T- and B-cell responses and previous exposure to hepatitis B virus in 'anti-HBc alone' patients.

A serologic response to hepatitis B virus (HBV) defined as 'anti-HBc alone' is commonly observed, but its significance remains unclear. This study aimed to define the relationship between 'anti-HBc alone' serostatus and HBV infection, including HBV-specific T- and B-cell memory responses. We enrolled 31 'anti-HBc alone' patients. Total HBV DNA and cccDNA were tested by nested polymerase chain reaction (PCR) analysis in liver samples from 22 'anti-HBc alone' patients vs controls (chronic or resolved HBV infection), followed by HBsAg/HBcAg immunohistochemical (IHC) staining. IFN-γ secretion by HBV-specific T cells was compared in individuals who were 'anti-HBc alone' (n = 27), resolved HBV (n = 21), chronic HBV (n = 24) and 12 healthy controls using enzyme-linked immunospot (ELISpot) assays. An HBsAg-IgG B-cell ELISpot assay was performed in 'anti-HBc alone' patients before and after one dose of recombinant HBsAg vaccine. The majority (23/31, 74.2%) of the 'anti-HBc alone' individuals were co-infected with HCV. Infrequent intrahepatic total HBV DNA (2/22, 9.1%) and cccDNA (1/22, 4.5%) were detected in biopsies; HBsAg and HBcAg IHC staining was negative. HBV-specific T-cell responses were similar between 'anti-HBc alone' individuals and HBV resolvers. Circulating HBV-memory B-cell responses were detected in all 'anti-HBc alone' individuals, consistent with an HBsAg-specific memory pool. After one HBV vaccine dose, increased anti-HBs antibody levels were observed, accompanied by an expansion of HBsAg-specific memory B cells (P = 0.0226). 'Anti-HBc alone' individuals showed HBV-specific T-cell and memory B-cell responses typical of previous viral exposure and protective memory, suggesting a resolved infection.

Expression and biological activities of bovine interleukin 4: effects of recombinant bovine interleukin 4 on T cell proliferation and B cell differentiation and proliferation in vitro

Interleukin 4 (IL-4) is a pleotropic cytokine affecting a wide range of cell types in both the mouse and the human. These activities include regulation of the growth and differentiation of both T and B lymphocytes. The activities of IL-4 in nonprimate, nonmurine systems are not well established. Herein, we demonstrate in the bovine system that IL-4 upregulates production of IgM, IgG1, and IgE in the presence of a variety of costimulators including anti-IgM, Staphylococcus aureus cowan strain I, and pokeweed mitogen. IgE responses are potentiated by the addition of IL-2 to IL-4. Culture of bovine B lymphocytes with IL-4 in the absence of additional costimulators resulted in the increased surface expression of CD23 (low-affinity Fc epsilon RII), IgM, IL-2R, and MHC class II in a dose-dependent manner. IL-4 alone increased basal levels of proliferation of bulk peripheral blood mononuclear cells but in the presence of Con A inhibited proliferation. In contrast to the activities of IL-4 in the murine system, proliferation of TH1- and TH2-like clones was inhibited in a dose-dependent manner as assessed by antigen-or IL-2-driven in vitro proliferative responses. These observations are consistent with the role of IL-4 as a key player in regulation of both T and B cell responses.

