Ligand-stimulated signaling events in immature CD4+CD8+ thymocytes expressing competent T-cell receptor complexes.

During thymic selection of the developing T-cell repertoire, the fate of individual CD4+CD8+ thymocytes is determined by the specificity of the T-cell antigen receptors (TCRs) they express. Paradoxically, most CD4+CD8+ thymocytes express few TCR molecules, and those they express are essentially incapable of transducing intracellular signals as measured by intracellular calcium mobilization. However, both TCR number and calcium-signaling capability are significantly induced in CD4+CD8+ thymocytes when the cells are released from intrathymic inhibitory signals that are mediated by their CD4 molecules. Here, the response to ligand engagement of TCR on "induced" CD4+CD8+ thymocytes that have been released from CD4-mediated inhibition was examined and was found to result in internalization of surface TCR complexes and rephosphorylation of zeta chains of the TCR complex. In addition, a proportion of induced CD4+CD8+ thymocytes were found to fragment their DNA upon ligand engagement. Thus, this study describes early events in immature CD4+CD8+ thymocytes resulting from TCR-mediated signals.     

Subcellular localization of T-cell receptor complexes containing tyrosine-phosphorylated zeta proteins in immature CD4+CD8+ thymocytes.

The T-cell antigen receptor (TCR) is a complex of at least six different proteins (alpha, beta, gamma, delta, epsilon, and zeta) that is assembled in the endoplasmic reticulum (ER) and transported to the cell surface. Unlike mature T cells, most immature CD4+CD8+ thymocytes retain within the ER and degrade greater than 90% of some of the TCR components they synthesize, resulting in low surface expression of TCR complexes. The few surface TCR complexes that most immature CD4+CD8+ thymocytes do express are only marginally capable of transducing signals mobilizing intracellular calcium. The inverse relationship with TCR expression and function suggested that phosphorylated zeta (P-zeta) molecules might function in CD4+CD8+ thymocytes either as an ER retention signal for newly synthesized TCR complexes or as a negative regulatory modification of TCR complexes present on the cell surface. The present study sought to evaluate these two possibilities by determining the subcellular location of TCR complexes containing P-zeta chains. We found that, unlike unmodified zeta chains, all P-zeta chains in CD4+CD8+ thymocytes existed in assembled TCR complexes and that all TCR complexes containing P-zeta molecules had undergone carbohydrate processing events indicative of transit through the Golgi apparatus. These results demonstrate that P-zeta chains are exclusively associated with mature TCR complexes, excluding the possibility that P-zeta serves as an ER retention signal in immature thymocytes. Although we could not directly determine the representation of P-zeta chains among surface TCR complexes, we found that 60-70% of surface TCR complexes on immature CD4+CD8+ thymocytes were associated with tyrosine-phosphorylated protein(s) and that this percentage was inversely correlated with their signaling competence. These results support the concept that tyrosine phosphorylation serves as a negative regulatory modification of certain TCR-associated proteins.     

