Intratracheal Delivery Of Alloantigen Research Articles

Depletion of Alveolar Macrophages Abrogates Prolongation of Cardiac Allograft Survival Induced by Intratracheal Delivery of Alloantigen

We previously showed that pretreatment with intratracheal delivery (ITD) of alloantigen induced prolonged cardiac allograft survival and generated regulatory cells in mice. In this study, we examined the role of alveolar macrophages (AM) in our ITD model. Some CBA mice were given ITD of C57BL/6 splenocytes and underwent transplantation of C57BL/6 hearts 7 days later. In others, AM were depleted with clodronate-loaded liposomes 3 days before ITD. In adoptive transfer studies, whole splenocytes were obtained from ITD-treated CBA mice and administered to naïve CBA secondary recipients, which were given C57BL/6 hearts immediately afterward. Interleukin-10 concentrations in bronchoalveolar lavage fluid were assessed by enzyme-linked immunosorbent assays. Immunohistologic and flow cytometric studies were performed after ITD. C57BL/6 splenocytes given by ITD were ingested by AM in 2 days and undetectable in paratracheal lymph nodes or spleen tissue. CBA mice given ITD of C57BL/6 splenocytes had markedly prolonged allograft survival (median survival time [MST], 86 days), whereas naïve CBA mice rejected allografts acutely (MST, 8 days). AM-depleted, ITD-treated mice also rejected allografts (MST, 5.5 days). Naïve secondary recipients given adoptive transfer of splenocytes from ITD-treated mice had prolonged allograft survival (MST, >100 days), whereas secondary recipients given adoptive transfer of splenocytes from AM-depleted, ITD-treated mice rejected the grafts (MST, 15.5 days). Interleukin-10 expression in bronchoalveolar lavage fluid was down-regulated in AM-depleted mice compared with naïve mice. AM have an important role in the induction of regulatory cells in our model of ITD of alloantigen.

Various Costimulatory Pathways Are Essential for Induction of Regulatory Cells by Intratracheal Delivery of Alloantigen

Background We previously reported that intratracheal delivery of alloantigen induced regulatory cells in a mouse heart transplantation model. We investigated the roles of costimulatory pathways in the induction of regulatory cells by intratracheal delivery of alloantigen. Methods CBA (H-2 k) mice were pretreated with intratracheal delivery of splenocytes (1 × 10 7) from C57BL/10 (H-2 b) mice and administration of monoclonal antibodies (mAb) specific for programmed death (PD)-1 and its ligands, programmed death-ligand (PD-L)1 and PD-L2, CD70, CD134 ligand (CD134L), CD153, CD137L, or receptor activator of nuclear factor-κB (NF-κB) (RANK). Seven days later, naive CBA mice underwent adoptive transfer of splenocytes (5 × 10 7) from the pretreated CBA mice and transplantation of C57BL/10 heart. Results Adoptive transfer of splenocytes from CBA mice that had been pretreated with intratracheal delivery of C57BL/10 splenocytes significantly prolonged the survival of C57BL/10 allograft (median survival time [MST], 68 days) as compared with adoptive transfer from untreated CBA mice (MST, 12 days). Concomitant administration of control immunoglobulin (Ig)G, anti-PD-L2 mAb, or anti-CD137L along with intratracheal delivery did not significantly affect the prolongation (MST, 72, 68, and 65 days, respectively). In contrast, anti-PD-1, anti-PD-L1, anti-CD70, anti-CD134L, anti-CD153, or anti-RANK mAb abrogated the prolongation induced by adoptive transfer from the pretreated mice with intratracheal delivery (MST, 18, 17, 16, 14, 10, and 18 days, respectively). Conclusion The PD-1/PD-L1, CD27/CD70, CD134/CD134L, CD30/CD153, and tumor necrosis factor (TNF)–related activation-induced cytokine (TRANCE)/RANK interactions are independently required for generation of regulatory cells by intratracheal delivery of alloantigen.

