Lck signaling inhibition causes improvement in clinical features of psoriatic inflammation through reduction in inflammatory cytokines in CD4+ T cells in imiquimod mouse model.

Psoriasis is a chronic dermal inflammatory disease with a world-wide prevalence in which different immune/non-immune cells, e.g. T cells, macrophages, neutrophils, and keratinocytes play a decisive role. These immune cells interact among themselves by releasing multiple mediators which eventually cause characteristic psoriatic plaques in the skin. T cells are reported to be significant contributors to psoriatic inflammation through release of multiple cytokines which are controlled by several kinases, one of them being Lymphocyte-specific protein tyrosine kinase (Lck). Lck has been reported to be crucial for expression/production of several key inflammatory cytokines though modulation of several other kinases/transcription factors in T cells. Therefore, in this investigation, effect of Lck inhibitor, A-770041 was examined on PLCγ, p38MAPK, NFATc1, NFkB and STAT3, TNF-α, IFN-γ, Foxp3, IL-17A, in CD4+ T cells in imiquimod (IMQ)-induced psoriatic inflammation in mice. Results from the present study exhibit that p-Lck expression is enhanced in CD4+ T cells of IMQ-treated mice which is concomitant with enhanced clinical features of psoriatic inflammation (ear/back skin thickness, MPO activity, acanthosis/leukocytic infiltration) and molecular parameters (enhanced expression of p-Lck, p-PLCγ, p-p38-MAPK, NFATc1, p-NFkB, TNF-α, IFN-γ, p-STAT3, and IL-17A in CD4+ T cells). Inhibition of Lck signaling led to amelioration of clinical features of psoriasis through attenuation of Th1/Th17 immune responses and upregulation of Treg cells in IMQ-treated mice. In summary, current investigations reveal that Lck signaling is a crucial executor of inflammatory signaling in CD4+ T cells in the context of psoriatic inflammation. Therefore, Lck inhibition may be pursued as an effective strategy to counteract psoriatic inflammation.

LCK over-expression drives STAT5 oncogenic signaling in PAX5 translocated BCP-ALL patients.

The PAX5 gene is altered in 30% of BCP-ALL patients and PAX5 chromosomal translocations account for 2-3% of cases. Although PAX5 fusion genes significantly affect the transcription of PAX5 target genes, their role in sustaining leukemia cell survival is poorly understood. In an in vitro model of PAX5/ETV6 leukemia, we demonstrated that Lck hyper-activation, and down-regulation of its negative regulator Csk, lead to STAT5 hyper-activation and consequently to the up-regulation of the downstream effectors, cMyc and Ccnd2. More important, cells from PAX5 translocated patients show LCK up-regulation and over-activation, as well as STAT5 hyper-phosphorylation, compared to PAX5 wt and PAX5 deleted cases. As in BCR/ABL1 positive ALL, the hyper-activation of STAT5 pathway can represent a survival signal in PAX5 translocated cells, alternative to the pre-BCR, which is down-regulated. The LCK inhibitor BIBF1120 selectively reverts this phenomenon both in the murine model and in leukemic primary cells. LCK inhibitor could therefore represent a suitable candidate drug to target this subgroup of ALL patients.

Identification of the dichotomous role of age-related LCK in calorie restriction revealed by integrative analysis of cDNA microarray and interactome.

Among the many experimental paradigms used for the investigation of aging, the calorie restriction (CR) model has been proven to be the most useful in gerontological research. Exploration of the mechanisms underlying CR has produced a wealth of data. To identify key molecules controlled by aging and CR, we integrated data from 84 mouse and rat cDNA microarrays with a protein-protein interaction network. On the basis of this integrative analysis, we selected three genes that are upregulated in aging but downregulated by CR and two genes that are downregulated in aging but upregulated by CR. One of these key molecules is lymphocyte-specific protein tyrosine kinase (LCK). To further confirm this result on LCK, we performed a series of experiments in vitro and in vivo using kidneys obtained from aged ad libitum-fed and CR rats. Our major significant findings are as follows: (1) identification of LCK as a key molecule using integrative analysis; (2) confirmation that the age-related increase in LCK was modulated by CR and that protein tyrosine kinase activity was decreased using a LCK-specific inhibitor; and (3) upregulation of LCK leads to NF-κB activation in a ONOO(-) generation-dependent manner, which is modulated by CR. These results indicate that LCK could be considered a target attenuated by the anti-aging effects of CR. Integrative analysis of cDNA microarray and interactome data are powerful tools for identifying target molecules that are involved in the aging process and modulated by CR.

