Cytokine modulation of TH1/TH2 phenotype differentiation in directly alloresponsive CD4+ human T cells.

Organ graft rejection is caused by the recognition of allogeneic MHC molecules by recipient T cells by two different pathways. The indirect pathway of alloreactivity requires the presentation of MHC peptides from the graft by autologous APC, as with conventional antigen. The direct pathway, on the other hand, requires the recognition of foreign MHC on foreign cells. The regulatory mechanisms for this component of alloreactivity have not been extensively studied. We show here that the T cell response activated by alloantigens in the direct pathway is similarly constrained and modulated by cytokines, as has been shown for classic antigen presentation. Thus, the inclusion of IL-2 or TGF-beta in MLC performed with purified responder T cells resulted in outgrowth of cells secreting IL-2 and IFN-gamma, whereas addition of IL-4, IL-10, or anti-TGF-beta encouraged outgrowth of cells secreting IL-4 and IL-10. T cells alloactivated via the direct pathway and then cloned in IL-2 alone secreted IL-4 and IL-10 as well as IFN-gamma and IL-2 (Th0 phenotype). Established clones remained susceptible to cytokine modulation, such that IL-4 and IL-10 decreased their secretion of IL-2 and IFN-gamma, whereas TGF-beta suppressed IL-4 and IL-10 secretion. The first alterations of Th0 toward Th1 or Th2 phenotypes could already be observed after only a very brief exposure to cytokines of 48 hr, followed by extended culture with IL-2 alone. These results confirm that human T cells with Th1 and Th2 phenotypes, recognizing alloantigen via the direct pathway, derive from the same IL-2-secreting precursor and can be manipulated by cytokines in an analogous fashion to conventional antigen-reactive cells. These findings may have implications for manipulating the direct pathway of alloantigen recognition in human organ transplantation.

Role of three quantitatively dominant endogenous peptides from HLA-DRB1*0401 molecules in class II specific alloreactivity.

Alloreactivity remains an important barrier to organ transplantation and is caused by T cell recognition of foreign histocompatibility antigens (HAg) in two ways: (1) indirect recognition, in which processed HAg peptides are presented by self MHC like any other foreign antigen, and (2) direct recognition, where the foreign MHC itself is recognized in contravention of the T cell recognition rule of self restriction. Whereas the role of endogenous peptides in direct MHC class I specific recognition is now established, their role in class II specific direct alloreactivity remains controversial, since no defined endogenous peptide has been shown to be required for alloreactivity. That mutations resulting in defective antigen processing impair class II specific allostimulation, however, suggests that the endogenous pathway is important for class II as well as class I alloreactivity. We attempted to establish the importance of endogenous peptides for alloreactivity by identifying common sequences of peptides bound by DR molecules of an HLA-DRB1*0401 homozygous B cell line. Peptides corresponding to three of these (calreticulin, HLA class I and an unidentified molecule) were used to restimulate established allospecific HLA-Dw4 reactive T cell clones, as well as to sensitize allogeneic T cells de novo in vitro. Xenogeneic chinese hamster ovary (CHO) cells coexpressing the relevant DR allele together with CD80 were used as antigen presenting cells. The role of CD80 could be determined on these cells because (1) they are xenogeneic and (2) they do not express B7 family members bound by CTLA-4Ig.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of the antifungal azoles itraconazole, fluconazole, ketoconazole and miconazole on cytokine gene expression in human lymphoid cells.

The antifungal azole drugs itraconazole (itra; R51,211), fluconazole (flu; UK-49,858), ketoconazole (keto) and micronazole (mico) have been investigated for their suppressive influence on the gene expression of the immunoregulatory cytokines IL2, IL4, IL9, GM-CSF, TNF-alpha, IFN-gamma, as well as both chains of the IL2 receptor in human PBMC and of the cytokines in the human keratinocyte cell line HaCat 17.5. The results obtained in Northern blot analysis were compared with the effects of the established immunosuppressant drug CSA and the new immunosuppressive drug FK 506, as well as the cytokine TGF-beta, which is also immunosuppressive. While 1 microgram/ml CSA and 0.1 microgram/ml FK 506 completely suppressed PHA-stimulated accumulation of mRNA for IL2, IL4, IL9, GM-CSF, TNF-alpha and IFN-gamma in PBMC, flu, keto and TGF-beta failed to inhibit any (except TNF-alpha blocked by TGF-beta). Itra and mico did suppress accumulation of mRNA, but unlike CSA and FK 506, only at high doses (10 micrograms/ml) and after extended incubation (24 h). None of the drugs nor TGF-beta suppressed the expression of the IL2R-alpha and IL2R-beta genes or TNF-alpha-stimulated cytokine gene expression in keratinocytes. Itra and mico, 1 mg/ml (achievable serum level), caused only slight inhibition of the cytokines in PBMC after 6 and 24 h of incubation. These results demonstrate that the mode of action of the azoles is different from CSA and FK 506.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin 10 is a human T cell growth factor in vitro.