Effects of Immunosuppressants on Induction of Regulatory Cells After Intratracheal Delivery of Alloantigen

We previously reported that intratracheal delivery (ITD) of alloantigen generated regulatory cells in mice. Here, we examined the effect of various doses of conventional immunosuppressants (FK506, cyclosporine A, azathioprine, mycophenolate mofetil, and rapamycin) on inducing regulatory cells in our model. CBA mice (primary recipients) were given C57BL/6 splenocytes by ITD and either no additional treatment or various doses of an immunosuppressant. Seven days later, splenocytes from these mice were adoptively transferred into naive secondary CBA recipients that underwent C57BL/6 cardiac grafting the same day. Adoptive transfer from primary recipients given ITD of splenocytes alone induced prolonged allograft survival in secondary recipients (median survival time [MST], 50 days), suggesting that regulatory cells were generated. When ITD of alloantigen was combined with daily administration of 0.1 mg/kg FK506 or 0.2 mg/kg rapamycin, graft survival was similarly prolonged (MST 55 and 50 days, respectively). When combined with 20 or 40 mg/kg MMF or 0.4 mg/kg rapamycin, the majority of recipients demonstrated indefinite survival (MST, >100 days in all groups). When ITD of alloantigen was combined with 0.3, 0.5, or 1.0 mg/kg FK506; 5, 10, or 25 mg/kg cyclosporine A; or 1.0 or 2.0 mg/kg azathioprine, allografts were rejected acutely (MST 7-13 days). Generation of regulatory cells by ITD of alloantigen was facilitated by mycophenolate mofetil and high doses of rapamycin but abrogated by cyclosporine A, azathioprine, and high doses of FK506. Low doses of rapamycin and of FK506 did not interfere with generation of regulatory cells.

Interleukin-10 but not Transforming Growth Factor-β is Essential for Generation and Suppressor Function of Regulatory Cells Induced by Intratracheal Delivery of Alloantigen

We previously reported that intratracheal delivery of alloantigen-induced regulatory cells in mouse heart-transplantation model. Here, we investigated roles of interleukin (IL)-10 and transforming growth factor (TGF)-beta in induction and effector phases of the regulatory cells. CBA mice were pretreated with intratracheal delivery of C57BL/10 splenocytes and administration of neutralizing anti-IL-10 or anti-TGF-beta monoclonal antibody (mAb). Seven days after the pretreatment, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes from the pretreated mice and underwent heart grafting from C57BL/10 mice. To determine roles of these cytokines in the effector phase of the regulatory cells, anti-IL-10 or anti-TGF-beta mAb was administered weekly into the secondary recipients after the adoptive transfer. Adoptive transfer of splenocytes from CBA mice that had been pretreated with intratracheal delivery of C57BL/10 splenocytes significantly prolonged the survival of C57BL/10 allograft (median survival time [MST] 68 days) as compared with adoptive transfer from untreated CBA mice (MST 12 days). In the induction phase, anti-IL-10 mAb abrogated development of the regulatory cells that afforded prolonged allograft survival in the secondary recipients (MST 20 days), whereas anti-TGF-beta mAb did not abrogate it (MST 88 days). In the effector phase, anti-IL-10 mAb abrogated prolonged allograft survival afforded by adoptive transfer of the regulatory cells in the secondary recipients (MST 27 days), whereas anti-TGF-beta mAb did not abrogate suppressor function of the regulatory cells (MST 53 days). IL-10 but not TGF-beta was required for generation and suppressor function of the regulatory cells induced by intratracheal delivery of alloantigen.

Programmed death-1-programmed death-L1 interaction is essential for induction of regulatory cells by intratracheal delivery of alloantigen.