CD8 co-receptor promotes susceptibility of CD8+ T cells to transforming growth factor-ß (TGF-ß)-mediated suppression.

CD8+ T cell function depends on a finely orchestrated balance of activation/suppression signals. While the stimulatory role of the CD8 co-receptor and pleiotropic capabilities of TGF-ß have been studied individually, the influence of CD8 co-receptor on TGF-ß function in CD8+ T cells is unknown. Here, we show that while CD8 enhances T cell activation, it also enhances susceptibility to TGF-ß-mediated immune suppression. Using Jurkat cells expressing a full-length, truncated or no αßCD8 molecule, we demonstrate that cells expressing full-length αßCD8 were highly susceptible, αßCD8-truncated cells were partially susceptible, and CD8-deficient cells were completely resistant to suppression by TGF-ß. Additionally, we determined that inhibition of Lck rendered mouse CD8+ T cells highly resistant to TGF-ß suppression. Resistance was not associated with TGF-ß receptor expression but did correlate with decreased Smad3 and increased Smad7 levels. These findings highlight a previously unrecognized third role for CD8 co-receptor which appears to prepare activated CD8+ T cells for response to TGF-ß. Based on the important role which TGF-ß-mediated suppression plays in tumor immunology, these findings unveil necessary considerations in formulation of CD8+ T cell-related cancer immunotherapy strategies.

Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation.

Brain cancers frequently recur or progress as focal masses after treatment with ionizing radiation. Radiation used to target gliomas may expand the cancer stem cell population and enhance the aggressiveness of tumors; however, the mechanisms underlying the expansion of cancer stem cell population after radiation have remained unclear. In this study, we show that LCK (lymphocyte-specific protein tyrosine kinase) is involved in the fractionated radiation-induced expansion of the glioma-initiating cell population and acquisition of resistance to anticancer treatments. Fractionated radiation caused a selective increase in the activity of LCK, a Src family non-receptor tyrosine kinase. The activities of other Src family kinases Src, Fyn, and Lyn were not significantly increased. Moreover, knockdown of LCK expression with a specific small interfering RNA (siRNA) effectively blocked fractionated radiation-induced expansion of the CD133(+) cell population. siRNA targeting of LCK also suppressed fractionated radiation-induced expression of the glioma stem cell marker proteins CD133, Nestin, and Musashi. Expression of the known self-renewal-related proteins Notch2 and Sox2 in glioma cells treated with fractionated radiation was also downregulated by LCK inhibition. Moreover, siRNA-mediated knockdown of LCK effectively restored the sensitivity of glioma cells to cisplatin and etoposide. These results indicate that the non-receptor tyrosine kinase LCK is critically involved in fractionated radiation-induced expansion of the glioma-initiating cell population and decreased cellular sensitivity to anticancer treatments. These findings may provide pivotal insights in the context of fractionated radiation-based therapeutic interventions in brain cancer.

Cloning, characterization, and expression pattern of Atlantic halibut (Hippoglossus hippoglossus L.) CD4-2, Lck, and ZAP-70.

As known from mammalia, the co-receptors CD4 or CD8 associate with a lymphocyte cell-specific kinase (Lck) upon T-cell activation. Lck phosphorylates tyrosine residues within the CD3 chains, providing docking sites for a 70 kDa zeta-associated-protein (ZAP-70), a tyrosine protein kinase important for T-cell signaling. The sequences of a CD4-like gene (CD4-2), Lck, and ZAP-70 were cloned, characterized, and the relative expression pattern was explored in several organs of Atlantic halibut (Hippoglossus hippoglossus L.). Important structural features, as a signal peptide, two Ig-like domains followed by a connecting peptide, a transmembrane region, and a CxC motif within the cytoplasmic tail were conserved within the predicted halibut CD4-2 protein. The deduced halibut Lck protein sequence was found to be composed of a N-terminal Src homology (SH) 4 domain, required for membrane attachment and CD4/CD8 binding, SH3 and SH2 adapter domains, and a SH1 domain followed by a regulatory C-terminal tail (COOH-domain). Tyrosine residues important in Lck activation were conserved within the SH1 and COOH-domain. Structural features of ZAP-70 as tandem SH2 domains and a C-terminal SH1 domain were predicted within the halibut ZAP-70 sequence, having the highest level of conservation within these regions. Several important phosphorylation sites found to play a critical role in T-cell antigen receptor signaling in mammalian were conserved. The overall expression pattern of the three genes was highly similar, showing the highest mRNA level of all three genes in thymus. Some expression was seen in spleen, anterior and posterior kidney, gills, and fin, as seen for other halibut T-cell markers. This study will enable further experiments on halibut T-cell signaling and activation, and enhance understanding about the development of immunological memory T-cells of halibut.

