Nasrin Nabavi

B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: Application to autoimmune disease therapy

CD4 T helper precursor cells mature along two alternative pathways, Th1 and Th2. Here we show that these pathways are differentially activated by two costimulatory molecules, B7-1 and B7-2. Using anti-B7 antibodies, this developmental step was manipulated both in vitro and in vivo in experimental allergic encephalomyelitis (EAE). Anti-B7-1 reduced the incidence of disease while anti-B7-2 increased disease severity. Neither antibody affected overall T cell induction but rather altered cytokine profile. Administration of anti-B7-1 at immunization resulted in predominant generation of Th2 clones whose transfer both prevented induction of EAE and abrogated established disease. Since co-treatment with anti-IL-4 antibody prevented disease amelioration, costimulatory molecules may directly affect initial cytokine secretion. Thus, interaction of B7-1 and B7-2 with shared counterreceptors CD28 and CTLA-4 results in very different outcomes in clinical disease by influencing commitment of precursors to a Th1 or Th2 lineage.

Selective CD28pYMNM mutations implicate phosphatidylinositol 3-kinase in CD86-CD28-mediated costimulation

CD28 costimulatory signals are required for lymphokine production and T cell proliferation. CD28 signaling recruits the intracellular proteins PI 3-kinase, ITK, and GRB-2/SOS. PI 3-kinase and GRB-2/SOS bind the CD28 cytoplasmic pYMNM motif via SH2 domains. We generated CD28 pYMNM mutants and found that Y191 mutation (Y191CD28F) disrupted both PI 3-kinase and GRB-2 binding, while M194 mutation (M194CD28C) disrupted only PI 3-kinase binding. Both mutants still bound ITK. We have assessed the ability of these selective mutants to support IL-2 production upon TCR zeta/CD3 ligation in the presence of CHO-CD86 (B7-2) cells. Both Y191CD28F and M194CD28C mutants failed to generate IL-2. These data directly implicate PI 3-kinase in CD28-mediated costimulation leading to IL-2 secretion. Wortmannin, an inhibitor of PI 3-kinase, induced cell apoptosis and as such was unsuitable for use in this study.

In vitro induction of T cell anergy by blocking B7 and early T cell costimulatory molecule ETC-1/B7-2

APC-associated B7 and ETC-1/B7-2 are two major costimulatory molecules for full activation of T lymphocytes during auto- and allogeneic immune responses. In this report, we further examine the role of the two molecules in murine CD4+ T cell activation and anergy development. As suggested in antibody blocking studies, optimal activation of CD4+ T cells in response to anti-CD3 stimulation requires collaborative signaling through the two molecules. Simultaneous blockade of B7 and ETC-1/B7-2 renders CD4+ T cells unresponsive to anti-CD3 restimulation. PCR analysis and cytokine reconstitution studies show that the observed unresponsiveness is correlated to a significant reduction of Th1-type cytokine production, suggesting B7 and ETC-1/B7-2-mediated costimulatory signaling may be specifically active in regulation of the function of the Th1 subset.

Cloning of an interleukin-4 inducible gene from cytotoxic T lymphocytes and its identification as a lipase

Interleukin-4 (IL-4) has been demonstrated to be an important lymphokine for the generation of cytotoxic T lymphocytes (CTLs). Here we describe an IL-4 inducible gene specifically expressed in CTLs. By sequence homology, this gene is likely to be the mouse homolog of pancreatic lipase. Oocyte translation of in vitro transcribed mRNA results in the expression of a protein with lipase activity, and Northern analysis of various tissues and a large panel of hematopoietic cell types demonstrates that this gene is expressed only in the pancreas and CTLs. Lysates of CTLs grown in IL-4, but not in IL-2, exhibit lipase activity. Furthermore, Northern analysis of CTLs grown in the presence of IL-4 for as little as 5 days demonstrates a marked induction of lipase mRNA, which correlates with enhanced cytolysis by these cells. These results suggest that this lipase may have an important role in CTL effector function.

