Mechanism of portal venous tolerant long-term MHC Class I L(d)-specific skin graft survival in transgenic 2CF1 mice.

Administration of alloantigen via the portal vein (PV) in non-transgenic animals has been shown to promote immunologic tolerance and enhance transplant allograft survival. The underlying mechanisms remain unclear. In 2C x dm2 F1 (2CF1) transgenic mice, the monoclonal antibody, 1B2, identifies specific 2C TCR transgenic CD8+ T cells that are cytotoxic against Class I MHC L(d). In these mice, the specific response by these cells to L(d+) skin grafts after PV administration of L(d+) antigen was determined. Saline (control) or allogeneic C57BL/6 x BALB/c F1 (CB6F1) spleen cells (25 x 10(6)), which differ from 2CF1 only at L(d), were injected PV into 2CF1 mice. One week later, CB6F1 tail skin was transplanted onto the dorsum of these 2CF1 mice. Skin graft rejection was defined as >50% loss of the graft. Parallel experiments were performed in non-transgenic littermates [B6F1 (C57BL/6 x dm2)]. FACS analysis of 2CF1 peripheral blood for 1B2+, CD4+, and CD8+ T cells was performed 2 days before PV injection (9 days prior to skin grafting), 5 days after PV injection (2 days prior to skin grafting), and 7, 14, 21, 28, and 60 days after skin grafting. FACS analysis of nai;ve, saline control, and CB6F1 PV-treated 2CF1 thymocytes was also performed. Responsiveness of saline (control)-treated and PV-treated 2CF1 splenocytes was measured by in vitro cytotoxic T lymphocyte (CTL). All CB6F1 skin grafts were rejected in <14 days by PV saline controls. However, a single PV injection of donor L(d+) CB6F1 cells was sufficient to induce indefinite CB6F1 (L(d+)) skin allograft survival in 100% of non-transgenic B6F1 and transgenic 2CF1 (anti-L(d)) TCR transgenic recipients. FACS analysis of 1B2+ T cells demonstrated that PV injection of donor antigen followed by a CB6F1 skin graft led to a 70% decrease in peripheral donor-reactive 1B2+ CD8+ T cells by day 7, while central thymocytes were unchanged. CTL of 2CF1 splenocytes following PV CB6F1 demonstrated that they were hyporesponsive to L(d) compared to saline-treated 2CF1 splenocytes. Despite recovery of peripheral CD8+ T cells to near normal levels by 60 days post-transplantation, skin graft survival persisted indefinitely. Administration of specific PV antigen results in exquisite long-term L(d+) skin allograft acceptance. This tolerance induction is related to a significant peripheral deletion of donor-reactive 1B2+ CD8+ transgenic T cells and anergy of the residual T cells.

Margenthaler JA ，Landeros K ，Kataoka M ，Flye MW ... -  《TRANSPLANT IMMUNOLOGY》

Donor-specific antigen transfusion-mediated skin-graft tolerance results from the peripheral deletion of donor-reactive CD8+ T cells.

The mechanism of donor-specific transfusion (DST)-induced long-term skin-graft survival is examined in 2CF1 (2C x dm2) transgenic and B6F1 (C57BL/6 x dm2) nontransgenic mice in which CB6F1 (Balb/c x B6) DST and donor skin grafts differ from 2CF1 or B6F1 recipients only at major histocompatibility complex class I Ld. Saline (control) or allogeneic CB6F1 spleen cells were injected intravenously into 2CF1 and B6F1 mice. One week later, CB6F1 tail skin was transplanted onto the dorsum of these mice. Fluorescence-activated cell sorter analysis (flow cytometric analysis) of peripheral blood was performed 2 days before DST, 5 days after DST, and 7, 14, 21, 28, and 75 days after skin grafting. Splenocyte responsiveness was measured by in vitro mixed lymphocyte culture and cytotoxic T lymphocyte. Cytokine protein production (interleukin [IL]-2 and interferon-gamma) was measured by enzyme-linked immunosorbent assay. Whereas all CB6F1 skin grafts in control saline-treated 2CF1 and B6F1 mice were rejected, 100% of 2CF1 and B6F1 pretreated with CB6F1 DST accepted the class I Ld disparate donor skin indefinitely. DST followed by a CB6F1 skin graft led to a significant deletion of donor-reactive CD8+ T cells by fluorescence-activated cell sorter analysis and decreased production of the inflammatory cytokines IL-2 and interferon-gamma. The hyporesponsiveness of residual CD8+ T cells in mixed lymphocyte culture and cytotoxic T lymphocyte to Ld after DST was restored to normal by IL-2. These findings demonstrate that administration of DST uniformly results in long-term Ld+ skin-allograft acceptance. This tolerance induction is related to both a significant decrease in donor-reactive CD8+ transgenic T cells and anergy of the residual CD8+ T cells.

