Jacqueline M. Slavik

An autoimmune disease-associated CTLA-4 splice variant lacking the B7 binding domain signals negatively in T cells.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in downregulating T cell responses. A number of autoimmune diseases have shown genetic linkage to the CTLA-4 locus. We have cloned and expressed an alternatively spliced form of CTLA-4 that has genetic linkage with type I diabetes in the NOD mice. This splice variant of CTLA-4, named ligand-independent CTLA-4 (liCTLA-4), lacks exon2 including the MYPPPY motif essential for binding to the costimulatory ligands B7-1 and B7-2. Here we show that liCTLA-4 is expressed as a protein in primary T cells and strongly inhibits T cell responses by binding and dephosphorylating the TcRzeta chain. Expression of liCTLA-4, but not full-length CTLA-4 (flCTLA-4), was higher in memory/regulatory T cells from diabetes-resistant NOD congenic mice compared to susceptible NOD mice. These data suggest that increased expression and negative signaling delivered by the liCTLA-4 may regulate development of T cell-mediated autoimmune diseases.

Genetic Analysis of Human Traits In Vitro: Drug Response and Gene Expression in Lymphoblastoid Cell Lines

Lymphoblastoid cell lines (LCLs), originally collected as renewable sources of DNA, are now being used as a model system to study genotype–phenotype relationships in human cells, including searches for QTLs influencing levels of individual mRNAs and responses to drugs and radiation. In the course of attempting to map genes for drug response using 269 LCLs from the International HapMap Project, we evaluated the extent to which biological noise and non-genetic confounders contribute to trait variability in LCLs. While drug responses could be technically well measured on a given day, we observed significant day-to-day variability and substantial correlation to non-genetic confounders, such as baseline growth rates and metabolic state in culture. After correcting for these confounders, we were unable to detect any QTLs with genome-wide significance for drug response. A much higher proportion of variance in mRNA levels may be attributed to non-genetic factors (intra-individual variance—i.e., biological noise, levels of the EBV virus used to transform the cells, ATP levels) than to detectable eQTLs. Finally, in an attempt to improve power, we focused analysis on those genes that had both detectable eQTLs and correlation to drug response; we were unable to detect evidence that eQTL SNPs are convincingly associated with drug response in the model. While LCLs are a promising model for pharmacogenetic experiments, biological noise and in vitro artifacts may reduce power and have the potential to create spurious association due to confounding.

CD28/CTLA-4 and CD80/CD86 families: signaling and function.

T cell stimulation in the absence of a second, costimulatory signal can lead to anergy or the induction of cell death. CD28 is a major T cell costimulatory receptor, the coengagement of which can prevent anergy and cell death. The CD28 receptor is a member of a complex family of polypeptides that includes at least two receptors and two ligands. Cytotoxic lymphocyte-associated molecule-4 (CTLA-4, CD152) is the second member of the CD28 receptor family. The ligands or counterreceptors for these two proteins are the B7 family members, CD80 (B7-1) and CD86 (B7-2). This article reviews the CD28/CTLA4 and CD80/CD86 families, and outlines the functional outcomes and biochemical signaling pathways recruited after CD28 ligation.

TIM-4 Expressed on APCs Induces T Cell Expansion and Survival

TIM (T cell, Ig, mucin) proteins can regulate T cell immune responses. Tim-4 mRNA is not expressed in T cells, but exclusively in APCs. Tim-4 is a ligand for Tim-1 and Tim-4.Ig fusion protein was shown to either inhibit or expand T cells. However, the molecular basis for such opposite effects was not defined. By generating mAbs, we show that expression of Tim-4 protein is restricted to CD11c+ and CD11b+ cells and is up-regulated upon activation. We show that Tim-4 specifically phosphorylates Tim-1 and induces T cell expansion by enhancing cell division and reducing apoptosis. Tim-4 also induces the phosphorylation of signaling molecules LAT, Akt, and ERK1/2 in T cells. Tim-4, expressed on APCs, is a costimulatory molecule that promotes T cell expansion and survival by cross-linking Tim-1 on T cells.

CD80 and CD86 Are Not Equivalent in Their Ability to Induce the Tyrosine Phosphorylation of CD28