Arsenic interferes with the signaling transduction pathway of T cell receptor activation by increasing basal and induced phosphorylation of Lck and Fyn in spleen cells.

Arsenic is known to produce inhibition as well as induction of immune cells proliferative responses depending on the doses as one of its mechanisms of immunotoxicity. Here we evaluate the effect of arsenic exposure on the activation of splenic mononuclear cells (SMC) in male CD57BL6N mice. Intra-gastric exposure to arsenic (as sodium arsenite) for 30 days (1, 0.1, or 0.01 mg/kg/day), reduced the proportion of CD4+ cells and the CD4+/CD8+ ratio in the spleen, increasing the proportion of CD11b+ cells. Arsenic exposure did not modify the proportion of B cells. SMC showed an increased level of phosphorylation of lck and fyn kinases (first kinases associated to TCR complex when activated). Although normal levels of apoptosis were observed on freshly isolated SMC, an increase in apoptotic cells related with the increase in phosphorylation of lck and fyn was observed when SMC were activated with Concanavalin-A (Con-A). Arsenic exposure reduced the proliferative response of SMC to Con-A, and also reduced secretion of IL-2, IL-6, IL-12 and IFNgamma. No effect was observed on IL-4, and IL-10 secretion. The same effects were observed when SMC of exposed animals were activated with anti-CD3/CD28 antibodies for 24 h, but these effects were transitory since a recovery, up to control levels or even higher, were observed after 72 h of stimulation. This study demonstrates that repeated and prolonged exposure to arsenic alters cell populations and produces functional changes depending on the specific activation pathway, and could be related with the phosphorylation status of lck and fyn kinases.

T-cell lymphomas in T-cell-specific Pten-deficient mice originate in the thymus.

Phosphatase and tensin homolog deleted on chromosome 10 (Pten) is a tumor suppressor protein whose loss of lipid phosphatase activity is associated with lymphomagenesis. We made use of the Cre-loxP system to delete Pten expression in Lck- or CD4-expressing T-lineage cells. Mice initially showed modest thymic hyperplasia and subsequently developed expanding and infiltrating T-cell lymphomas, leading to a premature death within 5 to 23 weeks. Frequently, all thymocyte and peripheral T-cell populations displayed phenotypes characteristic for immature developing thymocyte precursors and shared elevated levels of clonally rearranged T-cell receptor (TCR) beta chains. In concert, CD2, CD5, CD3epsilon and CD44, proteins associated with increased expression and signaling capacity of both the immature pre-TCR and the mature alphabetaTCR, were more abundantly expressed, reflecting a constitutive state of activation. Although most T-cell lymphomas had acquired the capability to infiltrate the periphery, not all populations left the thymus and expanded clonally exclusively in the thymus. In line with this, only transplantation of thymocytes with infiltrating capacity gave rise to T-cell lymphoma in immunodeficient recipients. These results indicate that T-cell-specific Pten deletion during various stages of thymocyte development gives rise to clonally expanding T-cell lymphomas that frequently infiltrate the periphery, but originate in the thymus.

Atorvastatin restores Lck expression and lipid raft-associated signaling in T cells from patients with systemic lupus erythematosus.

Loss of tolerance to self-Ags in patients with systemic lupus erythematosus (SLE), a prototypic autoimmune disease, is associated with dysregulation of T cell signaling, including the depletion of total levels of lymphocyte-specific protein kinase (Lck) from sphingolipid-cholesterol-enriched membrane microdomains (lipid rafts). Inhibitors of 3-hyroxy-3-methylgluteryl CoA reductase (statins) can modify the composition of lipid rafts, resulting in alteration of T cell signaling. In this study, we show that atorvastatin targets the distribution of signaling molecules in T cells from SLE patients, by disrupting the colocalization of total Lck and CD45 within lipid rafts, leading to a reduction in the active form of Lck. Upon T cell activation using anti-CD3/anti-CD28 in vitro, the rapid recruitment of total Lck to the immunological synapse was inhibited by atorvastatin, whereas ERK phosphorylation, which is decreased in SLE T cells, was reconstituted. Furthermore, atorvastatin reduced the production of IL-10 and IL-6 by T cells, implicated in the pathogenesis of SLE. Thus, atorvastatin reversed many of the signaling defects characteristic of SLE T cells. These findings demonstrate the potential for atorvastatin to target lipid raft-associated signaling abnormalities in autoreactive T cells and provide a rationale for its use in therapy of autoimmune disease.

