The CD1 family of major histocompatibility complex (MHC)-like molecules specializes in presenting lipid and glycolipid antigens to alpha/beta T lymphocytes, but little is known about the size of the CD1-restricted T cell population or the frequency of T lymphocytes specific for a given glycolipid antigen. Here, we report the generation and use of mouse CD1d1-glycolipid tetramers to visualize CD1d-restricted T cells. In contrast with previous BIAcore-based estimates of very short half-lives for CD1d-glycolipid complexes, we found that the dissociation rate of several different CD1d-glycolipid complexes was very slow. Fluorescent tetramers of mouse CD1d1 complexed with alpha-galactosylceramide (alphaGalCer), the antigen recognized by mouse Valpha14-Jalpha281/Vbeta8 and human Valpha24-JalphaQ/Vbeta11 natural killer T (NKT) cell T cell receptors (TCRs), allowed us for the first time to accurately describe, based on TCR specificity, the entire population of NKT cells in vivo and to identify a previously unrecognized population of NK1.1-negative "NKT" cells, which expressed a different pattern of integrins. In contrast, natural killer (NK) cells failed to bind the tetramers either empty or loaded with alphaGalCer, suggesting the absence of a CD1d-specific, antigen-nonspecific NK receptor. Mouse CD1d1-alphaGalCer tetramers also stained human NKT cells, indicating that they will be useful for probing a range of mouse and human conditions such as insulin-dependent diabetes mellitus, tumor rejection, and infectious diseases where NKT cells play an important role.

To define the phenotype and T cell receptor (TCR) repertoire of CD1d-dependent T cells, we compared the populations of T cells that persisted in major histocompatibility complex (MHC)-deficient mice, which lack mainstream T cells, with those from MHC/CD1d doubly deficient mice, which lack both mainstream and CD1d-dependent T cells. Surprisingly, up to 80% of the CD1d-dependent T cells were stained by tetramers of CD1d/alpha-galactosylceramide, which specifically identify the previously described CD1d autoreactive Valpha14-Jalpha18/Vbeta8 natural killer (NK) T cells. Furthermore, zooming in on the CD1d-dependent non-Valpha14 T cells, we found that, like Valpha14 NK T cells, they mainly expressed recurrent, CD1d autoreactive TCR families and had a natural memory phenotype. Thus, CD1d-restricted T cells differ profoundly from MHC-peptide-specific T cells by their predominant use of autoreactive and semiinvariant, rather than naive and diverse, TCRs. They more closely resemble other lineages of innate lymphocytes such as B-1 B cells, gammadelta T cells, and NK cells, which express invariant or semiinvariant autoreactive receptors. Finally, we demonstrate that the MHC-restricted TCR repertoire is essentially non-cross-reactive to CD1d. Altogether, these findings imply that lipid recognition by CD1d-restricted T cells may have largely evolved as an innate rather than an adaptive arm of the mouse immune system.

Given the importance of the Rho GTPase family member Rac1 and the Rac1/Cdc42 effector PAK1 in T-cell activation, we investigated the requirements for their activation by the T-cell receptor (TCR). Rac1 and PAK1 activation required the tyrosine kinases ZAP-70 and Syk, but not the cytoplasmic adaptor Slp-76. Surprisingly, PAK1 was activated in the absence of the transmembrane adaptor LAT while Rac1 was not. However, efficient PAK1 activation required its binding sites for Rho GTPases and for PIX, a guanine nucleotide exchange factor for Rho GTPases. The overexpression of ssPIX that either cannot bind PAK1 or lacks GEF function blocked PAK1 activation. These data suggest that a PAK1-PIX complex is recruited to appropriate sites for activation and that PIX is required for Rho family GTPase activation upstream of PAK1. Furthermore, we detected a stable trimolecular complex of PAK1, PIX and the paxillin kinase linker p95PKL. Taken together, these data show that PAK1 contained in this trimolecular complex is activated by a novel LAT- and Slp-76-independent pathway following TCR stimulation.