Margenthaler JA ，Kataoka M ，Flye MW 《TRANSPLANTATION》

Peripheral tolerance in transgenic mice expressing class I MHC L(d) only on cardiac cells.

The fate of autoreactive T cells exposed to extrathymic self-antigen is examined in a double transgenic (DTG) mouse [(L(d+) cardiacx2C)F1], where cardiac myocytes alone express L(d) and T cells express an antigen receptor (2C TCR) against L(d). Naïve cardiac L(d+) single transgenic (STG) mice (before breeding with 2C) and DTG mice were examined for evidence of autoimmunity. The L(d+) STG hearts were then transplanted to syngeneic L(d-) wild type C57BL/6 to evaluate the heart's immunogenicity. L(d+) skin grafts were transplanted to non-transgenic B6, transgenic 2C, STG, and DTG mice. Phenotype analysis of peripheral 1B2+(identifies 2C T cells), CD4+, and CD8+ T cells was performed by FACS. In vitro MLC and CTL, with and without the addition of IL-2 and suppressor cell assays, were evaluated. Neither STG nor DTG hearts developed any evidence of autoimmunity by histology. In contrast, B6 mice rejected the L(d+) STG heart in 17+/-9.7 days (P<0.01), while a syngeneic B6 heart transplant was accepted indefinitely. Survival of L(d+) skin grafts was prolonged in both STG and DTG mice. FACS quantitation revealed that while there was no deletion of peripheral 2C cells in the DTG, these 2C T cells did have a significantly reduced proliferative and cytotoxic response to H-2L(d). Restoration of the proliferative and cytotoxic response of the DTG cells by the addition of IL-2 was consistent with a state of anergy. These findings suggest that the expression of extrathymic class I MHC expression alone did not trigger autoimmune reactions but that the T cells can be rendered anergic to the specific 'self' antigen.

Margenthaler JA ，Kataoka M ，Flye MW 《TRANSPLANT IMMUNOLOGY》

Intrathymic alloantigen-mediated, tolerant, completely major histocompatibility complex-mismatched mouse hearts are specifically rejected by adoptively transferred anti-class I L(d+)-specific 2C cells.

Tolerance to cardiac allografts can be induced in mice and rats by the injection of donor alloantigen into the thymus in combination with a CD4 T-cell-depleting antibody. CD8(+) cells in these animals are hyporesponsive to graft-specific alloantigens. Most of the CD8(+) T cells in the transgenic 2C mouse express a T-cell receptor specific for the class I major histocompatibility complex L(d+) locus. This study was designed to determine whether the adoptive transfer of these 2C T cells could precipitate rejection of a tolerant, completely major histocompatibility complex-mismatched L(d+) or L(d-) heart. C57BL/6 mice (L(d-)) were given 10 x 10(6) cells of BALB/c (L(d+)) or dm2 (BALB/c background lacking L(d) [L(d-)]) splenocytes intrathymically and GK1. 5 (10 mg/kg) intraperitoneally. Twenty-one days later, BALB/c or dm2 hearts were transplanted. On the day of transplantation or after long-term allograft acceptance, recipients received naive 2C cells or 2C cells sensitized by in vitro mixed lymphocyte culture with BALB/c (L(d+)). Mean survival time of BALB/c cardiac allografts in untreated C57BL/6 mice was 7.3 days, although 73% of the mice that were pretreated with BALB/c splenocytes IT plus GK1.5 accepted the donor antigen-specific heart allografts indefinitely. All recipients that were pretreated with the intrathymic plus GK1.5 and that were injected with naive 2C cells at the time of heart transplantation experienced rejection of the BALB/c (L(d+)), but not the dm2 (L(d-)) hearts. In contrast, naive 2C cells could not reject tolerant (>30 days acceptance) BALB/c (L(d+)) hearts. 2C cells sensitized in vitro against L(d) were able to reject established BALB/c hearts but could not reject the L(d-) dm2 hearts. L(d)-specific 2C T-cell receptor transgenic T cells that are adoptively transferred to recipients will precipitate the rejection of accepted hearts that express class I L(d+) in mice rendered tolerant by an intrathymic injection of alloantigen plus anti-CD4 monoclonal antibodies.