Microarray analysis of selenium-depleted and selenium-supplemented mice.

Nutritional selenium deficiency is associated with Keshan disease in humans and white muscle disease in ruminant livestock. In this study, mice were fed a selenium-deficient diet for three generations. Female mice from the third depleted generation of these mice were given water containing either no added selenium or 0.1 or 1.0 ppm selenium as sodium selenate; DNA microarrays were used to compare gene expression in the muscle from mice fed the selenium diets to that from mice remaining on the depleted diet. The most prominent expression increases were observed with Ptger2 (a prostaglandin E receptor), Tcrb-V13 (a T-cell receptor beta), Tcf-7 (a T-cell transcription factor), and Lck (lymphocyte protein tyrosine kinase), and the major consistent decrease was Vav2, an oncogene in mice consuming the selenium containing diets.

A novel link between Lck, Bak expression and chemosensitivity.

Protein kinases are critically involved in signaling pathways that regulate cell growth, differentiation, activation, and survival. Lck, a member of the Src family of protein tyrosine kinases, plays a key role in T-lymphocyte activation and differentiation. However, under certain conditions Lck is also involved in the induction of apoptosis. In this issue of Oncogene, Samraj et al. used the Lck-defective JCaM1.6 cell line to demonstrate the critical role of Lck in the apoptotic response of T-cell leukemia cells to several chemotherapeutic drugs. They further showed that Lck controls the mitochondrial death pathway by regulating proapoptotic Bak expression. This chemosensitizing effect of Lck is independent of T-cell receptor signaling and does not require the kinase activity of Lck. These findings demonstrate that Lck might be part of two independent signaling pathways leading to either cell proliferation or apoptosis, and reveal a hitherto unrecognized link between Lck, Bak, and chemosensitivity of human leukemic cells.

Regulation of expression and function of Lck tyrosine kinase by high cell density.

For many types of cells, an increase in cell density leads to characteristic changes in intracellular signalling and cell function. It is unknown, however, whether cell density affects the function of T lymphocytes. It is presented here that aggregation of Jurkat T cells, murine thymocytes or human peripheral blood T cells, results in gradual modification of the Lck tyrosine kinase. Within one hour of aggregation, Lck in the detergent-insoluble lipid raft fraction is dephosphorylated mainly at the carboxy-terminal tyrosine. Further aggregation leads to gradual loss of Lck protein from both lipid raft and non-raft fractions which is accompanied by increased protein ubiquitination, a process that is more evident in the detergent-soluble fraction. In contrast, the expression of LAT, which like Lck distributes to raft and non-raft membrane, or Csk, a kinase with a structure similar to Lck, is not affected by cell aggregation. Dephosphorylation of lipid raft-associated Lck, albeit with reduced kinetics, is observed in aggregated Jurkat CD45-deficient cells as well, suggesting involvement of additional tyrosine phosphatases. Changes in Lck structure and expression correlate with reduced ability of aggregated cells to fully activate protein tyrosine phosphorylation after stimulation of the TCR, and with changes in the activation of down-stream signalling cascades.

Optimisation and evaluation of a high-throughput mammalian protein expression system.

Transient transfection of mammalian cells with episomal vectors is a very useful method for producing high levels of recombinant proteins. Transient systems remove the need for the laborious and time-consuming process of creating stable cell lines. Here, we describe the optimisation and evaluation of a high-throughput transient expression system in HEK293-EBNA cells. The process was developed for the expression of 10 constructs simultaneously in deep-well plates and subsequent purification using 96-well plate affinity chromatography. This enabled multiple combinations of different constructs, vectors, and expression conditions to be studied in parallel.

Adjuvant, adoptive immunotherapy with tumor infiltrating lymphocytes plus interleukin-2 after radical hepatic resection for colorectal liver metastases: 5-year analysis.

