Induction And Mechanism Of Materno-fetal Immune Tolerance By Adoptive Transfer Of The Paternal Antigen-anergic CD4～+T Cells

The embryo expresses paternal antigens foreign to the mother, and therefore has been viewed as allograft. Pregnancy constitutes a major challenge to the maternal immune system, as it has to tolerate the persistence of paternal alloantigen. Induction of the maternal immuno-tolerance to embryo is a main purpose for the maintenance of materno-fetal relationship. In organ transplantation, it has been shown that the anergic T cells generated in vivo and in vitro can be transferred as suppresser cells to prevent allograft rejection. However, to our knowledge, the capacity of the paternal antigen-anergic T cells to suppress maternal immune rejection to the allogeneic fetus in vivo has been less known.The antigen-specific T cell activation is a critical step in the rejection of transplanted allografts. T-cell activation depends on ligation of T-cell receptor (TCR) by specific antigen-MHC complex as well as costimulatory signals delivered by interaction of the certain adhesion molecules expressed on T cells and antigen-presenting cells (APC). The recognition of TCR upon the antigen peptide presented by MHC on APC in the absence of a costimulatory signal would induce a state of T-cell anergy in vitro as well as in vivo. Among a series of costimulatory pathways examined so far, the interaction of CD28 or CTLA-4 on T cells to CD80 and CD86 molecules on APC is the most critical pathway that determines whether the TCR-stimulated T cells become activated or anergic. This knowledge has been successfully used in animal models to induce the anergic T cells and prevent allograft rejection by blocking CD80 and CD86, thereby leading to long-term graft survival.In the present study, we employed a murine abortion-prone model in which CBA/J females mated by DBA/2 males have a high incidence of fetal resorptions, whereas a similar (H-2^k × H-2^d) matings, CBA/J females mated by BALB/c males, have normal and low resorption rates. The CD4⁺ T cells from non-pregnant CBA/J females were co-cultured with inactivated DBA/2 splenocytes in the presence of anti-CD80 and CD86 mAbs to generate the paternal antigen-anergic CD4⁺ T cells in vitro. Thereafter,these anergic CD4⁺ T cells were adoptively transferred into the abortion-prone CBA pregnant mice so as to investigate effects of paternal antigen-anergic CD4⁺ T cells on pregnant outcome of the abortion-prone matings and possible mechanisms.Part I Preparation and characterization of the paternal antigen-anergic CD4⁺ T cellsObjective To prepare the paternal antigen-anergic CD4⁺ T cells and identify its characteristics in vitro.Methods To prepare the paternal antigen-anergic T cells, CD4⁺ T cells freshly isolated from non-pregnant CBA females' splenocytes by negative selective magnetic beads, were co-cultured with the mitomycin C-treated DBA/2 splenocytes in the presence of anti-CD80/CD86 mAbs. The anergic CD4⁺ T cells were restimulated with inactivated DBA/2 splenocytes, immobilized anti-CD3mAb, and ConA or PMA plus ionomycin. To reverse the anergic state, recombinant murine IL-2 was added to the secondary MLR. The specificity and dose/contact-dependent manner of suppressive effects of the anergic CD4⁺ T cells on freshly isolated CD4⁺ T cells were identified by mixed lymphocyte reaction (MLR) and transwell test, respectively. RT-PCR and ELISA were respectively applied for assessing GRAIL mRNA transcription level and expression of Thl/Th2 cytokines in the anergic CD4⁺ T cells.Results The anergic CD4⁺ T cells failed to proliferate in response to immobilized anti-CD3mAb and paternal antigen recharge, but could proliferate in response to ConA or PMA plus ionomycin at a level similar to that of the freshly isolated CD4⁺ T cells. The anergy of the CD4⁺ T cells could be reversed