Although interleukin (IL)-10 inhibited lymphocyte proliferation in mixed lymphocyte cultures (MLC) and blocked stimulation of alloreactive T cell clones (TCC) by peripheral blood mononuclear cells (PBMC), the cells surviving culture with IL-10 showed enhanced viability. A minority of IL-2-dependent T cell lines, moreover, incorporated tritiated thymidine when cultured with IL-10 alone; their proliferation with IL-10 was dose-dependent, prevented by addition of neutralizing antisera to IL-10 but not to IL-2 and/or IL-4 and observed both shortly (4 days) and later (7-10 days) after T cell allostimulation. Examination of the proliferative responses to IL-10 of a panel of TCC revealed heterogeneity of responsiveness: whereas only one of five CD8+ TCR2 (T cell receptor alpha, beta)-TCC proliferated with IL-10, three of five CD4+ TCR2-TCC proliferated, one of them strongly. In contrast, all three TCR1(gamma delta)-TCC tested responded to IL-10, albeit rather weakly. These results therefore suggest that in addition to its well-established inhibitory action on T cell activation, IL-10 may also exert positive influences on clonal expansion of subsets of preactivated T cells.

Induction of hyporesponsiveness in human lymphocytes-T despite their expression of both the co-receptor CD28 and its ligand B7.

A majority (42/62) of TCR2+ interleukin 2-dependent human T lymphocyte clones was found concordantly to express not only the CD28 co-receptor structure at the cell surface but also its ligand B7. Interactions between CD28 and B7 can have important consequences for T cell activation, particularly in providing "signal 2" to prevent the induction of anergy caused by stimulation via the antigen receptor only ("signal 1"). However, despite the expression of co-receptor and ligand on the same cell surface, it remained possible to induce hyporesponsiveness in T cell clones either when using CD3 antibodies to deliver signal 1 or when using other T cell clones as stimulators. Thus, the potential for intraclonal autostimulation via CD28/B7 is apparently insufficient to prevent downregulation of responsiveness in these two systems.

Requirements for stimulation or anergy induction in alloreactive human T cell clones.

The "two signal" concept for T cell activation is widely accepted. Signal 1 is commonly delivered via the antigen receptor, and signal 2 via accessory interactions. Delivery of both signals results in activation, signal 1 alone in induction of hyporesponsiveness. The nature of signal 1 in alloreactivity is not completely clear; most evidence suggests that a complex of foreign major histocompatibility complex molecules and their bound peptides is recognized. Interactions between B7 (CD80) ligand and CD28/CTLA-4 receptors are currently considered the most important sources of signal 2. Xenogeneic cells transfected with human genes provide useful stimulators for dissecting signals 1 and 2 in alloreactivity. We show here that the majority of DR-specific alloreactive T cell clones (TCC) fails to recognize Chinese hamster ovary (CHO) cells transfected with human DR, whether or not these are cotransfected with genes for CD80 or LFA-3. Stimulation was not observed even in the presence of a pool of peptides isolated by low pH release from B cell line (BCL)-derived DR molecules, or in the presence of synthetic peptides corresponding to the sequences of the three most commonly identified endogenous peptides. Lack of recognition was observed both in failure to stimulate proliferation and in failure to induce anergy. However, one TCC was identified which responded weakly to DR+ CHO cells, and for this clone, the presence of either CD80 or LFA-3 strongly enhanced proliferative responses. Anergy was not induced, even in the absence of CD80. Immobilized HLA-DR molecules purified from a BCL also failed to stimulate proliferation, but unlike the CHO transfectants, they did induce anergy. Stimulation with BCL also induced anergy if CD80-dependent interactions were blocked with soluble CTLA-4-Ig receptor. These results are consistent with the model that DR molecules expressed in the absence of appropriate peptide are simply not recognized by most alloreactive T cells, whereas DR molecules containing appropriate bound peptide are recognized as signal 1 and induce anergy. CTLA-4-Ig blocking confirms that CD80-dependent interactions can be important in preventing anergy induction, but that they are not always necessary is illustrated by the existence of a single clone which recognized DR molecules on CHO transfectants, giving very weak proliferation without CD80, and nonetheless no anergy induction.

Human gamma delta T cells are resistant to induction of anergy but not to induction of cell death in vitro.

A condition of hyporesponsiveness can be induced in certain mature human alpha beta (TCR2) cells relatively easily by their stimulation in the absence of costimulatory signals (signal 1 alone). This state of "anergy" has been implicated in tolerance to self and transplanted organs as well as tumors and may represent an important regulatory component of immune responsiveness. Little is known about whether the same condition applies to gamma delta (TCR1) cells. We therefore undertook to investigate anergy induction in TCR1 cell clones using several approaches known to induce this state in TCR2 cells. First, TCR1 clones were found not to be anergized by culture on immobilized CD3 monoclonal antibody (mAb), while the majority of TCR2 clones were anergized. Second, blocking of autocrine proliferation (stimulated in TCR1 or TCR2 clones by mitogen in the presence of accessory cells) using CTLA-4-lg, a soluble B7 family counterreceptor resulted in anergy induction in TCR2 cells but not TCR1 cells, although experiments with CHO cells transfected with B7-1 (CD80) genes confirmed that these TCR1 clones were responsive to costimulation with B7. Third, blocking mitogen-induced proliferation with anti-IL 2 receptor antibodies and anti-IL 2 antisera resulted in anergy induction in TCR2 but not TCR1 cells. Fourth, stimulation with the calcium ionophore ionomycin also anergized TCR2 but not TCR1 cells. In all four systems, but especially in the latter, stimulation by signal 1 alone resulted in high levels of cell death (> 50%) which was similar for both TCR1 and TCR2 cells. Therefore, these data may reflect a high level of resistance to tolerance induction (manifested as proliferative anergy) but not to clonal deletion (manifested as stimulation-dependent cell death) on the part of TCR1 cells.