Tumor cells expressing major histocompatibility complex class II and B7 activation molecules stimulate potent tumor-specific immunity.

In previous studies we have shown that the highly malignant mouse SaI sarcoma can be converted into an immunogenic tumor that is immunologically rejected by the autologous host if the tumor cells are transfected with and express syngeneic major histocompatibility complex (MHC) class II genes. Tumor cells expressing class II heterodimers truncated for the cytoplasmic regions of the alpha and beta chains, however, are as malignant as wild-type class II- tumors. These studies have contributed to the hypothesis that T-cell activation requires two signals: the engagement of the MHC class II/peptide complex of the antigen-presenting cell (APC) by the T cell receptor for antigen of the responding T cell and the transmittal of a second, or costimulatory, signal by the APC to the responding T cell. In this report we show that induction of tumor-specific immunity is facilitated by delivery of a costimulatory signal provided by the B7 activation molecule. Mice challenged with SaI cells bearing truncated class II molecules and transfected with B7 cDNA are immune to the transfectants and are protected against a challenge of wild-type class II-B7- ascites or solid SaI tumor. The induced immunity requires CD4+ T cells and is specific for the immunizing sarcoma cells. These results highlight the critical role of the B7 costimulatory pathway in stimulating long-term, tumor-specific immunity that is effective against high doses of challenging wild-type tumor and suggest a strategy for enhancing tumor rejection.

Cytoplasmic components of natural killer cells limit the growth of Cryptococcus neoformans.

Murine natural killer (NK) cell‐mediated inhibition of growth of a yeast‐like target cell, Cryptococcus neoformans, was completely abrogated by blocking the effector cell secretory process with monensin. Therefore, further studies were performed to determine the ability of various cytoplasmic fractions of NK cells to mediate inhibition of cryptococcal growth. Percoll‐fractionated homogenates of rat LGL tumor cells demonstrated that the granule‐containing fractions plus three additional sets of less dense cytoplasmic fractions displayed anti‐cryptococcal activity; whereas only the cytoplasmic granule‐containing fractions had cytotoxic activity against YAC‐1 tumor cell and sheep erythrocyte targets. Maximal cryptococcal growth inhibition induced by LGL granules occurred after a 1 h incubation, required the presence of Ca2+ (1.0 mM) or Mg2+ (0.5 mM or 5.0 mM), and was completely abrogated in the presence of rabbit anti‐LGL granule IgG. Cytolysin, the granule component which mediates tumor cell and sheep erythrocyte lysis, effectively limited the growth of cryptococci. Since Percoll gradient fractionation of the LGL homogenates demonstrated three separate peaks of anti‐cryptococcal activity other than the granule peak, it is possible that the cytolysin‐containing granules are not the only subcellular component of NK cells playing a role in inhibition of C. neoformans growth.

Identification of an IL-4-Inducible Gene Expressed in Differentiating Lymphocytes and Male Germ Cells

Interleukin 4 (IL-4) is a cytokine that is involved in the differentiation of B and T lymphocytes. In this report, we describe the identification of a novel gene, N.52, which was cloned from the murine pre-B cell line R8205 grown in the presence of IL-4 for 48 hr. Although N.52 expression is detectable at low levels in unstimulated R8205 cells, the level of N.52 dramatically increases after only .4 hr exposure to IL-4 and remains at a high .level up to 48 hr. Although N.52 expression is low or absent in normal spleen B and T cells, its expression can be induced by the differentiation signals delivered by LPS in B cells and by Con A in T-cell hybrids. While N.52 mRNA is absent in all highly differentiated organs, it is detectable in stem cell harboring lymphoid tissues such as bone marrow, fetal liver, and thymus. Furthermore, N.52 mRNA is expressed at strikingly high levels in the testis, specifically in differentiating male germ cells. It is induced by differentiation signals triggered by the combination of cyclic AMP and retinoic acid in teratocarcinoma F9 cells. Taken together, these data suggest that N.52 is a developmentally regulated gene whose expression in cells of the immune and reproductive systems may be controlled by stimuli that induce differentiation.