The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck

CD4 molecules on the surface of T lymphocytes greatly augment the sensitivity and activation process of these cells, but how it functions is not fully understood. Here we studied the spatial organization of CD4, and its relationship to T-cell antigen receptor (TCR) and the active form of Src kinase p56lck (Lck) using single and dual-color photoactivated localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM). In nonactivated T cells, CD4 molecules are clustered in small protein islands, as are TCR and Lck. By dual-color imaging, we find that CD4, TCR, and Lck are localized in their separate clusters with limited interactions in the interfaces between them. Upon T-cell activation, the TCR and CD4 begin clustering together, developing into microclusters, and undergo a larger scale redistribution to form supramolecluar activation clusters (SMACs). CD4 and Lck localize in the inner TCR region of the SMAC, but this redistribution of disparate cluster structures results in enhanced segregation from each other. In nonactivated cells these preclustered structures and the limited interactions between them may serve to limit spontaneous and random activation events. However, the small sizes of these island structures also ensure large interfacial surfaces for potential interactions and signal amplification when activation is initiated. In the later activation stages, the increasingly larger clusters and their segregation from each other reduce the interfacial surfaces and could have a dampening effect. These highly differentiated spatial distributions of TCR, CD4, and Lck and their changes during activation suggest that there is a more complex hierarchy than previously thought.For helper T cells, CD4 has been termed a coreceptor based on its important role in antigen recognition class II major histocompatibility complex (MHC)–peptide complexes by the αβ T-cell receptor (TCR) as well as in signal transduction. Indeed, CD4 significantly increases T-cell sensitivity to antigen upon activation (1–4). This ability of CD4 to enhance antigen recognition has often been connected to the fact that the N-terminal Ig domain of CD4 has specific affinity to invariant sites on MHC class II molecules (5, 6). It has been suggested that CD4 stabilizes the molecular complex of TCR and peptide–MHC (pMHC) by binding to the same MHC either simultaneously with the TCR (7) or shortly after TCR–pMHC engagement (2, 3). However, from more recent 2D measurements, CD4 blockades showed no effect on the stability of TCR binding to agonist peptide–MHC complexes in a synapse (8). In terms of signal transduction, the role of CD4 has been studied based on the binding ability of a cysteine motif in the cytoplasmic tail of CD4 to Src kinase p56lck (Lck) (9), which is responsible for the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) sequences in TCR–CD3 complex as the earliest observable biochemical event during T-cell activation (10). It has been proposed that CD4 mainly contributes to the sensitivity of T cells by facilitating the recruitment of Lck to TCR–CD3s that are actively engaged in ligand recognition (11, 12). Nevertheless, the absence of CD4 does not preclude T cells from being generated at the thymus or being activated by TCR–pMHC engagement (13, 14).It is now well appreciated that spatial reorganization and distribution of some of the membrane receptors and signaling molecules is one of the critical regulating mechanisms in T-cell activation. The molecular assembly and clusters such as supramolecular activation clusters (SMACs) (15) of immunological synapse (IS) (14), microclusters (16–20), and their roles in T-cell signaling have been widely studied. More recently, the presence and unique roles of smaller-sized protein clusters, termed “nanoclusters” or “protein islands,” of TCR–CD3 complex (21–24), linker for activation of T cell (LAT) (21, 22, 24, 25), Lck (26), and other signaling molecules (24) were revealed by electron microscopy and the newly available superresolution fluorescence microscopy.Considering that the TCR–CD3 complex, CD4, and Lck are constitutively expressed in nonactivated T cells, it is highly likely that the interaction dynamics between these components would also be controlled spatially during the T-cell activation process. Here, we studied the relative molecular distribution of these molecules using single- and dual-color photoactivated localization microscopy (PALM) (27) and direct stochastic optical reconstruction microscopy (dSTORM) (28, 29) in live and fixed T cells for both nonactivated and activating conditions. The corresponding spatial analyses were also used to quantitatively determine the sizes, degree of clustering, and degree of interactions of these clusters. We found that CD4 is also expressed in preclustered structures, separate from TCR–CD3 and LAT, and composed of three to six molecules per cluster. The interactions between these molecules occurred only in the interfaces between the clusters. Upon T-cell activation, the TCR–CD3 and CD4 molecules increased the size of their own clusters without appreciable mixing. Instead, their molecular segregation increased, whereas the T cell develops a synapse structure, often in the SMAC or “bull’s eye” pattern, with the TCR–CD3 in the central supramolecular activation cluster (cSMAC) with the CD4 and Lck clusters localizing around it. These observed clustering behaviors accompanying reorganization of spatial distributions of CD4, Lck, and TCR might be a general and effective mechanism to activate and regulate the T-cell signaling by controlling the magnitude of interfacial interactions between signaling components in each cluster.     

Negative selection of CD4+ CD8+ thymocytes by T-cell receptor peptide antagonists.

Antigen-induced activation of T cells can be specifically inhibited by antigen analogs that have been termed T-cell receptor peptide antagonists. These antagonists appear to act by inducing the formation of nonstimulatory or partially stimulatory complexes between T-cell receptors and the major histocompatibility complex molecules presenting the peptides. Herein, we have investigated the effect of T-cell receptor peptide antagonists on thymocyte negative selection. First, peptide antagonists were identified for the cytochrome c-specific T-cell clone AD10. These peptides were then tested for their ability to induce negative selection in an in vitro model system using thymocytes from mice transgenic for the AD10 T-cell receptor. Though unable to induce mature T-cell activation, the T-cell receptor peptide antagonists induced deletion of CD4+ CD8+ thymocytes. These results suggest that negative selection of CD4+ CD8+ thymocytes can be induced by T-cell receptor interactions of a lower affinity than those required for mature T-cell activation.     

Molecular characterization of T-cell antigen receptor expression by subsets of CD4- CD8- murine thymocytes.

Precursors of all T-lineage cells are found in a population of thymocytes that lack the CD4 and CD8 surface glycoproteins. These "double-negative" thymocytes are markedly heterogeneous in their expression of other surface markers and include cells at various stages of development. In this study, CD4- CD8- adult murine thymocytes were separated into subsets based on the expression of the "heat stable antigen" (HSA) and of Ly 1 (CD5). The sorted subsets were analyzed directly (without prior expansion in culture) for T-cell antigen receptor (TcR) gene rearrangement and mRNA expression and for TcR and CD3 cell-surface protein expression. Very little surface CD3 or TcR expression was detected on the major HSA+ Ly 1low subset. However, the HSA+ Ly 1high, HSA- Ly 1high, and HSA- Ly 1low subsets all contained cells with surface expression of CD3 and TcR. In contrast to previous studies, we found no subset that exclusively expressed either the alpha beta or gamma delta heterodimer, although the ratio of alpha beta+ to gamma delta+ varied widely. Two of these three subsets (HSA- Ly 1low and HSA- Ly 1high) showed very high usage of V beta 8 gene products in the alpha beta heterodimer, but nevertheless included approximately equal to 15% non-V beta 8 alpha beta forms. All CD4- CD8- subsets were found to have extensively rearranged their TcR gamma genes and to express gamma mRNA. Expression of a high ratio of mature [1.3 kilobases (kb)] to truncated (1.0 kb) beta message and presence of alpha message was largely restricted to subsets with TcR alpha beta surface expression.     