Programmed death (PD)-1 has been implicated in peripheral tolerance. The authors investigated the roles of PD-1 and its ligands, PD-L1 and PD-L2, in the induction of regulatory cells by intratracheal delivery of alloantigen. CBA (H-2k) mice were pretreated with intratracheal delivery of C57BL/10 (H-2b) splenocytes and administration of monoclonal antibody (mAb) specific for PD-1, PD-L1, or PD-L2. Seven days later, C57BL/10 hearts were transplanted into the pretreated CBA mice. Some naive CBA mice underwent adoptive transfer of splenocytes from the pretreated CBA mice and transplantation of C57BL/10 heart. Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 7 days). Pretreatment with intratracheal delivery of C57BL/10 splenocytes prolonged graft survival significantly (MST, 65 days). Administration of control immunoglobulin (Ig) G or anti-PD-L2 mAb did not significantly affect the prolongation (MST, 72 and 68 days, respectively). In contrast, anti-PD-1 or anti-PD-L1 mAb abrogated the prolongation (MST, 18 and 17 days, respectively). Adoptive transfer from mice pretreated with intratracheal delivery of alloantigen plus control IgG or anti-PD-L2 mAb prolonged survival of C57BL/10 grafts in secondary CBA recipients (MST, 72 and 56 days, respectively). However, concurrent administration of anti-PD-1 or anti-PD-L1 mAb abrogated prolonged survival after the adoptive transfer (MST, 14 and 20 days, respectively). PD-1-PD-L1 interaction was essential for induction of regulatory cells by intratracheal delivery of alloantigen.

CD27/CD70, CD134/CD134 ligand, and CD30/CD153 pathways are independently essential for generation of regulatory cells after intratracheal delivery of alloantigen.

We investigated whether blockade of tumor necrosis factor receptor-ligand pathways could generate regulatory cells induced by intratracheal delivery of alloantigen. CBA (H-2k) mice were pretreated with intratracheal delivery of splenocytes (1x10(7)) from C57BL/10 (H-2b) mice and intraperitoneal administration of monoclonal antibody (mAb) specific for CD70, CD134 ligand (CD134L), CD153, or CD137L. Seven days later, C57BL/10 hearts were transplanted into pretreated CBA mice. Some naive CBA mice underwent adoptive transfer of splenocytes (5x10(7)) from pretreated CBA mice and transplantation of a C57BL/10 heart on the same day. Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST] 12 days). Pretreatment with intratracheal delivery of C57BL/10 donor splenocytes prolonged graft survival significantly (MST 84 days). Mice given intratracheal delivery of alloantigen plus anti-CD70, anti-CD134L, or anti-CD153 mAb, but not those given intratracheal delivery of alloantigen plus anti-CD137L mAb, rejected their graft acutely (MST 16, 14, 10, and 65 days, respectively). Adoptive transfer of splenocytes from mice pretreated with intratracheal delivery of alloantigen plus anti-CD70, CD134L, or CD153 mAb did not prolong survival of C57BL/10 cardiac grafts in naive secondary CBA recipients (MST 14, 11, and 11 days, respectively), whereas adoptive transfer of splenocytes from mice given intratracheal delivery of alloantigen plus anti-CD137L mAb did (MST 75 days). The CD27/CD70, CD134/CD134L, and CD30/CD153 pathways are independently required for generation of regulatory cells in our model.

Prolonged survival of fully allogeneic cardiac grafts in naive mice and those with sensitization induced by antigen delivery through the respiratory tract

Nonresponsiveness to protein antigen induced by administration of the antigen through the alimentary or respiratory tract is well known as mucosal tolerance. We previously reported in this Journal that donor splenocytes delivered orally induce prolonged survival of fully allogeneic cardiac grafts and generate regulatory cells when delivered with a nondepleting anti-CD4 monoclonal antibody. Therefore, in this study we examined whether intratracheal delivery of alloantigen could induce prolonged survival of fully mismatched allogeneic cardiac grafts and regulatory cells.

Role of the CTLA4 pathway in hyporesponsiveness induced by intratracheal delivery of alloantigen.