BACKGROUND AND OBJECTIVES: Conventional chemotherapy has not proven effective in improving long-term results of surgery for liver metastases from colorectal cancer. We assessed the usefulness of immunotherapy with tumor infiltrating lymphocytes (TIL) plus Interleukin-2 (IL-2) as adjuvant treatment. METHODS: Between 1995 and 1998, 47 patients were enrolled onto a prospective protocol; 25 entered the treatment group (A) and 22 entered the control group (B). All patients had undergone radical liver resection. TIL obtained from surgical specimens from group A patients were cultured and activated in vitro with IL-2, then reinfused into the patients with IL-2. We investigated pre- and post-IL-2 stimulation expression of T cell receptor (TCR) zeta- and epsilon-chains, p56(lck), Fas, and Fas-L by TIL immunostaining. RESULTS: Fourteen patients from group A (56%) received immunotherapy; 14 from group B (60%) underwent conventional chemotherapy, and the remaining 19 patients did not receive any treatment. No significant differences between the two groups were found in the actuarial and disease-free survival (DSF) rates after 1, 3, and 5 years. After IL-2 exposure, TCR zeta-chain expression significantly increased (P = 0.001); An increase in TCR epsilon-chain expression (P = 0.04), and p56(lck) (P = 0.03) was detected; TCR epsilon-chain expression was significantly increased in disease-free patients compared to those who relapsed (P = 0.04). Fas-L expression was correlated with the TCR epsilon-chain and p56(lck) levels (P = 0.05). CONCLUSIONS: Our data suggest that we are still a long way from being able to propose TIL + IL-2 treatment as an effective adjuvant therapy. However, the results confirm that the biological indicators examined could play an important role in modulating immunitary response against tumor cells.

Enrichment of lck in lipid rafts regulates colocalized fyn activation and the initiation of proximal signals through TCR alpha beta.

Recent results provide insight into the temporal and spatial relationship governing lck-dependent fyn activation and demonstrate TCR/CD4-induced activation and translocation of lck into lipid rafts and the ensuing activation of colocalized fyn. The prediction follows that directly targeting lck to lipid rafts will bypass the requirement for juxtaposing TCR and CD4-lck, and rescue cellular activation mediated by Ab specific for the constant region of TCRbeta chain. The present study uses a family of murine IL-2-dependent CD4(+) T cell clonal variants in which anti-TCRCbeta signaling is impaired in an lck-dependent fashion. Importantly, these variants respond to Ag- and mAb-mediated TCR-CD4 coaggregation, both of which enable the coordinated interaction of CD4-associated lck with the TCR/CD3 complex. We have previously demonstrated that anti-TCRCbeta responsiveness in this system correlates with the presence of kinase-active, membrane-associated lck and preformed hypophosphorylated TCRzeta:zeta-associated protein of 70 kDa complexes, a phenotype recapitulated in primary resting CD4(+) T cells. We show in this study that forced expression of wild-type lck achieved the same basal composition of the TCR/CD3 complex and yet did not rescue anti-TCRCbeta signaling. In contrast, forced expression of C20S/C23S-mutated lck (double-cysteine lck), unable to bind CD4, rescues anti-TCRCbeta proximal signaling and cellular growth. Double-cysteine lck targets lipid rafts, colocalizes with >98% of cellular fyn, and results in a 7-fold increase in basal fyn kinase activity. Coaggregation of CD4 and TCR achieves the same outcome. These results underscore the critical role of lipid rafts in spatially coordinating the interaction between lck and fyn that predicates proximal TCR/CD3 signaling.

Superantigen stimulation reveals the contribution of Lck to negative regulation of T cell activation.

The conventional paradigm of T cell activation through the TCR states that Lck plays a critical activating role in this signaling process. However, the T cell response to bacterial superantigens does not require Lck. In this study we report that not only is Lck dispensable for T cell activation by superantigens, but it actively inhibits this signaling pathway. Disruption of Lck function, either by repression of Lck gene expression or by selective pharmacologic inhibitors of Lck, led to increased IL-2 production in response to superantigen stimulation. This negative regulatory effect of Lck on superantigen-induced T cell responses required the kinase activity of Lck and correlated with early TCR signaling, but was independent of immunological synapse formation and TCR internalization. Our data demonstrate that the multistage role of Lck in T cell signaling includes the activation of a negative regulatory pathway of T cell activation.

Selective activation of Src family kinases and JNK by low levels of chromium(VI).