Ligation of either CD80 (B7-1) or CD86 (B7-2), two principal ligands for CD28, is thought to skew the immune response toward Th1 or Th2 differentiation. We have examined early signal transduction pathways recruited following T cell stimulation with either CD80 or CD86. Purified human peripheral T cells or Jurkat T cells were stimulated with Chinese hamster ovary (CHO) cells expressing either human CD80 (CHO-CD80) or human CD86 (CHO-CD86) or with anti-CD28 monoclonal antibody (mAb). In the presence of phorbol 12-myristate 13-acetate, both CHO-CD80 and CHO-CD86, like anti-CD28 mAb, were capable of stimulating cytokine production from both human peripheral T cells and Jurkat T cells. Both CHO-CD80 and CHO-CD86, in the presence of anti-CD3 mAb, costimulated NFAT-dependent transcriptional activation. Several intracellular signaling proteins, such as CBL and VAV, were phosphorylated on tyrosine in response to CD80, CD86, and anti-CD28 mAb. Surprisingly, although stimulation of Jurkat T cells with either CHO-CD80 or anti-CD28 mAb resulted in robust tyrosine phosphorylation of CD28 itself, ligation with CHO-CD86 was unable to induce detectable CD28 tyrosyl phosphorylation over a range of stimulation conditions. In addition, the association of phosphoinositide 3-kinase with CD28 and enhanced tyrosine phosphorylation of phospholipase Cγ were seen after anti-CD28 mAb and CHO-CD80 stimulation but to a much lesser extent after CHO-CD86 stimulation. Thus, ligation of CD28 with either CD80 or CD86 leads to shared early signal transduction events such as the tyrosine phosphorylation of CBL and VAV, to NFAT-mediated transcriptional activation, and to the costimulation of interleukin-2 and granulocyte-macrophage colony-stimulating factor production. However, CD80 and CD86 also induce distinct signal transduction pathways including the tyrosine phosphorylation of CD28 and phospholipase Cγ1 and the SH2-dependent association of phosphoinositide 3-kinase with CD28. These quantitative, if not qualitative, differences between signaling initiated by these two ligands for CD28 may contribute to functional differences (e.g. Th1 or Th2 differentiation) in T cell responses.

Uncoupling p70 s6 Kinase Activation and Proliferation: Rapamycin-Resistant Proliferation of Human CD8 + T Lymphocytes

Rapamycin is a fungal macrolide that inhibits the proliferation of T cells. Studies in both animals and humans have found that rapamycin significantly reduces graft rejection. However, though CD8+ T cells are involved in graft infiltration and rejection, little is known regarding the effects of rapamycin on CD8+ human T cell responses. In this study, we examined the mechanism of rapamycin-induced inhibition of Ag-driven activation of CD8+ T cells. Surprisingly, a heterogeneous proliferative response in the presence of rapamycin was observed among different Ag-specific CD8+ T cell clones; this was also observed in CD8+ peripheral blood T cells activated with TCR cross-linking ex vivo. Inhibition of T cell proliferation by rapamycin was controlled by both the strength of signal delivered through the Ag receptor as well as the specific costimulatory signals received by the T cell. Rapamycin-resistant proliferation occurred despite inhibition of p70s6 kinase activity. Moreover, rapamycin-resistant proliferation of the CD8+ T cell clones was blocked by anti-IL-2 Abs, suggesting that while some of the parallel pathways triggered by IL-2R signaling are sensitive to the effects of rapamycin, others account for the Ag-driven rapamycin resistance. These data provide a new framework for examining the specific mechanism of action of rapamycin in human disease.

Reduced Self-Reactivity of an Autoreactive T Cell After Activation with Cross-reactive Non–Self-Ligand

Autoreactive CD4+ T lymphocytes are critical to the induction of autoimmune disease, but because of the degenerate nature of T cell receptor (TCR) activation such receptors also respond to other ligands. Interaction of autoreactive T cells with other non–self-ligands has been shown to activate and expand self-reactive cells and induce autoimmunity. To understand the effect on the autoreactivity of naive cross-reactive T cells of activation with a potent nonself ligand, we have generated a TCR transgenic mouse which expresses a TCR with a broad cross-reactivity to a number of ligands including self-antigen. The activation of naive transgenic recombination activating gene (Rag)2 − / − T cells with a potent non–self-ligand did not result in a enhancement of reactivity to self, but made these T cells nonresponsive to the self-ligand and anti-CD3, although they retained a degree of responsiveness to the non–self-ligand. These desensitized cells had many characteristics of anergic T cells. Interleukin (IL)-2 production was selectively reduced compared with interferon (IFN)-γ. p21ras activity was reduced and p38 mitogen-activated protein kinase (MAPK) was relatively spared, consistent with known biochemical characteristics of anergy. Surprisingly, calcium fluxes were also affected and the anergic phenotype could not be reversed by exogenous IL-2. Therefore, activation with a hyperstimulating non–self-ligand changes functional specificity of an autoreactive T cell without altering the TCR. This mechanism may preserve the useful reactivity of peripheral T cells to foreign antigen while eliminating responses to self.

Allelic Variation of MHC Structure Alters Peptide Ligands to Induce Atypical Partial Agonistic CD8+ T Cell Function

T cell receptors recognize small changes in peptide ligands leading to different T cell responses. Here, we analyzed a panel of HLA-A2–Tax11-19 reactive T cell clones to examine how small allelic variations of MHC molecules could alter the functional outcome of antigen recognition. Similar to the effects induced by antigenic altered peptide ligands, weak or partial agonistic T cell functions were identified in individual T cell clones with the recognition of MHC-altered peptide ligands (MAPLs). Interestingly, one subtype of HLA-A2 molecules induced an unusual type of partial agonistic function; proliferation without cytotoxicity. Modeling of crystallographic data indicated that polymorphic amino acids in the HLA-A2 peptide binding groove, especially the D-pocket, were responsible for this partial agonism. Reciprocal mutations of the Tax peptide side chain engaging the D-pocket indeed restored the agonist functions of the MHC–peptide complex. Whereas early intracellular signaling events were not efficiently induced by these MAPLs, phosphorylated c-Jun slowly accumulated with sustained long-term expression. These data indicate that MAPLs can induce atypical partial agonistic T cell function through structural and biochemical mechanisms similar to altered peptide ligands.