Otomo N ，Motoyama K ，Yu S ，Shimizu Y ，Margenthaler J ，Tu F ，Flye MW ... -  《SURGERY》

The immunologic function of 1B2+ double negative (CD4-CD8-) T cells in the 2C transgenic mouse.

2C mice bearing the cytotoxic TCR for class I L(d) on a C57BL/6 (B6) background have a preponderance of 1B2+CD8+ T cells directed against L(d). These naive CD8+ T cells are not directly cytotoxic without prior in vivo or in vitro activation. However, after in vitro sensitization, they become highly cytotoxic and will acutely and specifically reject a tolerant L(d+) BALB/c heart graft. Anti-lymphocyte serum (ALS) treatment eliminates CD4+ and CD8+ cells and a large double negative (CD4-CD8-) 1B2+ non-cytotoxic transgenic cell population remains. The immunological function of this unique peripheral population of T cells is investigated in the 2C transgenic mouse. To determine the activation characteristics of the 2C CD4-CD8- T cells, 2C peripheral T cells were analyzed for 1B2+, CD8+, and CD4+ marker by FACS before and 48-h after 0.5 cc ALS i.p. Similarly, in vitro, the response of these 2C CD4-CD8- T cells remaining after deletion of mature CD4+ and CD8+ T cells with ALS plus complement were evaluated by mixed lymphocyte culture and cytotoxic T lymphocyte after 7 days culture with BALB/c, IL-2, or BALB/c + IL-2. Parallel experiments were performed with control non-transgenic B6 mice. Following in vitro culture with BALB/c + IL-2, 2C CD4-CD8- T cells were injected into B6 mice with a tolerant BALB/c heart (tolerization via anti-CD4 mAb and intrathymic BALB/c) to determine their immunogenicity. While peripheral T cells in control B6 mice have <5% CD4-CD8- cells, transgenic 2C mice have a significantly increased percentage at 29 to 35% (P < 0.01). After the deletion of CD4+ and CD8+ T cells with either in vivo or in vitro ALS, 2C CD4-CD8- T cells increased to 96 to 99%. After 7-day culture, the 2C CD4-CD8- T cells decreased again to 33 to 38%. Simultaneously, 2C CD8+ T cells decreased from 56 to 62% to 0.1 to 3% after ALS treatment, but again increased to 61 to 70% after in vitro culture. Untreated 2C cells responded to IL-2 or BALB/c antigen equally well. However, after ALS treatment, CD4-CD8- T cells responded to IL-2 and IL-2 plus antigen, but not BALB/c antigen alone. Finally, CD4-CD8- T cells cultured for 7 days with BALB/c + IL-2 rejected the tolerant BALB/c heart in 5.3 +/- 0.3 days. In the periphery of transgenic 2C mice is a unique CD4-CD8- population of T cells bearing the transgenic specific marker 1B2. These non-cytotoxic cells can be optimally stimulated to develop marked specific L(d) cytotoxicity in parallel with the expression of the CD8+ epitope.

Margenthaler JA ，Flye MW 《JOURNAL OF SURGICAL RESEARCH》