THE ROLE OF B CELLS IN THE T CELL DEPENDENT RELEASE OF HUMAN B CELL GROWTH FACTOR

Quiescent human B cells are postulated to go through activation and proliferation phases before undergoing differentiative phase for immunoglobulin secretion. The present studies address some of the aspects of activation and proliferation phase of normal human B cells. The definitions of signals responsible for B cell activation and proliferation resulted in the development of a highly specific, reproducible B cell growth factor (BCGF) assay. This BCGF bioassay utilizes activation by rabbit anti-human IgM-antibody. The functional specificity of this assay for measuring BCGF activity was demonstrated by the finding that target B cells proliferated but did not differentiate. The factor specificity was determined by specific absorption of BCGF by anti-IgM activated B cells. This assay was utilized for the studies of T-B cell collaboration and the essential function of monocytes in the production and/or release of B cell growth factor in a syngeneic in vitro system. It is apparent that highly purified T cells are poor producers of BCGF by themselves and require monocytes to secrete significant quantities of BCGF upon PHA stimulation. Macrophage soluble factor, Interleukin 1, is capable of replacing monocyte function for the release of BCGF by activated T cells. In our studies, B cells are incapable to function as accessory cells to replace monocyte function. Normal B cells are also not capable of producing BCGF under our experimental observations. However, the addition of these B cells at an optimum cell density (T:B ratio 1:1) doubles the monocyte dependent release of BCGF by syngeneic T cells. The augmentative role of B cells is expanded to understand the mechanism of BCGF release by T cells. It is observed from our studies that DR antigen of B cell surface is involved in the release of BCGF. The functional difference between DR of B cells and monocytes is observed as IL-1 could replace DR-treated monocytes whereas failed to replace DR-treated B cells for the release of BCGF by T cells. This functional difference may be attributed to the reported microheterogeneity in DR of B cells and monocytes. The addition of irradiated B cells increased the monocyte dependent T cell proliferation, suggesting the increase of T cell pool for BCGF release. In summary, the development of a biological assay specific for B cell growth factor led to the delineation of an interesting role of B cells in the release of its own growth factor by T cells. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Mechanisms by which nonsense codons downregulate T-cell receptor transcripts

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that degrades aberrant mRNAs harboring premature termination codons (PTCs). Two out of three T-cell receptor β (TCRβ) transcripts carry PTCs as a result of error-prone programmed rearrangements that occur at this locus during lymphocyte maturation. PTCs decrease TCRβ mRNA levels to a much greater extent than mRNAs transcribed from non-rearranging genes. This robust decrease in TCRβ mRNA levels is not a unique characteristic of the T-cell environment or the TCRβ promoter. The simplest explanation for this is that PTC-bearing TCRβ mRNAs elicit a stronger NMD response. An alternative explanation is NMD collaborates with another mechanism to dramatically decrease PTC-bearing TCRβ mRNA levels. ^ In my dissertation, I investigated the molecular mechanism behind the strong decrease in TCRβ mRNA levels triggered by PTCs. To determine the location of this response, I performed mRNA half-life analysis and found that PTCs elicited more rapid TCRβ mRNA decay in the nuclear fraction, not the cytoplasmic fraction. Although decay was restricted to the nuclear fraction, PTC-bearing TCRβ transcript levels were extremely low in the cytoplasm, a phenomenon that I named the nonsense-codon induced partitioning shift (NIPS). I established that NIPS shares several qualities with NMD, including its dependence on translation and NMD factors. Several lines of evidence suggested that NIPS results from PTCs eliciting retention of TCRβ transcripts in the nuclear fraction. This retention, as well as rapid TCRβ mRNA decay, most likely occurs in either the nucleoplasm or the outer nuclear membrane, based on analysis of nuclear and cytoplasmic markers in the highly purified nuclei I used for my studies. To further address the location of decay, I asked whether nuclear or cytoplasmic RNA decay factors mediated the destruction of PTC-bearing mRNAs. My results suggested that a nuclear component of the 3'-to-5' exosome, as well as an endonucleolytic activity, are involved in the destruction of PTC-containing TCRβ mRNAs. Individual endogenous NMD substrates had differential requirements for nuclear and cytoplasmic exonucleases. In summary, my results provide evidence that PTCs trigger multiple mechanisms involving multiple decay factors to remove and regulate mRNAs in mammalian cells. ^

