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Immunosuppression is an important treatment modality in transplantation and human diseases that are associated with aberrant T cell activation. There are considerable differences regarding the cellular processes targeted by the immunosuppressive drugs that are in clinical use. Drugs like azathioprine (Aza) mainly act by halting proliferation of fast dividing cells, whereas others like cyclosporine A (CsA) specifically target signaling pathways in T cells. Since the outcome of T cell responses critically depends on the quality and strength of costimulatory signals, this study has addressed the interplay between costimulation and the immunosuppressive agents CsA and Aza during the in vitro activation of human T cells. We used an experimental system that allows analyzing T cells activated in the presence of selected costimulatory ligands to study T cells stimulated via CD28, CD2, LFA-1, ICOS or 4-1BB. The mean inhibitory concentrations (IC(50)) for Aza and CsA were determined for the proliferation of T cells receiving different costimulatory signals as well as for T cells activated in the absence of costimulation. CD28 signals but not costimulation via CD2, 4-1BB, ICOS or LFA-1 greatly increased the IC(50) for CsA. By contrast, the inhibitory effects of Aza were not influenced by T cell costimulatory signals. Our results might have implications for combining standard immunosuppressive drugs with CTLA-4Ig fusion proteins, which act by blocking CD28 costimulation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

IL-1 receptor accessory protein (IL-1RAcP) is the second subunit required to form a functional receptor complex for IL-1α and β, IL-1F6, IL-1F8, IL1-F9 and IL-33. While it does not directly interact with the cytokines, IL-1RAcP is necessary to mediate signal transduction. We previously reported a monoclonal antibody with an unknown specificity, termed AY19, that was capable to induce a significant increase in the size of CFU-GM colonies when added to cultures of human cord blood CD34(+) hematopoietic progenitors. Here we demonstrate that AY19 mAb recognizes IL1-RAcP. We show that this adaptor molecule is significantly present on peripheral blood monocytes and lymphocytes including CD4(+) and CD8(+) T lymphocytes, B and NK cells. Interestingly, its expression is found increased on CD127(low)CD4(+)CD25(high) T cells when compared to CD127(low)CD4(+)CD25(-) T cell subset, suggesting that the level of IL-1RAcP membrane expression could allow to distinguish within CD127(low)CD4(+) T lymphocytes the CD25(high) T regulatory subset from conventional CD25(-) T lymphocytes. Functional studies reveal that addition of AY19 mAb enhances the proliferation of peripheral blood mononuclear cells (PBMC) obtained with mitogenic concentrations of PMA. Interestingly, we found that although AY19 mAb does not increase the optimal PBMC proliferation induced by a mitogenic pair of anti-CD2 mAbs it prolongs their time of proliferation. Thus, these results indicate that the anti-IL-1RAcP mAb AY19 exhibits unique functional properties by triggering co-stimulatory signals in lymphocytes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

For the purpose of establishing a new adoptive immunotherapy for bile duct carcinoma (BDC), we previously constructed two kinds of bispecific antibodies (bsAbs), anti-MUC1 x anti-CD3 (M x 3) and anti-MUC1 x anti-CD28 (M x 28), which activate T cells and form bridges between them and MUC1-expressing tumor cells. In our previous studies [Cancer Res. 56 (1996) 4205] specific targeting therapy (STT) consisting of i.v. administration of lymphokine activated killer cells with a T cell phenotype (T-LAK) sensitized with two kinds of bsAbs to human BDC-grafted severe combined immunodeficient (SCID) mice demonstrated remarkable inhibition of tumor growth. However, complete cures could not be obtained. In order to improve antitumor efficacy, we have paid attention to anti-CD2 monoclonal antibodies (mAbs), thought to play an important roles in signal transduction in T cell activation or control of T cell receptor (TCR)-driven activation. Therefore, we developed another bsAb, anti-MUC1 x anti-CD2 (M x 2), in order to examine if this would show synergism with the two previously described bsAbs. The combination of the three bsAbs (M x 3, M x 28 and M x 2 bsAbs) showed highest cytotoxicity against MUC1-expressing BDC cells when given simultaneously with peripheral blood mononuclear cells (PBMCs) or T-LAK cells in vitro. When 2 x 10(7) T-LAK cells sensitized with different combinations of bsAbs were administered four times i.v. to BDC-grafted SCID mice, the best therapeutic result was obtained with a combination of all three bsAbs. These results indicate usefulness of combination of three bsAbs for targeting cancer immunotherapy.