The authors previously reported that intratracheal delivery (ITD) of donor alloantigen induced donor-specific hyporesponsiveness to C57BL/10 cardiac allografts in CBA recipients and that blockade of the B7 pathways abrogated that hyporesponsiveness. In this study, the authors used a CD28-deficient model to evaluate which signal, either through CD28 or cytotoxic T-lymphocyte-associated antigen (CTLA4), is involved in the induction of hyporesponsiveness. Seven days before transplantation of hearts from C3H/HeJ (H2k) mice into C57BL/6 (H2b) or CD28-deficient (C57BL/6 background) mice, the transplant recipients were given ITD of donor splenocytes (1 x 10(7)), alone or in combination with human CTLA4-immunoglobulin (Ig) (200 microg). ITD of C3H splenocytes induced donor-specific hyporesponsiveness to C3H cardiac grafts in C57BL/6 recipients (graft median survival time [MST], 40 days). Administration of CTLA4-Ig concurrently with ITD abrogated the prolonged allograft survival (MST, 12 days). Interestingly, ITD of C3H splenocytes induced prolonged survival of C3H allografts in CD28-deficient recipients (MST, 55 days). Furthermore, administration of CTLA4-Ig combined with ITD of C3H splenocytes abrogated the prolonged survival of C3H allografts in CD28-deficient recipients (MST, 7 days), whereas recipients given isotype-control antibody in combination with ITD of splenocytes had prolonged survival of C3H allografts (MST, 58 days). Taken together, the authors' findings indicate that a signal through CTLA4, rather than through CD28, plays an important role in the induction of hyporesponsiveness by ITD of alloantigen in this model.

B7/CTLA4 pathway is essential for generating regulatory cells after intratracheal delivery of alloantigen in mice.

The mechanism of hyporesponsiveness induced by intratracheal (IT) delivery of alloantigen was examined and its effect on cardiac graft survival was assessed in studies in mice. In CBA (H2 ) mice, donor splenocytes were given by IT delivery 7 days before transplantation of a C57BL/10 (H2 ) heart. To determine whether regulatory cells were involved in hyporesponsiveness, splenocytes from mice given IT delivery of alloantigen and antibodies for B7-1, B7-2, or CTLA4 were adoptively transferred to naïve secondary recipients 7 days after delivery; those recipients underwent heart transplantation the same day. Effects on cell proliferation and cytokine production of splenocytes from mice given IT delivery of alloantigen were examined in mixed leukocyte cultures (MLC). Cardiac graft survival was significantly prolonged in mice given IT delivery of alloantigen (median survival time [MST], 81 days); those given syngeneic splenocytes rejected grafts acutely (MST, 7 days; P<0.05). Adoptive transfer of splenocytes also significantly prolonged survival of cardiac grafts in secondary recipients (MST, 62 days). When B7-1, B7-2, or CTLA4 antibody was combined with IT delivery of alloantigen in the first recipient, all grafts were rejected within 14 days in second recipients after adoptive transfer. In mixed leukocyte cultures, splenocytes from these mice did not respond to alloantigen and production of interleukin-4 and interleukin-10 was increased. Donor splenocytes delivered IT induced hyporesponsiveness and regulatory cells in our animal model, and such induction was dependent on B7-1, B7-2, and CTLA4 signals.

FK 506 abrogates induction of regulatory cells after intratracheal delivery of alloantigen

IT HAS BEEN reported that one conventional immunosuppressant FK 506, prolongs allograft survival. However, the effects of the drug on interactions leading to induction of regulatory mechanisms remain unclear. We previously reported that intratracheal delivery of donor splenocytes could induce prolonged survival of a fully allogeneic cardiac graft, resulting in the generation of regulatory cells. Using this model, we examined the influence of FK 506 on the induction of regulatory cells after intratracheal delivery of alloantigen.

CD25+ regulatory cells generated by intratracheal delivery of alloantigen

CD25+ regulatory cells generated by intratracheal delivery of alloantigen