PIM KINASE INHIBITION: SINGLE AGENT AND COMBINATION APPROACHES IN B-CELL LYMPHOMA

Proviral integration site for Moloney murine leukemia virus (Pim) kinases are Ser/Thr/Tyr kinases. They modulate B-cell development but become oncoproteins and promote cancer development once overexpressed. Containing three isoforms, Pim-1, -2 and -3 are known to phosphorylate various substrates that regulate transcription, translation, cell cycle, and survival pathways in both hematological and solid tumors. Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma. Elevated Pim kinase levels are common in MCL, and it negatively correlates with patient outcome. SGI-1776 is a small molecule inhibitor selective for Pim-1/-3. We hypothesize that SGI-1776 treatment in MCL will inhibit Pim kinase function, and inhibition of downstream substrates phosphorylation will disrupt transcriptional, translational, and cell cycle processes while promoting apoptosis. SGI-1776 treatment induced moderate to high levels of apoptosis in four MCL cell lines (JeKo-1, Mino, SP-53 and Granta-519) and peripheral blood mononuclear cells (PBMCs) from MCL patients. Phosphorylation of transcription and translation regulators, c-Myc and 4E-BP1 declined in both model systems. Additionally, levels of short-lived Mcl-1 mRNA and protein also decreased and correlated with decline of global RNA synthesis. Collectively, our investigations highlight Pim kinases as viable drug targets in MCL and emphasize their roles in transcriptional and translational regulation. We further investigated a combination strategy using SGI-1776 with bendamustine, an FDA-approved DNA-damaging alkylating agent for treating non-Hodgkin’s lymphoma. We hypothesized this combination will enhance SGI-1776-induced transcription and translation inhibition, while promoting bendamustine-triggered DNA damage and inducing additive to synergistic cytotoxicity in B-cell lymphoma. Bendamustine alone resulted in moderate levels of apoptosis induction in MCL cell lines (JeKo-1 and Mino), and in MCL and splenic marginal zone lymphoma (a type of B-cell lymphoma) primary cells. An additive effect in cell killing was observed when combined with SGI-1776. Expectedly, SGI-1776 effectively decreased global RNA and protein synthesis levels, while bendamustine significantly inhibited DNA synthesis and generated DNA damage response. In combination, intensified inhibitory effects in DNA, RNA and protein syntheses were observed. Together, these data suggested feasibility of using Pim kinase inhibitor in combination with chemotherapeutic agents such as bendamustine in B-cell lymphoma, and provided foundation of their mechanism of actions in lymphoma cells.

Molecular studies of human high grade B cell non -Hodgkin's lymphomas

The non-Hodgkin's B cell lymphomas are a diverse group of neoplastic diseases. The incidence rate of the malignant tumors has been rising rapidly over the past twenty years in the United States and worldwide. The lack of insight to pathogenesis of the disease poses a significant problem in the early detection and effective treatment of the human malignancies. These studies attempted to investigate the molecular basis of pathogenesis of the human high grade B cell non-Hodgkin's lymphomas with a reverse genetic approach. The specific objective was to clone gene(s) which may play roles in development and progression of human high grade B cell non-Hodgkin's lymphomas.^ The messenger RNAs from two high grade B cell lymphoma lines, CJ and RR, were used for construction of cDNA libraries. Differential screening of the derived cDNA libraries yielded a 1.4 kb cDNA clone. The gene, designated as NHL-B1.4, was shown to be highly amplified and over-expressed in the high grade B cell lymphoma lines. It was not expressed in the peripheral blood lymphoid cells from normal donors. However, it was inducible in peripheral blood T lymphocytes by a T cell mitogen, PHA, but could not be activated in normal B cells by B cell mitogen PMA. Further molecular characterization revealed that the gene may have been rearranged in the RR and some other B cell lymphoma lines. The coding capacity of the cDNA has been confirmed by a rabbit reticulocyte lysate and wheat germ protein synthesis system. A recombinant protein with a molecular weight of approximate 30 kDa was visualized in autoradiogram. Polyclonal antisera have been generated by immunization of two rabbits with the NHL-B1.4 recombinant protein produced in the E. coli JM109. The derived antibody can recognize a natural protein with molecular weight of 49 kDa in cell lysate of activated peripheral T lymphocytes of normal donors and both the cell lysate and supernatant of RR B cell lymphoma lines. The possible biologic functions of the molecule has been tested preliminarily in a B lymphocyte proliferation assay. It was found that the Q-sepharose chromatograph purified supernatant of COS cell transfection could increase tritiated thymidine uptake by B lymphocytes but not by T lymphocytes. The B cell stimulatory activity of the supernatant of COS cell tranfection could be neutralized by the polyclonal antisera, indicating that the NHL-B1.4 gene product may be a molecule with BCGF-like activity.^ The expression profiles of NHL-B1.4 in normal and neoplastic lymphoid cells were consistent with the current B lymphocyte activation model and autocrine hypothesis of high grade B cell lymphomagenesis. These results suggested that the NHL-B1.4 cDNA may be a disease-related gene of human high grade B cell lymphomas, which may codes for a postulated B cell autocrine growth factor. ^

Pathways leading to apoptosis resistance in a murine B -cell lymphoma cell system

