difference in MST of secondary skin grafts between recipients of fresh (19 days) and stored corneal grafts (18 days) was recorded. According to our results, the ability of corneal tissue to immunize both multiple minor H mismatched, as well as only H-Y mismatched host, was not influenced by storage in a tissue culture.

Differential Regulation of Rejection of Small Intestinal and Skin Allografts in Rats by Injection of Antibodies to ICAM-1 or the Integrins α4, αL, or β2

Female Lewis (LEW) rats received orthotopic small intestinal transplantation (SIT), or tail skin grafts from female (Lewis × Brown Norway)F1 (LBNF1) rats, along with peritransplant portal venous (pv) infusion of LBNF1 bone marrow-derived dendritic cells derived from male donors. All animals received im injection with cyclosporin A (5 mg/kg) for 3 consecutive days following transplantation. In some cases rats received intravenous injections, at 2-day intervals, with 1 mg of monoclonal antibodies to ICAM-1 or the integrins α4, αL, or β2, or combinations of these reagents. Cells were harvested from the recipient rats at different times posttransplantation, and single cell suspensions were analyzed by FACS for expression of CD3+, CD4+, CD8+, αβTcR+, and γδTcR+cells. Other tissue samples were used for histopathological assessment of rejection. We also investigated donor-specific and third-party (Wistar–Furth, Wi) restimulation of host lymphocytes from MLN, PLN, and PP for production of different cytokinesin vitro.Of the various antibodies tested, only anti-α4, but not anti-αL, -β2, nor -ICAM-1 led to further increased graft survival of LBNF1 SIT beyond that seen with pv-infused cells alone (30 days vs 19 days), while the combination of anti-αL(or β2) and ICAM-1 produced further significantly increased survival of skin grafts (30 days vs 21 days). For both SIT and skin-grafted animals increased graft survival was associated with decreased production of IL-2 and IFN-γ and increased production of IL-4 and IL-10 from tissues local to the graft (PP and draining LN, respectively), with less significant alterations in tissues distant to the graft (PLN for SIT, and MLN for skin grafts). While, as reported previously, pv-immunized SIT rats showed increased γδTCR+cells within the SIT in association with increased graft survival, treatment with anti-α4diminished this increase in γδTCR+cells, while simultaneously increasing SIT survival. Nevertheless, the bias toward increased IL-10 production, and decreased IFN-γ production, from cells of animals showing increased survival was maintained. These data suggest that local graft infiltration with γδTCR+cells following pv immunization is not necessary for prolongation of survival in this model system, although functional changes in the local cytokine milieu may be important.

Presentation of the HPV16E7 Protein by Skin Grafts Is Insufficient to Allow Graft Rejection in an E7-Primed Animal

The E7 transforming protein of Human Papillomavirus type 16 (HPV16) is expressed in the skin of a line of FVB mice transgenic for the E6 and E7 open reading frames of HPV16 driven from the αA crystallin promoter (FVBαAcryHPV16E6E7). We have transferred skin from FVBαAcryHPV16E6E7 mice to naive or E7-primed syngeneic FVB recipients to assess whether the E7 protein of HPV16 can function as a minor transplantation antigen (MTA) and promote skin graft rejection. FVB mice did not reject E7 expressing tail or flank skin grafts. E7 immunized FVB × C57BL/6J mice recipients of FVBαAcryHPV16E6E7 × C57BL/6J skin generated humoral and DTH responses to E7in vivoand E7-specific CTL precursors in the spleen, but failed to reject E7 expressing tail skin grafts by 100 days posttransfer. Thus although HPV16 E7 + ve mesenchymal and endodermal tumors can be eliminated by an E7-specific immune response, the same protein is unable to act as a MTA and promote graft rejection when expressed in skin cells. Lack of rejection of grafts expressing MTAs such as E7 may be relevant to the immunology of epithelial tumors expressing tumor-specific antigens and to our understanding of the immunology of diseases of the skin.

A Synthetic CD4-CDR3 Peptide Analog Enhances Bone Marrow Engraftment Across Major Histocompatibility Barriers