T cell antigen receptor (TCR) stimulation induces tyrosine phosphorylation of many intracellular proteins, including the proto-oncogene Vav, which is expressed exclusively in hematopoietic and trophoblast cells. Vav is critical for lymphocyte development and activation. Overexpression of Vav in Jurkat T cells leads to potentiation of TCR-mediated IL-2 gene activation. However, the biochemical function of Vav is unknown. Here, we demonstrate that the major induced tyrosine phosphoprotein associated with Vav is the hematopoietic cell-specific SLP-76. The Vav SH2 domain is required for this interaction and for TCR-mediated Vav tyrosine phosphorylation. Similar to Vav, overexpression of SLP-76 markedly potentiates TCR-mediated NF-AT and IL-2 gene activation. Furthermore, overexpression of both Vav and SLP-76 synergistically induces basal and TCR-stimulated NF-AT activation. These results suggest that a signaling complex containing Vav and SLP-76 plays an important role in lymphocyte activation.

Stimulation of antigen receptors in T and B cells leads to the activation of the Src and Syk families of protein tyrosine kinases (PTK). These PTKs subsequently phosphorylate numerous intracellular substrates, including the 95-kD protooncogene product Vav. Vav is essential for both T and B cell development and T and B cell antigen receptor-mediated signal transduction. After receptor ligation, Vav associates with phosphorylated Syk and ZAP-70 PTKs, an interaction that depends upon its SH2 domain. Here we demonstrate that a point mutation of tyrosine 315 (Y315F) in ZAP-70, a putative Vav SH2 domain binding site, eliminated the Vav- ZAP-70 interaction. Moreover, the Y315 mutation impaired the function of ZAP-70 in antigen receptor signaling. Surprisingly, this mutation also resulted in marked reduction in the tyrosine phosphorylation of ZAP-70, Vav, SLP-76, and Shc. These data demonstrate that the Vav binding site in ZAP-70 plays a critical role in antigen receptor-mediated signal transduction.

Naive CD4+ T helper cells (Th) differentiate into one of two well-defined cell types during immune responses. Mature Th1 and Th2 cells regulate the type of response as a consequence of the unique cytokines that they secrete. CD4 serves a prominent role in potentiating antigen recognition by helper T cells. We have examined the role of CD4 in peripheral T cell differentiation by studying helper T cells from mice with a congenital defect in CD4 expression. After protein immunization or infection with Leishmania major, CD4-deficient mice were incapable of mounting antigen-specific Th2 responses, but retained their Th1 potency. CD4-deficient, T cell receptor transgenic T cells were also incapable of Th2 differentiation after in vitro activation. Expression of a wild-type CD4 transgene corrected the Th2 defect of CD4-deficient mice in all immune responses tested. To investigate the role of the cytoplasmic domain, mice reconstituted with a truncated CD4 molecule were also studied. Expression of the tailless CD4 transgene could not rescue the Th2 defect of CD4-deficient mice immunized with protein or CD4-deficient transgenic T cells activated in vitro, raising the possibility that the cytoplasmic domain of CD4 may influence Th2 generation. Expression of the tailless transgene was, however, capable of restoring Th2 development in CD4-deficient mice infected with L. major or CD4-deficient transgenic T cells activated in the presence of recombinant IL-4, demonstrating that the cytoplasmic domain is not absolutely required for Th2 development. Together, these results demonstrate a previously undescribed role of the CD4 molecule. The requirement for CD4 in Th2 maturation reflects the importance of molecules other than cytokines in the control of helper T cell differentiation.

The angiogenesis inhibitor sunitinib is a tyrosine kinase inhibitor that acts mainly on the VEGF and PDGF pathways. We have previously shown that sunitinib is sequestered in the lysosomes of exposed tumor and endothelial cells. This phenomenon is part of the drug-induced resistance observed in the clinic. Here, we demonstrate that when exposed to light, sequestered sunitinib causes immediate destruction of the lysosomes, resulting in the release of sunitinib and cell death. We hypothesized that this photoactivation of sunitinib could be used as a vaso-occlusive vascular-targeting approach to treating cancer. Spectral properties of sunitinib and its lysosomal accumulation were measured in vitro. The human A2780 ovarian carcinoma transplanted onto the chicken chorioallantoic membrane (CAM) and the Colo-26 colorectal carcinoma model in Balb/c mice were used to test the effects of administrating sunitinib and subsequently exposing tumor tissue to light. Tumors were subsequently resected and subject to immunohistochemical analysis. In A2780 ovarian carcinoma tumors, treatment with sunitinib+light resulted in immediate specific angio-occlusion, leading to a necrotic tumor mass 24 h after treatment. Tumor growth was inhibited by 70% as compared with the control group (**P<0.0001). Similar observations were made in the Colo-26 colorectal carcinoma, where light exposure of the sunitinib-treated mice inhibited tumor growth by 50% as compared with the control and by 25% as compared with sunitinib-only-treated tumors (N≥4; P=0.0002). Histology revealed that photoactivation of sunitinib resulted in a change in tumor vessel architecture. The current results suggest that the spectral properties of sunitinib can be exploited for application against certain cancer indications.