The aberrant activation of signal transduction pathways has long been linked to uncontrolled cell proliferation and the development of cancer. The activity of one such signaling module, the Mitogen-Activated Protein Kinase (MAPK) pathway, has been implicated in several cancer types including pancreatic, breast, colon, and lymphoid malignancies. Interestingly, the activation of MAP-Kinase-Kinase-Kinase proteins often leads to the additional activation of NF-κB, a transcription factor that acts as a cell survival signal through its control of antiapoptotic genes. We have investigated the role of a specific dimer form of the NF-κB transcription factor family, NF-κB1 (p50) homodimers, in its control of the proto-oncogene, Bcl-2, and we have identified the MEK/ERK (MAPK) signaling cascade as a mediator of NF-κB1 activity. ^ Two murine B cell lymphoma cell lines were used for these studies: LY-as, an apoptosis proficient line with low Bcl-2 protein expression and no nuclear NF-κB activity, and LY-ar, a nonapoptotic line with constitutive p50 homodimer activity and 30 times more Bcl-2 protein expression than LY-as. Experiments modulating p50 activity correlated the activation of p50 homodimers with Bcl-2 expression and additional gel shift experiments demonstrated that the Bcl-2 P1 promoter had NF-κB sites with which recombinant p50 was able to interact. In vitro transcription revealed that p50 enhanced the production of transcripts derived from the Bcl-2 P1 promoter. These data strongly suggest that Bcl-2 is a target gene for p50-mediated transcription and suggest that the activation of p50 homodimers contributes to the expression of Bcl-2 observed in LY-ar cells. ^ Studies of upstream MAPK pathways that could influence NF-κB activity demonstrated that LY-ar cells had phosphorylated ERK proteins while LY-as cells did not. Treatment of LY-ar cells with the MEK inhibitors PD 98059, U0126, and PD 184352 led to a loss of phosphorylated ERK, a reversal of nuclear p50 homodimer DNA binding, and a decrease in the amount of Bcl-2 protein expression. Similarly, the activation of the MEK/ERK pathway in LY-as cells by phorbol ester led to Bcl-2 expression that could be blocked by PD 98059. Furthermore, treatment of LY-ar cells with TNFα, an IKK activator, did not change the suppressive effect of PD 98059 on p50 homodimer activity, suggesting an IKK-independent pathway for p50 homodimer activation. Lastly, all three MEK inhibitors sensitized LY-ar cells to radiation-induced apoptosis. ^ These data indicate that the activation of the MEK/ERK MAP-Kinase signaling pathway acts upstream of p50 homodimer activation and Bcl-2 expression in this B cell lymphoma cell system and suggest that the activation of MEK/ERK may be a key step in the progression of lymphoma to advanced-staged disease. Other researchers have used MEK inhibitors to inhibit cell growth and sensitize a number of tumors to chemotherapies. In light of our data, MEK inhibitors may additionally be useful clinically to radiosensitize cancers of lymphoid origin. ^

Characterization of inhibitory and stimulatory forms of the murine natural killer cell receptor 2B4

The receptor 2B4 belongs to the Ig superfamily and is found on the surface of all murine natural killer (NK) cells as well as T cells displaying non-MHC-restricted cytotoxicity. Previous studies have suggested that 2B4 is an activating molecule because cross-linking of this receptor results in increased cytotoxicity and γ-interferon secretion as well as granule exocytosis. However, it was recently shown that the gene for 2B4 encodes two different products that arise by alternative splicing. These gene products differ solely in their cytoplasmic domains. One form has a cytoplasmic tail of 150 amino acids (2B4L) and the other has a tail of 93 amino acids (2B4S). To determine the function of each receptor, cDNAs for 2B4S and 2B4L were transfected into the rat NK cell line RNK-16. Interestingly, the two forms of 2B4 had opposing functions. 2B4S was able to mediate redirected lysis of P815 tumor targets, suggesting that this form represents an activating receptor. However, 2B4L expression led to an inhibition of redirected lysis of P815 targets when the mAb 3.2.3 (specific for rat NKRP1) was used. In addition, 2B4L constitutively inhibits lysis of YAC-1 tumor targets. 2B4L is a tyrosine phosphoprotein, and removal of domains containing these residues abrogates its inhibitory function. Like other inhibitory receptors, 2B4L associates with the tyrosine phosphatase SHP-2. Thus, 2B4L is an inhibitory receptor belonging to the Ig superfamily.