Inhaled hexavalent chromium (Cr(VI)) promotes pulmonary disease and lung cancer through poorly defined mechanisms. These mechanisms were studied in A549 lung epithelial cells to investigate the hypothesis that nontoxic Cr(VI) exposures selectively activate cell signaling that shifts the balance of gene transcription. These studies demonstrated that nontoxic doses of Cr(VI) (10 microM) increased reactive oxygen species and selectively activated c-Jun N-terminal kinase (JNK), relative to ERK or p38 MAP kinase. In contrast, only toxic, nonselective levels of exogenous oxidants stimulated JNK. However, JNK activation in response to Cr(VI) and exogenous H(2)O(2) (1 mM) shared requirements for intracellular thiol oxidation, activation of Src family kinases, and p130(cas) (Cas). Cr(VI) did not mimic H(2)O(2)-mediated stimulation of JNK in fibroblasts containing only Src and did not activate Src or Yes in A549 cells. Instead, Fyn and Lck were activated in A549 cells, indicating activation of specific Src family kinases in response to Cr(VI). Finally, Cr(VI) was demonstrated to directly activate purified Fyn in vitro and the majority of this activation did not require oxidant generation. These data suggest that nontoxic levels of Cr(VI), which can shift patterns of gene transcription, are selective in their activation of cell signaling and that Cr(VI) can directly activate Src family kinases independently of reactive oxygen species generation.

Allelic exclusion of the TCR alpha-chain is an active process requiring TCR-mediated signaling and c-Cbl.

Phenotypic allelic exclusion at the TCRalpha locus is developmentally regulated in thymocytes. Many immature thymocytes express two cell surface alpha-chain species. Following positive selection, the vast majority of mature thymocytes and peripheral T cells display a single cell surface alpha-chain. A posttranslational mechanism occurring at the same time as positive selection and TCR up-regulation leads to this phenotypic allelic exclusion. Different models have been proposed to explain the posttranslational regulation of the alpha-chain allelic exclusion. In this study, we report that allelic exclusion is not regulated by competition between distinct alpha-chains for a single beta-chain, as proposed by the dueling alpha-chain model, nor by limiting CD3 zeta-chain in mature TCR(high) thymocytes. Our data instead favor the selective retention model where the positive selection signal through the TCR leads to phenotypic allelic exclusion by specifically maintaining cell surface expression of the selected alpha-chain while the nonselected alpha-chain is internalized. The use of inhibitors specific for Lck and/or other Src kinases indicates a role for these protein tyrosine kinases in the signaling events leading to the down-regulation of the nonselectable alpha-chain. Loss of the ubiquitin ligase/TCR signaling adapter molecule c-Cbl, which is important in TCR down-modulation and is a negative regulator of T cell signaling, leads to increased dual alpha-chain expression on the cell surface of double-positive thymocytes. Thus, not only is there an important role for TCR signaling in causing alpha-chain allelic exclusion, but differential ubiquitination by c-Cbl may be an important factor in causing only the nonselected alpha-chain to be down-modulated.

Optimal colocalization of TCR and CD8 as a novel mechanism for the control of functional avidity.

The improved efficacy of high avidity CTL for clearance of virus has been well-documented. Thus, elucidation of the mechanisms that confer the increased sensitivity to peptide ligand demonstrated by high avidity CTL is critical. Using CTL lines of high and low avidity generated from a TCR transgenic mouse, we have found that functional avidity can be controlled by the expression of CD8alphaalpha vs CD8alphabeta and the ability of CTLs to colocalize the TCR and CD8 in the membrane. Colocalization of these molecules was mediated by lipid rafts and importantly, raft disruption resulted in the conversion of high avidity CTL into a lower functional avidity phenotype. These novel findings provide insights into the control of functional avidity in response to viral infection.

TCR signals mediated by Src family kinases are essential for the survival of naive T cells.

The role of TCR signals triggered by recognition of self MHCs in maintaining the survival of naive peripheral T cells remains controversial. Here we examine the role of the Src family kinases, p56(lck) (Lck) and p59(fyn) (Fyn), in the survival of naive T cells. We show that long term survival requires a combination of signals transduced by Src family kinases and signals through the IL-7R. In the absence of either one, naive T cells die slowly, but if both signals are removed, cell loss is greatly accelerated. The TCR signal can be mediated by either Fyn or Lck at wild-type levels of expression, but not by Lck alone if expressed suboptimally. The disappearance of T cells in the absence of Fyn and Lck was associated with a complete loss of TCRzeta-chain phosphorylation and down-regulation of CD5, both of which are also MHC contact dependent, indicating that the Src family kinases are critical for transducing a TCR-MHC survival signal.