No abstract

Inducible costimulatory molecule (ICOS) plays a pivotal role in T cell activation and Th1/Th2 differentiation. ICOS blockade has disparate effects on immune responses depending on the timing of blockade. Its role in transplantation immunity, however, remains incompletely defined. We used a vascularized mouse cardiac allograft model to explore the role of ICOS signaling at different time points after transplantation, targeting immune initiation (early blockade) or the immune effector phase (delayed blockade). In major histocompatibility-mismatched recipients, ICOS blockade prolonged allograft survival using both protocols but did so more effectively in the delayed-treatment group. By contrast, in minor histocompatibility-mismatched recipients, early blockade accelerated rejection and delayed blockade prolonged graft survival. Alloreactive CD4 + T cell expansion and alloantibody production were suppressed in both treatment groups, whereas only delayed blockade resulted in suppression of effector CD8 + T cell generation. After delayed ICOS blockade, there was a diminished frequency of allospecific IL-10-producing cells and an increased frequency of both IFN-γ-and IL-4-producing cells. The beneficial effects of ICOS blockade in regulating allograft rejection were seen in the absence of CD28 costimulation but required CD8 + cells, cytotoxic T lymphocyte antigen-4, and an intact signal transducer and activator of transcription-6 pathway. These data define the complex functions of the ICOS-B7h pathway in regulating alloimmune responses in vivo.

The inability to reproducibly induce robust and durable transplant tolerance using CD28-B7 pathway blockade is in part related to the persistence of alloreactive effector/memory CD8+ T cells that are less dependent on this pathway for their cellular activation. We studied the role of the novel T cell costimulatory pathway, CD27-CD70, in alloimmunity in the presence and absence of CD28-B7 signaling. CD70 blockade prolonged survival of fully mismatched vascularized cardiac allografts in wild-type murine recipients, and in CD28-deficient mice induced long-term survival while significantly preventing the development of chronic allograft vasculopathy. CD70 blockade had little effect on CD4+ T cell function but prevented CD8+ T cell-mediated rejection, inhibited the proliferation and activation of effector CD8+ T cells, and diminished the expansion of effector and memory CD8+ T cells in vivo. Thus, the CD27-CD70 pathway is critical for CD28-independent effector/memory CD8+ alloreactive T cell activation in vivo. These novel findings have important implications for the development of transplantation tolerance-inducing strategies in primates and humans, in which CD8+ T cell depletion is currently mandatory.

Hairy roots were induced by inoculation of stems of sterile plants of Atropa belladonna with Agrobacterium rhizogenes. The axenic culture of the hairy roots isolated from the stems proliferated 60 fold as based on the initial fresh weight after one month of culture. The presence of atropine and scopolamine in hairy roots were examined by TLC and HPLC. Their amounts were analyzed by GLC. The results show that the amount of the two alkaloids in the axenic cultures was the same as or even higher than those of normal plants grown in the field.

ObjectiveTo determine the precise in vivo interaction between T-cell costimulatory blockade and conventional immunosuppression in transplantation. Summary Background DataBlocking B7 or CD154 T-cell costimulatory activation pathways prevents allograft rejection in small and large animal transplant models and is considered a promising strategy for clinical organ transplantation. MethodsA fully MHC-mismatched vascularized mouse cardiac allograft model was used to test the interactions between anti-CD154 or CTLA4Ig monotherapy and conventional immunosuppressive drugs in promoting long-term graft acceptance. The frequency of alloreactive T cell was measured by ELISPOT. Chronic rejection was examined by histology. ResultsCyclosporine, tacrolimus, and anti-IL-2R monoclonal antibody therapy abrogated the effect of a single-dose protocol of anti-CD154 therapy. In contrast, rapamycin acted synergistically with anti-CD154 therapy in promoting long-term allograft survival. The addition of calcineurin inhibitors did not abolish this synergistic effect. Intense CD154-CD40 blockade by a multiple-dose schedule of anti-CD154 resulted in long-term graft survival and profound alloreactive T-cell unresponsiveness and overcame the opposite effects of calcineurin inhibitors. CTLA4Ig induced long-term graft survival, and the effect was not affected by the concomitant use of any immunosuppressive drugs. ConclusionsThe widespread view that calcineurin inhibitors abrogate the effects of T-cell costimulatory blockade should be revisited. Sufficient costimulatory blockade and synergy induced by CD154 blockade and rapamycin promote allograft tolerance and prevent chronic rejection.