by the exogenous IL-2. The anergic CD4⁺ T cells could suppress the response of the freshly isolated CD4⁺ T cells upon paternal antigens stimulation in a dose-dependent and antigen-specific manner in primary MLR. Transcription of GRAIL mRNA was enhanced in the anergic CD4⁺ T cells. Expression of IL-2, IFN-γ, IL-4, IL-10 and TGF-β in the anergic CD4⁺ T cells decreased significantly compared to that of the freshly isolated CD4⁺ T cells (P<0.05).Conclusions The anergic CD4⁺ T cells could suppress the response of the freshly isolated CD4⁺ T cells upon paternal antigens stimulation in a dose-dependent, directcell-to-cell contact and antigen-specific manner. The anergic CD4⁺ T cells could act as suppressor cells in vitro, and their suppressive effects might manifest independent of cytokines, such as IL-4, IL-10 or TGF-|3.Part II The role of CD4⁺CD25⁺ regulatory T cells in the CD4⁺ T cell anergy inductionObjective To probe into the essential role of CD4⁺ CD25⁺ T cells in induction of the paternal antigen-anergic CD4⁺ T cells.Methods Flow cytometry and RT-PCR were respectively applied for assessing the subset of CD4⁺CD25⁺regulatory T cells, and the transcription of Foxp3, marker of regulatory T cell lineage, after anergy had been induced in CD4⁺ T cells. The CD4⁺CD25⁺, CD4⁺CD25^- T cells were purified from non-pregnant CBA female splenocytes by magnetic beads. The CD4⁺CD25⁺ T cells and CD4⁺CD25^-T cells in different ratios were mixed, and then co-cultured with the mitomycin C-treated DBA/2 splenocytes in absence or presence of anti-CD80/CD86 mAbs. IL-2 expression was assayed by ELISA.Results The CD4⁺CD25⁺ T subset was expanded in the anergic CD4⁺ T cells. Intracellular expression of CTLA-4 and transcription of Foxp3 in the anergic CD4⁺CD25⁺ T cell population was up-regulated. The CD25-depleted CD4⁺T cells had a higher and more sustained allo-responsiveness than did the whole CD4⁺ T cells. In the absence of anti-CD80/CD86 mAbs, both the whole CD4⁺ T cells and CD25-dpleted CD4⁺ T cells responded vigorously to the DBA/2 Ag restimulation. The CD25-dpleted CD4⁺ T cells that had been cultured with anti-CD80/CD86 mAbs had secondary Proliferative response in excess of the whole CD4⁺ T cells. The CD4⁺CD25⁺ T cells were essential for the anergy of CD4⁺ T cells induced via CD80/CD86 blockade.Conclusions The CD4⁺CD25⁺ T cells were required for induction of anergy to paternal antigen via costimulatory blockade. Presence of the CD4⁺CD25⁺ T cells facilitated induction of the CD4⁺ T cell anergy, and the CD4⁺CD25^- T cells could be rendered anergic by CD4⁺CD25⁺ T cells. Ligation of CD80 and CD86 on naive Tcells by the cell surface molecule CTLA-4 on CD4⁺CD25⁺ T cells results in suppression of naive T cell response upon the alloantigen stimulation.Part III Effect of the adoptively transferred paternal antigen-anergic CD4⁺ T cells on the pregnant outcome of abortion-prone matingsObjective To study the effect of the adoptively transferred paternal antigen-anergic CD4⁺ T cells on pregnant outcome of abortion-prone matings, and the mechanisms involved.Methods The abortion-prone CBA females mated with DBA/2 males were adoptively transferred with DBA/2 antigen-anergic CD4⁺ T cells, C57BL/6 antigen-anergic CD4⁺ T cells, and CD4⁺ T cells from non-pregnant CBA females on day 0-2 or day 4 of gestation via tail vein, CBA females mated with DBA/2 males without transfer as control. The CD4⁺ CD25⁺ T cells were obtained from spleens of non-pregnant CBA females, DBA/2 antigen-anergic CD4⁺ T cells, and CBA females mated with DBA/2 males by immuno-magnetic beads respectively, and then labeled with CFSE in vitro. The CFSE-labeled CD4⁺ CD25⁺ T cells were transferred to abortion-prone CBA females mated with DBA/2 males on day 0-2 of gestation. In 48 and 96 hours after the adoptive transfer, two-photon confocal microscopy was used to image the