Regulation of T cell receptor (TCR) β gene expression by CD3 complex signaling in immature thymocytes: Implications for TCRβ allelic exclusion

During αβ thymocyte development, clonotype-independent CD3 complexes are expressed at the cell surface before the pre-T cell receptor (TCR). Signaling through clonotype-independent CD3 complexes is required for expression of rearranged TCRβ genes. On expression of a TCRβ polypeptide chain, the pre-TCR is assembled, and TCRβ locus allelic exclusion is established. We investigated the putative contribution of clonotype-independent CD3 complex signaling to TCRβ locus allelic exclusion in mice single-deficient or double-deficient for CD3ζ/η and/or p56lck. These mice display defects in the expression of endogenous TCRβ genes in immature thymocytes, proportional to the severity of CD3 complex malfunction. Exclusion of endogenous TCRβ VDJ (variable, diversity, joining) rearrangements by a functional TCRβ transgene was severely compromised in the single-deficient and double-deficient mutant mice. In contrast to wild-type mice, most of the CD25+ double-negative (DN) thymocytes of the mutant mice failed to express the TCRβ transgene, suggesting defective expression of the TCRβ transgene similar to endogenous TCRβ genes. In the mutant mice, a proportion of CD25+ DN thymocytes that failed to express the transgene expressed endogenous TCRβ polypeptide chains. Many double-positive cells of the mutant mice coexpressed endogenous and transgenic TCRβ chains or more than one endogenous TCRβ chain. The data suggest that signaling through clonotype-independent CD3 complexes may contribute to allelic exclusion of the TCRβ locus by inducing the expression of rearranged TCRβ genes in CD25+ DN thymocytes.

Copresentation of natural HIV-1 agonist and antagonist ligands fails to induce the T cell receptor signaling cascade

It is not known how human immunodeficiency virus type 1 (HIV-1)-derived antagonist peptides interfere with intracellular activation of cytotoxic T lymphocytes (CTL). We identified Gag epitope variants in HIV-1-infected patients that act as antagonists of CTL responses to unmutated epitopes. We then investigated the effect that presentation of each variant has on the early events of T cell receptor (TCR) signal transduction. We found that altered peptide ligands (APL) failed to induce phosphorylation of pp36, a crucial adaptor protein involved in TCR signal transduction. We further investigated the effect that simultaneous presentation of APL and native antigen at low, physiological, peptide concentrations (1 nM) has on TCR signal transduction, and we found that the presence of APL can completely inhibit induction of the protein tyrosine phosphorylation events of the TCR signal transduction cascade.

T cell vaccination induces T cell receptor Vβ-specific Qa-1-restricted regulatory CD8+ T cells

Vaccination of mice with activated autoantigen-reactive CD4+ T cells (T cell vaccination, TCV) has been shown to induce protection from the subsequent induction of a variety of experimental autoimmune diseases, including experimental allergic encephalomyelitis (EAE). Although the mechanisms involved in TCV-mediated protection are not completely known, there is some evidence that TCV induces CD8+ regulatory T cells that are specific for pathogenic CD4+ T cells. Previously, we demonstrated that, after superantigen administration in vivo, CD8+ T cells emerge that preferentially lyse and regulate activated autologous CD4+ T cells in a T cell receptor (TCR) Vβ-specific manner. This TCR Vβ-specific regulation is not observed in β2-microglobulin-deficient mice and is inhibited, in vitro, by antibody to Qa-1. We now show that similar Vβ8-specific Qa-1-restricted CD8+ T cells are also induced by TCV with activated CD4+ Vβ8+ T cells. These CD8+ T cells specifically lyse murine or human transfectants coexpressing Qa-1 and murine TCR Vβ8. Further, CD8+ T cell hybridoma clones generated from B10.PL mice vaccinated with a myelin basic protein-specific CD4+Vβ8+ T cell clone specifically recognize other CD4+ T cells and T cell tumors that express Vβ8 and the syngeneic Qa-1a but not the allogeneic Qa-1b molecule. Thus, Vβ-specific Qa-1-restricted CD8+ T cells are induced by activated CD4+ T cells. We suggest that these CD8+ T cells may function to specifically regulate activated CD4+ T cells during immune responses.