In this report, we show that the Src-like adaptor protein (SLAP) plays an important role in thymocyte development. SLAP expression is developmentally regulated; it is low in CD4-CD8- thymocytes, it peaks in the CD4+CD8+ subset, and it decreases to low levels in more mature cells. Disruption of the SLAP gene leads to a marked upregulation of TCR and CD5 expression at the CD4+CD8+ stage. The absence of SLAP was also developmentally significant because it enhanced positive selection in mice expressing the DO11.10 transgenic T cell receptor. Moreover, SLAP deletion at least partially rescued the development of ZAP-70-deficient thymocytes. These results demonstrate that SLAP participates in a novel mechanism of TCR downregulation at the CD4+CD8+ stage and regulates positive selection.

Binding of a T cell to an appropriate antigen-presenting cell (APC) induces the rapid reorientation of the T cell cytoskeleton and secretory apparatus towards the cell-cell contact site in a T cell antigen receptor (TCR) and peptide/major histocompatibility complex-dependent process. Such T cell polarization directs the delivery of cytokines and cytotoxic mediators towards the APC and contributes to the highly selective and specific action of effector T cells. To study the signaling pathways that regulate cytoskeletal rearrangements in T lymphocytes, we set up a conjugate formation assay using Jurkat T cells as effectors and cell-sized latex beads coated with various antibodies as artificial APCs. Here, we report that beads coated with antibodies specific for the TCR-CD3 complex were sufficient to induce T cell polarization towards the bead attachment site, as judged by reorientation of the microtubule-organizing center (MTOC) and localized actin polymerization. Thus, these cytoskeletal changes did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-zeta and CD3epsilon, were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-zeta and once in CD3epsilon, revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling requirements.

CD28 is a cell surface molecule that mediates a costimulatory signal crucial for T cell proliferation and lymphokine production. The signal transduction mechanisms of CD28 are not well understood. Itk, a nonreceptor protein tyrosine kinase specifically expressed in T cells and mast cells, has been implicated in the CD28 signaling pathway because of reports that it becomes phosphorylated on tyrosines and associates with CD28 upon cross-linking of the cell surface molecule. To determine whether Itk plays a functional role in CD28 signaling, we compared T cells from Itk-deficient mice and control mice for their responses to CD28 costimulation. T cells defective in Itk were found to be fully competent to respond to costimulation. Whereas the CD3-mediated proliferative response was severely compromised in the absence of Itk, the calcineurin-independent CD28-mediated response was significantly elevated when compared with cells from control animals. The augmented proliferation was not due to increased production of interleukin-2. The results suggest that Itk has distinct roles in the CD3 versus the CD28 signaling pathways. By negatively regulating the amplitude of signaling upon CD28 costimulation, Itk may provide a means for modulating the outcome of T cell activation during development and during antigen-driven immune responses.

The arrival of the Delta variant of SARS-CoV-2 was associated with increased transmissibility and illness of greater severity. Reports of nosocomial outbreaks of Delta variant COVID-19 in acute care hospitals have been described but control measures varied widely.

To assess the role of exocytotic release in signaling by monoamines, we have disrupted the neuronal vesicular monoamine transporter 2 (VMAT2) gene. VMAT2-/- mice move little, feed poorly, and die within a few days after birth. Monoamine cell groups and their projections are indistinguishable from those of wild-type littermates, but the brains of mutant mice show a drastic reduction in monoamines. Using midbrain cultures from the mutant animals, amphetamine but not depolarization induces dopamine release. In vivo, amphetamine increases movement, promotes feeding, and prolongs the survival of VMAT2-/- animals, indicating that precise, temporally regulated exocytotic release of monoamine is not required for certain complex behaviors. In addition, the brains of VMAT2 heterozygotes contain substantially lower monoamine levels than those of wild-type littermates, and depolarization induces less dopamine release from heterozygous than from wild-type cultures, suggesting that VMAT2 expression regulates monoamine storage and release.

