THE IMMUNOSUPPRESSIVE DRUG FK778 INDUCES REGULATORY ACTIVITY IN STIMULATED HUMAN CD4+CD25- T CELLS

6 recipients of bm3 grafts demonstrated gradual and persistent loss of graft function over 100 days. Histological analysis revealed interstitial inflammatory infiltrates, edema and minimal fibrosis. In contrast, CD30/recipients of bm3 grafts maintained significantly better graft function over 100 days and displayed focal perivascular inflammation, more fibrosis and reduced numbers of apoptotic cells in the graft. Graft coronary artery disease (% affected vessels, stage of GCAD, % luminal narrowing, I:M ratio) and circulating alloantibody was also reduced in CD30-/reciepients. Conclusions: These results indicate that CD30 can modulate T cell proliferation as well as cytokine production in response to alloantigen. Moreover, these data demonstrate a role for CD30 costimulation in long

The induction of transplantation tolerance involves a T-cell mediated process of immune regulation. In clinical transplantation, the use of immunosuppressive drugs that promote or facilitate this process would be highly desirable. Here, we investigated the tolerance-promoting potential of the immunosuppressive drug FK778, currently under development for clinical therapy. Using a human allogeneic in vitro model we showed that, upon TCR triggering, FK778 induced a regulatory phenotype in CD4 + CD25 -T cells. Purified CD4 + CD25 -T cells primed in the presence of FK778, showed hyporesponsiveness upon restimulation with alloantigen in the absence of the drug. This anergic state was reversible by exogenous IL-2 and was induced independent of naturally occurring CD4 + CD25 + regulatory T cells. The FK778 induced anergic cells showed suppressor activity, were CD25 high , CD45RO + , CD27 -, CD62L -, and expressed CTLA-4, GITR and FoxP3. The cells revealed enhanced phosphorylation of STAT3. In conclusion, the new drug FK778 shows tolerizing potential through the induction of a regulatory T cell subset in CD4 + CD25 -T cells.

Abstract# 2483
Poster Board #-Session: P358-III Backgrounds: To tailor the immune suppression individually, understanding the mechanism of T cell activation is of great interest to minimize the advance effects of immunosuppressive agents after organ transplantation.
In this study, we investigated the potentiality of heme oxygenase-1, a homeostatic protein, in T cell activation.

Methods and results:
The expression of HO-1 was induced during the human CD4 cell activation upon CD3/CD28 stimulation in a dose-dependent manner. The expression levels of HO-1 were associated with the proliferation levels of CFSE-labelled CD4 cells and with higher levels of Bcl-xL and CD25 expression. Yeast two-hybrid and luciferase reporter assays showed that HO-1 could interact with CREB protein, the common transcription factor of Bcl-xL and CD25 expression, and could enhance CREB-mediated transcriptional activities. Phenotype analysis of HO-1 deficient mice showed that there were no significant differences in the amount of CD4+CD25+ and CD8+CD25+in the spleen or lymph nodes as compared to their wild-type littermate. Interestingly, HO-1-deficient CD4+ cells were resistant to Con-A or CD3/CD28 stimulation. The proliferation capabilities of CFSE-labelled HO-1-deficient CD4+ cells were significantly lower than wild-type cells. Although the expression of Bcl-xL and CD25 correlated strictly with the proliferation profile, the production of IL-2 was not affected by the HO-1 status of stimulated CD4+ cells. In addition, increase of HO-1 activity by TAT-mediated HO-1 protein transduction, a functional synthetic protein, was able to restore the expression levels of CD25 and the proliferation capabilities of HO-1-deficient CD4+ cells. Conclusions: Our data suggest that HO-1 plays a pivotal role in CD4+ T cell proliferation through enhancement of CREB activities on the transcription of CD25. Aims: Previously we showed that administration of a non-activating anti-CD28 single chain F-variable (scFv) Ab (αCD28) in conjunction with αCD154 (MR1) or CsA induces long-term allograft survival and donor-specific tolerance in mouse cardiac transplantation. Here we investigated whether graft acceptance with anti-CD28 targeting therapies is associated with upregulation of Treg cells in the spleen and in the graft. Methods: BALB/c recipients of C57BL/6 or BALB/c heterotopic cardiac transplants were untreated or received combined treatment of αCD28+MR1 or αCD28+CsA (scFv Ab, 200μg, d0-13; MR1, 250μg, d0; CsA, 400μg, d0-3). Donor and third-party skin grafting was performed at day 100. Graft infiltrating cells and splenocytes were harvested at D 10-12, and analyzed by FACS. On POD 100, intragraft gene expression was quantified by realtime RT-PCR (n≥2/group). Results: Graft survival >100d was seen in 8 of 11 mice with αCD28+MR1 and 9 of 12 mice with αCD28+CsA, vs. 9 days without treatment. At 100 days, recipients with surviving cardiac allografts demonstrated prolonged survival of donor skin (median 12 d with CsA, 41 d with MR1), but not third party (MST <8 days in both groups). On POD 10-12, FACs analysis revealed that the proportion of CD4+Foxp3+ in graft infiltrating cells was increased in anti-CD28 treated mice (αCD28+MR1: 5 %, αCD28+CsA: 3.4 %) as compared to untreated (0.9%) or naïve (0.3%) controls; in contrast, the CD4+ profile was similar in the spleen of all transplant groups. On POD 100, real-time RT-PCR revealed that in αCD28+MR1and αCD28+CsA-treated mice, intragraft foxp3 was at least 57 times higher than in isograft controls; CTLA-4, CD25, IFNγ, IL-2, IL-4, IL-10, and TGFβ-1 were also increased, from 1.8 to 33 times higher than in isograft controls.

Abstract# 2484 Poster Board #-Session
Conclusions: Presence of CD4+CD25+foxp3+ Treg cells in the graft during tolerance induction, and persistent expression of foxp3 and CTLA4 genes within an accepted allograft, suggest that emergence and persistence of Treg cells in the graft may be a critical immunomodulatory mechanism following perioperative anti-CD28 induction therapy. We postulate that these cells mediate durable peripheral donor-specific tolerance in situ, whereas, with this regimen, modulation of systemic anti-donor immunity is only sufficient to prolong donor skin survival.