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.

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.

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)-gamma. p21(ras) 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.

Results of a portfolio approach to intramural research funding at an academic medical center.

In response to stagnant Federal grant funding levels and to catalyze early stage or high-risk research not currently supported by the NIH, many academic medical centers (AMCs) provide supplemental intramural funding to faculty investigators. However, it can be challenging to decide how to deploy these funds for maximum impact. We conducted a retrospective, descriptive analysis to explore trends in applications and awards associated with an institution-wide intramural funding center at a major U.S. AMC. From 2010 to 2017, the Brigham Research Institute at Brigham and Women's Hospital awarded a total of 354 grants totaling over $9 million to affiliated researchers through six distinct and complementary grant programs. The number of applicants remained essentially stable, despite expansion of the funding program portfolio. Distribution of applicants and awardees by academic rank and gender generally reflected that of medical school faculty at large. This descriptive analysis demonstrates interest in a diverse range of intramural funding programs among AMC faculty, and a lack of overt rank or gender bias in the programs' awardees. However, it highlights the institution's need to better understand the amount of residual unmet demand for intramural funding; the degree to which underrepresented constituencies can and should be actively supported; and the "return on investment" of these grants.

An autoimmune disease-associated CTLA4 splice variant lacking the B7 binding domain signals negatively in T cells.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) plays a critical role in down-regulating T cell responses. A number of autoimmune diseases have shown genetic linkage to the CTLA4 locus. We have cloned and expressed an alternatively spliced form of CTLA4 that has genetic linkage with type 1 diabetes in NOD mice. This splice variant of CTLA4, named ligand-independent CTLA4 (liCTLA4), lacks exon 2 including the MYPPPY motif essential for binding to the costimulatory ligands B7-1 and B7-2. liCTLA4 is expressed as a protein in primary T cells and strongly inhibits T cell responses by binding and dephosphorylating the TcRzeta chain. Expression of liCTLA4, but not full length CTLA4 (flCTLA4), was higher in memory/regulatory T cells from diabetes resistant NOD congenic mice compared to susceptible NOD mice. Transgenic expression of liCTLA4 in autoimmune prone Ctla4 -/- mice inhibited spontaneous T cell activation and prevented early lethality in the Ctla4 -/- mice. Thus, increased expression and negative signalling delivered by the liCTLA4 may play a critical role in regulating the development of T cell-mediated autoimmune diseases.

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.

Multiple sclerosis.

Multiple sclerosis (MS) is a complex genetic disease associated with inflammation in the central nervous system (CNS) white matter and is thought to be mediated by autoimmune processes. Clonal expansion of B cells, their antibody products, and T cells, hallmarks of inflammation in the CNS, are found in MS. The association of the disease with major histocompatibility complex genes, the inflammatory white matter infiltrates, similarities with animal models, and the observation that MS can be treated with immunomodulatory and immunosuppressive therapies support the hypothesis that autoimmunity plays a major role in the disease pathology. This review discusses the immunopathology of MS with particular focus given to regulatory T cells and the role of B cells and antibodies, immunomodulatory therapeutics, and finally new directions in MS research, particularly new methods to define the molecular pathology of human disease with high-throughput examination of germline DNA haplotypes, RNA expression, and protein structures that will allow the generation of a new series of hypotheses that can be tested to develop better understandings and therapies for this disease.

Rapamycin-resistant proliferation of CD8+ T cells correlates with p27kip1 down-regulation and bcl-xL induction, and is prevented by an inhibitor of phosphoinositide 3-kinase activity.

Rapamycin inhibits the proliferation of many mammalian cell types, including lymphocytes, making the compound useful as an immunosuppressant. Rapamycin has also been a useful tool for studying signaling mechanisms regulating cellular proliferation. However, the effects of rapamycin remain poorly understood, and the precise mechanisms of clinical action remain elusive. Previously, we found that, depending on the strength of the signal delivered to the T cell via both the T cell receptor and the costimulatory molecule CD28, CD8+ T cells are capable of rapamycin-resistant proliferation. Here, we have further elucidated the mechanism of rapamycin-resistant proliferation of human CD8+ T cells. Under conditions where rapamycin inhibited proliferation, p27kip1 down-regulation was prevented, whereas under conditions resulting in rapamycin-resistant proliferation, p27kip1 was down-regulated. Further, T cell receptor/CD28-dependent induction of bcl-xL expression was not inhibited by rapamycin, which correlated with both rapamycin-resistant proliferation and increased cell survival. Moreover, an inhibitor of phosphoinositide 3-kinase activity was able to eliminate rapamycin-resistant proliferation of freshly isolated CD8+ human cells, strongly suggesting that phosphoinositide 3-kinase activity was required for the rapamycin-resistant proliferation of CD8+ T cells. The selective immunosuppressive effect of rapamycin in human CD8+ T cell populations could be predictive of a selective effect allowing cytotoxic responses during microbial infections where there are strong strengths of signals associated with high affinity T cell receptors and strong costimulatory second signals. In contrast, the weaker autoimmune and perhaps allogeneic responses can be selectively inhibited by the actions of rapamycin.

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.