Huntington's disease (HD) is a dominant disorder characterized by premature and progressive neurodegeneration. In order to generate an accurate model of the disease, we introduced an HD-like mutation (an extended stretch of 72-80 CAG repeats) into the endogenous mouse Hdh gene. Analysis of the mutation in vivo reveals significant levels of germline instability, with expansions, contractions and sex-of-origin effects in evidence. Mice expressing full-length mutant protein display abnormal social behaviour in the absence of acute neurodegeneration. Given that psychiatric changes, including irritability and aggression, are common findings in HD patients, our data are consistent with the hypothesis that some clinical features of HD may be caused by pathological processes that precede gross neuronal cell death. This implies that effective treatment of HD may require an understanding and amelioration of these dysfunctional processes, rather than simply preventing the premature death of neurons in the brain. These mice should facilitate the investigation of the molecular mechanisms that underpin the pathway from genotype to phenotype in HD.

Although recent studies have indicated that the major histocompatibility complex-like, beta2-microglobulin-associated CD1 molecules might function to present a novel chemical class of antigens, lipids and glycolipids, to alpha/beta T cells, little is known about the T cell subsets that interact with CD1. A subset of CD1d-autoreactive, natural killer (NK)1.1 receptor-expressing alpha/beta T cells has recently been identified. These cells, which include both CD4(-)CD8(-) and CD4(+) T cells, preferentially use an invariant Valpha14-Jalpha281 T cell receptor (TCR) alpha chain paired with a Vbeta8 TCR beta chain in mice, or the homologous Valpha24-JalphaQ/Vbeta11 in humans. This cell subset can explosively release key cytokines such as interleukin (IL)-4 and interferon (IFN)-gamma upon TCR engagement and may regulate a variety of infectious and autoimmune conditions. Here, we report the existence of a second subset of CD1d-restricted CD4(+) T cells that do not express the NK1.1 receptor or the Valpha14 TCR. Like the Valpha14(+) NK1.1(+) T cells, these T cells exhibit a high frequency of autoreactivity to CD1d, use a restricted albeit distinct set of TCR gene families, and contribute to the early burst of IL-4 and IFN-gamma induced by intravenous injection of anti-CD3. However, the Valpha14(+) NK1.1(+) and Valpha14(-) NK1.1(-) T cells differ markedly in their requirements for self-antigen presentation. Antigen presentation to the Valpha14(+) NK1.1(+) cells requires endosomal targeting of CD1d through a tail-encoded tyrosine-based motif, whereas antigen presentation to the Valpha14(-) NK1.1(-) cells does not. These experiments suggest the existence of two phenotypically different subsets of CD1d-restricted T cells that survey self-antigens loaded in distinct cellular compartments.

Absence of CD4 impairs the efficiency of T cell receptor (TCR) signaling in response to major histocompatibility complex (MHC) class II-presented peptides. Here we use mice carrying a conditional Cd4 allele to study the consequences of impaired TCR signaling after the completion of thymocyte development. We show that loss of CD4 decreases the steady-state proliferation of T cells as monitored by in vivo labeling with bromo-deoxyuridine. Moreover, T cells lacking CD4 compete poorly with CD4-expressing T cells during proliferative expansion after transfer into lymphopenic recipients. The data suggest that T cells compete with one another during homeostatic proliferation, and indicate that the basis of this competition is TCR signaling.

The T-cell antigen receptor (TCR) triggers a signaling cascade initiated by the tyrosine kinase Lck and requiring the proto-oncogene p95(vav). Vav is activated by Lck and can function as a guanine nucleotide exchange factor for the Rho-family GTPases, Rac1 and Cdc42. To investigate the involvement of these GTPases in TCR signaling, we focused on their well characterized effector, Pak1. This serine/threonine kinase is activated by GTP-bound Rac1 or Cdc42. However, its role in mediating downstream signaling events is controversial. We observed rapid, TCR-dependent activation of Pak1 and TCR-inducible association of Pak1 with Nck, which was tyrosine phosphorylated following stimulation. Pak1 activation occurred independently of Ras activation or calcium flux, but was dependent on the Lck tyrosine kinase, and was downstream of Vav and Cdc42. Dominant negative Pak1 or Nck specifically inhibited TCR-mediated activation of the nuclear factor of activated T cells (NFAT) transcription factor. TCR-mediated activation of Erk2 was also inhibited by dominant negative Pak. However, Pak1 activation was neither necessary nor sufficient for TCR-dependent c-Jun N-terminal kinase (JNK) activation. Therefore, Pak1 acts downstream of Vav and is required for activation of Erk2 and NFAT by a JNK-independent pathway. This is the first demonstration of a requirement for Pak to mediate the regulation of gene expression by an extracellular ligand.

Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)-, and interleukin 2-dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3zeta, ZAP-70, SH2 domain-containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling.

The Src family protein tyrosine kinases (PTKs), Lck and Fyn, are coexpressed in T cells and perform crucial functions involved in the initiation of T cell antigen receptor (TCR) signal transduction. However, the mechanisms by which Lck and Fyn regulate TCR signaling are still not completely understood. One important question is whether Lck and Fyn have specific targets or only provide functional redundancy during TCR signaling. We have previously shown that Lck plays a major role in the tyrosine phosphorylation of the TCR-zeta chain and the ZAP-70 PTK. In an effort to identify the targets that are specifically regulated by Fyn, we have studied the tyrosine phosphorylation of Pyk2, a recently discovered new member of the focal adhesion kinase family PTK. We demonstrated that Pyk2 was rapidly tyrosine phosphorylated following TCR stimulation. TCR-induced tyrosine phosphorylation of Pyk2 was selectively dependent on Fyn but not Lck. Moreover, in heterologous COS-7 cells, coexpression of Pyk2 with Fyn but not Lck resulted in substantial increases in Pyk2 tyrosine phosphorylation. The selective regulation of Pyk2 tyrosine phosphorylation by Fyn in vivo correlated with the preferential phosphorylation of Pyk2 by Fyn in vitro. Our results demonstrate that Pyk2 is a specific target regulated by Fyn during TCR signaling.

ZAP-70 is a nonreceptor protein tyrosine kinase that is essential for signaling via the T cell antigen receptor (TCR). ZAP-70 becomes phosphorylated and activated by LCK protein tyrosine kinase after interaction of its two NH2-terminal SH2 domains with tyrosine-phosphorylated subunits of the activated TCR. In this study, the localization of ZAP-70 was investigated by immunofluorescence and confocal microscopy. ZAP-70 was found to be localized to the cell cortex in a diffuse band under the plasma membrane in unstimulated T cells, and this localization was not detectably altered by TCR stimulation. Analysis of mutants indicated that ZAP-70 targeting was independent of its SH2 domains but required its active kinase domain. The specific compartmentalization of ZAP-70 suggests that it may interact with an anchoring protein in the cell cortex via its hinge or kinase domains. It is likely that the maintenance of high concentrations of ZAP-70 at the cell cortex, that only has to move a short distance to interact with phophorylated TCR subunits, facilitates rapid initiation of signaling by the TCR. In addition, as the major increase in tyrosine phosphorylation induced by the TCR also occurs at the cell cortex (Ley, S.C., M. Marsh, C.R. Bebbington, K. Proudfoot, and P. Jordan. 1994. J. Cell. Biol. 125:639-649), ZAP-70 may be localized close to its downstream targets.

Itk is a member of the Btk/Tec/Itk family of nonreceptor protein tyrosine kinases (PTKs), and has been implicated in T cell antigen receptor (TCR) signal transduction. Lck and Fyn are the Src-family nonreceptor PTKs that are involved in TCR signaling. To address the question of how these members of different families of PTKs functionally contribute to T cell development and to T cell activation, mice deficient for both Itk and either Lck or Fyn were generated. The Itk/Lck doubly deficient mice exhibited a phenotype similar to that of Lck-deficient mice. The phenotype of the Itk/Fyn doubly deficient mice was similar to that of Itk deficient mice. However the Itk/Fyn doubly deficient mice exhibited a more severe defect in TCR-induced proliferation of thymocytes and peripheral T cells than did mice deficient in either kinase alone. These data support the notion that Itk and Fyn both make independent contributions to TCR-induced T cell activation.