positioning of CFSE-labeled cells within the host spleen, uterus-draining lymph nodes, and at materno-fetal-interface. ELISA was applied to determine the secretion in vitro of Th1 and Th2 cytokines (IL-4, IL-10, TGF-β, TNF-α, IFN-γ) at the host materno-fetal interface, and then the embryo resorption rate was counted on day 14 of gestation. On day 9 of gestation, the host splenocytes composed of CFSE⁺ and CFSE" T cells were primed with DBA/2 splenocytes for 4 days in vitro, and then the expression of intracellular cytokines (IL-2, IL-4, IL-10 and IFN-γ) and membrane costimulatory molecules (CD80, CD86, CD28 and CTLA-4) in CFSE^+/-T cells was analyzed by flow cytometry. IDO activity in the cultured supernatants of the materno-fetal-interface tissues of the host was evaluated by HPLC.Results Adoptive transfer of the DBA/2 antigen-anergic CD4⁺ T cellssignificantly increased fetal survival of the CBA/JxDBA/2 matings at a level comparable to that of adoptive transfer of CD4⁺ T cells from BALB/c-mated CBA mice. In contrast, adoptive transfer of C57BL/6 antigen-anergic CD4⁺ T cells or CD4⁺ T cells from non-pregnant CBA females did not reduce significantly the embryo resorption rate in the abortion-prone matings. A decrease in the production of TNF-α and IFN-γ was accompanied by a parallel increase in the production of IL-4 and IL-10, as well as TGF-β at materno-fetal interface in the host after adoptive transfer of either the DBA/2 antigen-anergic CD4⁺ CD25⁺ T cells or CD4⁺ CD25⁺ T cells from BALB/c-mated CBA/J mice. In addition, adoptive transfer of both the paternal antigen-anergic CD4⁺ CD25⁺ T cells and CD4⁺ CD25⁺ T cells from BALB/c-mated CBA mice significantly reduced the embryo resorption rate in DBA/2-mated CBA females, too. Moreover, the transferred cells were localized in spleen, uterus-draining lymph node, as well as at maternal-fetal-interface of the pregnant host. Compared to transfer of CD4⁺ T cells from non-pregnant CBA/J mice, transfer of either paternal antigen-anergic CD4⁺ CD25⁺ T cells or CD4⁺ CD25⁺ T cells from BALB/c-mated CBA/J mice resulted in lower frequency of cells positive for IL-2, IFN-γ and CD28, and higher frequency of CTLA-4 and IL-10-producing cells in host CFSE^- T population, whereas the frequency of IL-4-producing cells did not show significant change. The IDO activity was up-regulated at materno-fetal-interface of the pregnant host after transferred with either the paternal antigen-anergic CD4⁺ CD25⁺ T cells or the CD4⁺ CD25⁺ T cells from BALB/c-mated CBA/J females.Conclusions Adoptive transfer of the paternal antigen-anergic CD4⁺ T cells restored the Th2 bias at the materno-fetal interface in abortion-prone matings, which was crucial to the maintenance of normal pregnancy, and induced the materno-fetal immuno-tolerance, thus leading to decrease in the resorption rate of abortion-prone matings to that of normal pregnancy. The transferred paternal antigen-anergic CD4⁺ T cells had an immunosuppressive effect on potentially reactive T cells of the host, made host naive T cells to bear the same characteristics in terms of the expression of intracellular cytokines and costimulatory molecules as anergic T cells, which induced host naive T cells tolerance to the paternal antigen, and successfully prevent from maternal rejection to the allogeneic fetus by infectious mechanisms. Therefore, the paternal antigen-anergic CD4⁺CD25⁺ T cells play a key role in materno-fetal immune tolerance induced by adoptive transfer of the DBA/2 antigen-anergic CD4⁺ T cells.In conclusion, our findings suggest that the transferred paternal antigen-anergic CD4⁺ T cells not only function as antigen-specific suppresser cells but also disable the host naive T cells, which co-suppress maternal rejection to the allogeneic fetus, thus resulting in the decrease of the embryo resorption rate of the abortion-prone matings.