Cross-lineage expression of Ig-β (B29) in thymocytes: Positive and negative gene regulation to establish T cell identity

Developmental commitment involves activation of lineage-specific genes, stabilization of a lineage-specific gene expression program, and permanent inhibition of inappropriate characteristics. To determine how these processes are coordinated in early T cell development, the expression of T and B lineage-specific genes was assessed in staged subsets of immature thymocytes. T lineage characteristics are acquired sequentially, with germ-line T cell antigen receptor-β transcripts detected very early, followed by CD3ɛ and terminal deoxynucleotidyl transferase, then pTα, and finally RAG1. Only RAG1 expression coincides with commitment. Thus, much T lineage gene expression precedes commitment and does not depend on it. Early in the course of commitment to the T lineage, thymocytes lose the ability to develop into B cells. To understand how this occurs, we also examined expression of well defined B lineage-specific genes. Although λ5 and Ig-α are not expressed, the μ0 and Iμ transcripts from the unrearranged IgH locus are expressed early, in distinct patterns, then repressed just before RAG1 expression. By contrast, RNA encoding the B cell receptor component Ig-β was found to be transcribed in all immature thymocyte subpopulations and throughout most thymocyte differentiation. Ig-β expression is down-regulated only during positive selection of CD4+CD8– cells. Thus several key participants in the B cell developmental program are expressed in non-B lineage-committed cells, and one is maintained even through commitment to an alternative lineage, and repressed only after extensive T lineage differentiation. The results show that transcriptional activation of “lymphocyte-specific” genes can occur in uncommitted precursors, and that T lineage commitment is a composite of distinct positive and negative regulatory events.

Kinetics of T cell receptor β, γ, and δ rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44+CD25+ Pro-T thymocytes

We performed a comprehensive analysis of T cell receptor (TCR) γ rearrangements in T cell precursors of the mouse adult thymus. Using a sensitive quantitative PCR method, we show that TCRγ rearrangements are present in CD44+CD25+ Pro-T thymocytes much earlier than expected. TCRγ rearrangements increase significantly from the Pro-T to the CD44−CD25+ Pre-T cell transition, and follow different patterns depending on each Vγ gene segment, suggesting that ordered waves of TCRγ rearrangement exist in the adult mouse thymus as has been described in the fetal mouse thymus. Recombinations of TCRγ genes occur concurrently with TCRδ and D-Jβ rearrangements, but before Vβ gene assembly. Productive TCRγ rearrangements do not increase significantly before the Pre-T cell stage and are depleted in CD4+CD8+ double-positive cells from normal mice. In contrast, double-positive thymocytes from TCRδ−/− mice display random proportions of TCRγ rearranged alleles, supporting a role for functional TCRγ/δ rearrangements in the γδ divergence process.

Major histocompatibility complex class I-restricted T cells are required for all but the end stages of diabetes development in nonobese diabetic mice and use a prevalent T cell receptor α chain gene rearrangement

Nonobese diabetic (NOD) mice develop insulin-dependent diabetes mellitus due to autoimmune T lymphocyte-mediated destruction of pancreatic β cells. Although both major histocompatibility complex class I-restricted CD8+ and class II-restricted CD4+ T cell subsets are required, the specific role each subset plays in the pathogenic process is still unclear. Here we show that class I-dependent T cells are required for all but the terminal stages of autoimmune diabetes development. To characterize the diabetogenic CD8+ T cells responsible, we isolated and propagated in vitro CD8+ T cells from the earliest insulitic lesions of NOD mice. They were cytotoxic to NOD islet cells, restricted to H-2Kd, and showed a diverse T cell receptor β chain repertoire. In contrast, their α chain repertoire was more restricted, with a recurrent amino acid sequence motif in the complementarity-determining region 3 loop and a prevalence of Vα17 family members frequently joined to the Jα42 gene segment. These results suggest that a number of the CD8+ T cells participating in the initial phase of autoimmune β cell destruction recognize a common structural component of Kd/peptide complexes on pancreatic β cells, possibly a single peptide.

The Syk and ZAP-70 SH2-containing tyrosine kinases are implicated in pre-T cell receptor signaling

An early stage in thymocyte development, after rearrangement of the β chain genes of the T cell receptor (TCR), involves expression of the pre-TCR complex and accompanying differentiation of CD4−CD8− double negative (DN) cells to CD4+CD8+ double positive (DP) cells. The ZAP-70 and Syk tyrosine kinases each contain two N-terminal SH2 domains that bind phosphorylated motifs in antigen receptor subunits and are implicated in pre-T receptor signaling. However, mice deficient in either ZAP-70 or Syk have no defect in the formation of DP thymocytes. Here we show that, in mice lacking both Syk and ZAP-70, DN thymocytes undergo β chain gene rearrangement but fail to initiate clonal expansion and are incapable of differentiating into DP cells after expression of the pre-TCR. These data suggest that the ZAP-70 and Syk tyrosine kinases have crucial but overlapping functions in signaling from the pre-TCR and hence in early thymocyte development.