AbstractThe efficacy of a synthetic peptide analog mimicking the CDR3-D1 domain of the CD4 molecule was investigated in murine models of allogeneic bone marrow engraftment after transplantation across major histocompatibility complex (MHC) barriers. A single dose of a CD4-CDR3 peptide analog was administered at the time of marrow transplantation to three different allogeneic mouse strain combinations after appropriate sublethal total body irradiation: (1) B10.BR → C57BL/6J (B6), a full allogeneic MHC difference; (2) (B6xDBA/2)F1 → (B6xCBA)F1 , a haploidentical MHC combination; and (3) B6.C-H2bm12 → B6-Ly5.2, involving only a MHC class II difference. Donor-host chimerism was assessed after 1 and 2 months posttransplantation by flow cytometric analysis of spleen and/or lymph node cells. Peptide-treated animals in all three strain combinations exhibited significantly enhanced donor lymphoid engraftment, which was similarly reflected in the total lymphocyte compartment and its T-cell (CD4+, CD8+) and B-cell subsets. In addition, peptide-treated mice in the haploidentical and MHC class II-mismatched strain combinations exhibited prolonged tolerance of both donor and syngeneic host-type tail skin grafts while rejecting third-party allogeneic grafts, thus supporting the reconstitution of immunocompetence in these chimeras. Lymphocytes from the peptide-treated haploidentical chimeric mice also displayed donor-specific tolerance upon stimulation in a one-way mixed lymphocyte reaction. In a 6-day colony-forming unit–granulocyte-macrophage (CFU-GM) assay to quantitate the level of hematopoietic cell engraftment in both the haploidentical and class II-disparate strain combinations, bone marrow cells from the peptide-treated mice exhibited significant increases in CFU-GM compared with the saline-treated control groups. Finally, early multiple treatments with the peptide after transplantation significantly enhanced donor chimerism in donor-presensitized recipient mice across the MHC class II barrier and proved to be significantly more effective than anti-CD4 monoclonal antibody treatment. These results indicate that the structure-based CD4-CDR3 peptide analog may represent a valuable approach to the inhibition of graft rejection after MHC-mismatched bone marrow transplantation.

Suppression of mouse skin allograft rejection by protein A-Yersiniae V antigen fusion peptide.

V antigen is an established virulence factor of Yersinia pestis, the causative agent of bubonic plague. Injection of homogenous staphylococcal protein A-V antigen fusion peptide into mice was previously found to suppress tumor necrosis factor-alpha and interferon-gamma necessary for generation of protective granulomas. Here, we show that BALB/c mice receiving daily intraperitoneal injections of 100 microg of control protein A initiated rejection of C57BL/6 mouse tail skin grafts after 6.2+/-1.1 days. This time doubled to 12.2+/-1.4 days upon similar administration of protein A-V antigen fusion peptide (P<0.001); times of total allograft retention remained constant. This finding indicates that V antigen can postpone inflammation known to be associated with recognition and destruction of foreign tissue by T lymphocytes.

TOLERANCE INDUCTION TO MULTIPLE MINOR HISTOINCOMPATIBLE CELLS BY ACTIVATED B CELLS IS ASSOCIATED WITH PREFERENTIAL ACTIVATION OF Th2 CELLS1

C3H/HeH or C57BL/6 mice were injected with resting or Escherichia coli lipopolysaccharide (LPS)-stimulated splenic B cells from adult B10.BR mice. Animals were grafted with tail skin grafts from B10.BR mice 36 hr later. Spleen cells were removed from these mice 7 days after grafting and challenged in tissue culture with irradiated B10.BR spleen cells or BALB/c cells. LPS blasts, but not naive B cells, induced an antigen-specific reduction in proliferation and IL-2 production from stimulated C3H/HeJ cells. The response obtained from C57BL/6 spleen responder cells was increased by this treatment. IL-4 production was either unchanged (C57BL/6) or enhanced (C3H/HeJ). Modification of the C3H/HeJ anti-B10.BR response by B blasts was not blocked by CTLA-4 Ig, although the increased response seen using MHC-incompatible (C57BL/6) spleen cells was inhibited by CTLA-4 Ig. B10.BR, but not BALB/c, skin graft survival in vivo was enhanced in C3H/HeJ recipients of B10.BR B blasts. In addition, in lymph nodes draining the graft site of C3H/HeJ mice injected with B10.BR LPS blasts, mRNA for IL-4 was detected by polymerase chain reaction. When similar studies were performed with B10.BR immune C3H/HeJ or C57BL/6 mice, no enhancement of graft survival in vivo, or decrease in proliferation/IL-2 production in vitro, was seen following prechallenge with B10.BR LPS blasts.

Skin allograft rejection.

Skin allograft rejection is a test of the competence of T lymphocytes to mediate in vivo tissue destruction, which in turn reflects their role in critical functions such as anti-viral and tumor immunity. The tail-skin graft procedure described here is useful predominantly because of the ease of preparation and resistance to ischemic (nonspecific) necrosis. Additionally, it is not necessary to sacrifice the donor mouse. However, rejection of tail-skin grafts should not be used to test for genetic homogeneity in breeding experiments or to detect minor histocompatibility (minor-H) antigens because tail skin is less sensitive than trunk skin in detecting such differences. The trunk-skin graft procedure detailed here more difficult to perform than the tail-skin graft. It requires extensive tissue preparation and is more susceptible to ischemic necrosis. Trunk-skin grafts are especially useful for detecting minimal histocompatibility differences.