Inducible costimulatory molecule (ICOS) plays a pivotal role in T cell activation and Th1/Th2 differentiation. ICOS blockade has disparate effects on immune responses depending on the timing of blockade. Its role in transplantation immunity, however, remains incompletely defined. We used a vascularized mouse cardiac allograft model to explore the role of ICOS signaling at different time points after transplantation, targeting immune initiation (early blockade) or the immune effector phase (delayed blockade). In major histocompatibility-mismatched recipients, ICOS blockade prolonged allograft survival using both protocols but did so more effectively in the delayed-treatment group. By contrast, in minor histocompatibility-mismatched recipients, early blockade accelerated rejection and delayed blockade prolonged graft survival. Alloreactive CD4 + T cell expansion and alloantibody production were suppressed in both treatment groups, whereas only delayed blockade resulted in suppression of effector CD8 + T cell generation. After delayed ICOS blockade, there was a diminished frequency of allospecific IL-10-producing cells and an increased frequency of both IFN-γ-and IL-4-producing cells. The beneficial effects of ICOS blockade in regulating allograft rejection were seen in the absence of CD28 costimulation but required CD8 + cells, cytotoxic T lymphocyte antigen-4, and an intact signal transducer and activator of transcription-6 pathway. These data define the complex functions of the ICOS-B7h pathway in regulating alloimmune responses in vivo.

We developed a new method for culture of isolated pollen. Using highly homogeneous populations of immature pollen grains of Nicotiana tabacum L. prepared by means of Percoll density gradient centrifugation, we could direct their developmental pathway by regulating certain culture conditions. When the pollen population was cultured in basal medium with glutamine, most pollen grains underwent normal maturation. On the other hand, when first cultured in basal medium without glutamine, most pollen grains did not mature but after transfer to medium with glutamine and sucrose began to divide. This method for inducing pollen cell division was possible only with midbinucleate pollen grains which are characterized by having no central vacuole and no or only a few starch grains. Evidently, some essential changes necessary for the embryogenic response can be induced by glutamine starvation only in pollen grains at a specific stage.

The mechanisms underlying physiological regulation of alloimmune responses remain poorly defined. We investigated the roles of cytokines, CTLA-4, CD25+ T cells, and apoptosis in regulating alloimmune responses in vivo. Two murine cardiac transplant models were used, B10.D2 (minor mismatch) and C57BL/6 (major mismatch), into BALB/c recipients. Recipients were wild type, STAT4−/− (Th1 deficient), or STAT6−/− (Th2 deficient) mice. Minor mismatched allografts were accepted spontaneously in ∼70% of wild type and STAT4−/− mice. By contrast, there was significantly shorter graft survival in minor mismatched STAT6−/− mice. Either the adoptive transfer of STAT4−/− splenocytes or the administration of IL-4Fc fusion protein into STAT6−/− mice resulted in long term graft survival. Blocking CTLA-4 signaling accelerated the rejection in all recipients, but was more pronounced in the minor combination. This was accompanied by an increased frequency of alloreactive T cells. Furthermore, CTLA-4 blockade regulated CD4+ or CD8+ as well as Th1 or Th2 alloreactive T cells. Finally, while anti-CD25 treatment prolonged graft survival in the major mismatched combination, the same treatment accelerated graft rejection in the minor mismatched group. The latter was associated with an increased frequency of alloreactive T cells and inhibition of T cell apoptosis. These data demonstrate that cytokine regulation, CTLA-4 negative signaling, and T cell apoptosis play critical roles in regulating alloimmunity, especially under conditions where the alloreactive T cell clone size is relatively small.