LO-CD2a/BTI-322, a rat anti human CD2 mAb, shows in vitro and in vivo immunosuppressive properties and induces T-cell depletion resulting partially from an antibody dependent cellular cytotoxicity (ADCC) mediated by NK cells. The aim of this paper is to study the in vitro effect of LO-CD2a/BTI-322 on NK cells, the majority of them also expressing the CD2 molecule. The addition of the mAb to purified naive NK cells induces apoptosis of CD2+ cells. The apoptosis is rapid, Fas ligand independent and completely inhibited by the calcium chelator EGTA, suggesting a fractricidal ADCC reaction and implying that NK cells are not resistant to lysis when used as target cells. At the end of the reaction, the CD2 - remaining cells are still capable of natural cytotoxicity against K562 cells, but at a lower rate than untreated cells.

Phosphorylation of the cAMP-response element binding protein CREB within 1 h of CD2 but not CD3 cross-linking of human PBMC was recently demonstrated. The absence of P-CREB following CD3 cross-linking was unexpected, as other laboratories reported increased phosphorylation of CREB following CD3 cross-linking of the Jurkat lymphocyte cell line. Due to Jurkat T-cells being IL-2-independent, it was postulated that IL-2 might provide a necessary co-stimulus for phosphorylation of CREB in primary lymphocytes. Therefore, P-CREB was evaluated following co-stimulation of human PBMC through the IL-2 and CD2 or CD3 receptors. IL-2 did not further augment phosphorylation of CREB following CD2 cross-linking. However, while neither IL-2 nor CD3 cross-linking alone induced P-CREB, a 4.5-fold increase in phosphorylation of CREB within 1 h of IL-2/CD3 co-stimulation was observed. Phosphorylation was not associated with the induction of cAMP, and inhibition of PKA signaling had no effect on P-CREB. Consistent with signal transduction through p56lck or p59fyn, inhibition of PTK signaling reduced phosphorylation 50%. Interestingly, inhibiting PKC signaling with calphostin C further increased P-CREB levels 3-fold over that observed in IL-2/CD3 co-stimulated cells not pretreated with a PKC inhibitor. In contrast to previous studies performed in the absence of exogenous IL-2, no increase in binding of CREB to a 32P-labeled oligonucleotide probe was observed by electrophoretic mobility shift assay. These data suggest that the IL-2 and CD3 signaling pathways provide a necessary and co-operative stimulus promoting phosphorylation of CREB following receptor cross-linking.

Natural resistance or susceptibility of host to infection with several intracellular pathogens, such as Mycobacterium, Salmonella and Leishmania, is controlled in mice by the expression of a single dominant gene locus designated Lsh/Ity/Bcg. Natural resistance-associated macrophage protein gene 1 (Nramp1) was isolated as a candidate gene. Nramp1 gene encodes a 60 kDa polypeptide with 10-12 potential transmembrane domains and an evolutionary conserved consensus transport motif. The present study shows that the human NRAMP1 gene is expressed in all established hematopoietic cell lines examined, including monocytes/macrophages and B- and T-lymphocytes. In contrast, cell type-specific expressions are observed in human peripheral blood leukocytes. NRAMP1 expression is very low level in granulocytes. B- and T-lymphocytes are equivalent in the level of NRAMP1 expression. Notable expression of NRAMP1 gene can be detected in the monocyte population. These results have important implications for the host defence mechanisms and the pathogenesis of intracellular pathogens which are recognized and ingested by the mononuclear phagocyte system including monocytes/macrophages.

The epithelium of the murine vagina contains a resident population of gamma delta T-cells that expresses a homogenous Vgamma4/Vdelta1 TCR lacking N-region junctional diversity, implying that these T-cells recognize a very limited array of antigenic structures. The vaginal gamma delta T-cells express a pattern of surface markers characteristic of memory/effector T-cells that have previously been activated. Although vaginal gamma delta T-cells do not express the major costimulatory molecules CD28 and CD2, they do proliferate in response to a systemically delivered anti gamma delta TCR stimulus. Vaginal gamma delta T-cells contain mRNA that encodes the keratinocyte growth factor raising the possibility that these cells play a role in the repair of vaginal epithelium following injury. While the antigen recognized by the vaginal gamma delta TCR is unknown, a model is proposed which attempts to relate some of the unusual phenotypic features of vaginal gamma delta T-cells to the physiological injury and shedding of vaginal epithelium that occurs during the estrous cycle.