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》

Immunogenicity of L(d+) transgenic mouse hearts.

C57BL/6 mice transfected with the L(d) gene coupled to the alpha-myosin heavy chain promoter result in transgenic mice with L(d) antigen expressed only on cardiac tissue. These transgenic animals allow the examination of immune reactivity against cardiac L(d) by "self" or by adoptively transferred L(d) specific 2C cells, and the response of nontransgenic C57BL/6 mice to the transplanted L(d+) heart. Naïve cardiac L(d+) transgenic mice were examined for evidence of L(d) "autoimmunity." Forty million fresh 2C cells or 2C cells sensitized in vitro for 7 days against Balb/c (L(d+)) + interleukin-2 were also given intravenously to L(d+) transgenic mice. At 5 and 12 days after injection, heart-infiltrating lymphocytes were analyzed by fluorescence-activated cell sorter. The L(d+) transgenic hearts were also transplanted to syngeneic L(d-) nontransgenic C57BL/6 to evaluate the heart's immunogenicity. Naïve L(d+) transgenic mice did not exhibit any evidence of lymphocytic infiltration on histologic examination. Adoptive transfer of either fresh or in vitro sensitized 2C cells was also unable to reject the native L(d+) heart in transgenic mice (100% of the mice survived long term [more than 60 days]). Sensitization of the L(d+) transgenic mice with a Balb/c skin graft and interleukin-2 pump infusion (7 days) beginning 1 day before 2C cell injection also did not promote rejection of the native L(d+) heart. However, fluorescence-activated cell sorter analysis did reveal that a significantly greater number of in vitro sensitized 2C cells homed to the L(d+), but not L(d-), heart after both 5 and 12 days (P <.01, P <.001). In contrast, C57BL/6 mice rejected the L(d+) (C57BL/6 background) transgenic heart in a mean survival time of 17 +/- 9.7 days (P <.01), whereas a syngeneic C57BL/6 heart transplant was accepted indefinitely. Lymphocytic infiltration consistent with rejection was present in all animals receiving an Ld+ transgenic heart transplant, whereas no infiltrate was present in those receiving a syngeneic C57BL/6 heart transplant. Although the class I L(d) transgene is not recognized in its native host, its immunogenicity is shown by the homing of anti-L(d) 2C cells to the heart in situ and rejection of L(d+) heart grafts when transplanted into syngeneic C57BL/6 mice.

Margenthaler JA ，Tu F ，Otomo N ，Shimizu Y ，Yu S ，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》

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》

Regional oral tolerance in transgenic 2C mice.

Antigen injected subcutaneously (SQ) results in a strong systemic immune response, whereas antigen infused orally or by the portal vein tends to induce tolerance. Nontransgenic C57BL/6J (H-2(b)), or B6, mice and transgenic 2C mice (that express a cytotoxic T cell receptor against major histocompatibility complex Class I L(d)) were used to investigate the regional and systemic responses to oral antigen. B6 (H-2(b)) and 2C (H-2(b)) mice were given either saline or BALB/cByJ (H-2(d), L(d+)) (BALB/c) spleen cells (SCs) (25 x 10(6)) by oral gavage (PO) on day 0. Injection of 10 x 10(6) BALB/c SCs SQ was performed after 7 days, followed by a footpad injection of 10 x 10(6) BALB/c SC on day 14. Specific footpad swelling was measured 24 hours later by means of a micrometer. Mixed lymphocyte culture was performed on splenic and mesenteric lymph node (MLN) lymphocytes. The percentage of splenic and MLN CD4+ and CD8+ T cells was quantitated by fluorescence activated cell sorter. Cytokine messenger RNA (mRNA) and protein was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. Whereas B6 mice were shown to have specifically decreased responsiveness to Balb/c cells in vivo and in vitro after oral Balb/c, 2C mice did not demonstrate any downregulation in delayed-type hypersensitivity responsiveness or splenic lymphocyte proliferation. However, MLN lymphocytes from 2C mice did demonstrate decreased proliferation against Balb/c after oral Balb/c gavage (P < .01). Subset percentages of naïve B6 (n=8) spleen and MLN lymphocytes were 50% to 52% for CD4+ and 46% to 47% for CD8+ T cells. These percentages were unchanged in B6 mice after PO Balb/c. Subset percentages of naïve 2C (n=6) spleen and MLN lymphocytes were 2% to 5% for CD4+ and 61% to 64% for CD8+ T cells. After PO Balb/c, MLN CD4+ and CD8+ T cells were unchanged, whereas splenic CD8+ T cells decreased to 44% to 48% and CD4+ T cells increased to 32% to 33% (P < .05, P < .05 vs naïve 2C spleen). After PO Balb/c, inflammatory interleukin (IL)-2 and interferon gamma mRNA and protein were increased in 2C spleen cells, whereas they were decreased in 2C MLN. Anti-inflammatory cytokine IL-4 and IL-10 mRNA and protein were increased in 2C MLN after PO Balb/c, whereas they were not detectable in 2C spleen cells. Systemic oral tolerance can be induced in B6 mice, whereas only regional mesenteric lymph node tolerance develops in transgenic 2C mice. The inability to induce systemic oral tolerance in 2C mice appears to be due to a significant increase in peripheral (splenic) CD4+ T cells.

Margenthaler JA ，Flye MW 《SURGERY》