Tyrosine kinase activity of CD4-associated p56lck may not be required for CD4-dependent T-cell activation.

The lymphoid-specific tyrosine kinase p56lck (Lck) is critical for the development and activation of T lymphocytes, and Lck kinase activity has been implicated in both T-cell antigen receptor/CD3- and CD4-mediated signaling. CD4-dependent T-cell activation has been demonstrated to be dependent upon the association of CD4 with Lck. To examine the role of the kinase activity of Lck in CD4-dependent T-cell activation, we have generated several kinase-deficient mutants of Lck. When transfected into CD4+ murine T-cell hybridoma cells, these mutants cause approximately 90% diminution in CD4-associated Lck kinase activity. Specifically, upon CD4 crosslinking there is decreased Lck autophosphorylation and decreased phosphorylation of an exogenous substrate. When CD4 is crosslinked to the T-cell antigen receptor-CD3 complex, decreased phosphorylation of associated substrates is also observed. In spite of this striking inhibition of Lck kinase function, cells expressing the kinase-deficient mutants demonstrate normal or enhanced CD4-dependent antigen responsiveness. These data demonstrate that the level of Lck kinase activity does not correlate with its CD4-associated function and suggest that the kinase activity of Lck may not be required for CD4-mediated signaling.     

CD5 acts as a tyrosine kinase substrate within a receptor complex comprising T-cell receptor zeta chain/CD3 and protein-tyrosine kinases p56lck and p59fyn.

T-cell antigens including CD2, CD4, CD6, CD8, and CD28 serve as coreceptors with the T-cell receptor (TCR)/CD3 complex in control of T-cell growth. The molecular basis by which these antigens fulfill this role has remained a major issue. An initial clue to this question came with our finding that the sensitivity of in vitro kinase labeling (specifically using protein-tyrosine kinase p56lck) allowed detection of a physical association between CD4-p56lck and the TCR/CD3 complexes. Another T-cell antigen, CD5, is structurally related to the macrophage scavenger receptor family and, as such, can directly stimulate and/or potentiate T-cell proliferation. In this study, we reveal that in Brij 96-based cell lysates, anti-CD5 antibodies coprecipitated TCR zeta chain (TCR zeta)/CD3 subunits as well as the protein-tyrosine kinases p56lck and p59fyn. Conversely, anti-CD3 antibody coprecipitated CD5, p56lck, and p59fyn. Indeed, anti-CD5 and anti-CD3 gel patterns were virtually identical, except for a difference in relative intensity of polypeptides. Anti-CD4 coprecipitated p56lck, p32, and CD3/TCR zeta subunits but precipitated less CD5, suggesting the existence of CD4-TCR zeta/CD3 complexes distinct from the CD5-TCR zeta/CD3 complexes. Consistent with the formation of a multimeric CD5-TCR zeta/CD3 complex, anti-CD5 crosslinking induced tyrosine phosphorylation of numerous T-cell substrates, similar to those phosphorylated by TCR zeta/CD3 ligation. Significantly, as for TCR zeta, CD5 was found to act as a tyrosine kinase substrate induced by TCR/CD3 ligation. The kinetics of phosphorylation of CD5 (t1/2 = 20 sec) was among the earliest of activation events, more rapid than seen for TCR zeta (t1/2 = 1 min). CD5 represents a likely TCR/CD3-associated substrate for protein-tyrosine kinases (p56lck or p59fyn) and an alternative signaling pathway within a multimeric TCR complex.     

Engagement of the external domains of CD45 tyrosine phosphatase can regulate the differentiation of immature CD4+CD8+ thymocytes into mature T cells.

Immature precursor cells are induced in the thymus to express clonotypic T-cell antigen receptors (TCRs) and to differentiate into mature T cells. Perhaps the least understood event which occurs during intrathymic development is the positive selection of immature CD4+CD8+ thymocytes for differentiation into mature CD4+ and CD8+ T cells based on the TCR specificity individual thymocytes express. TCR expression by CD4+CD8+ thymocytes is quantitatively regulated by CD4-mediated activation of p56lck protein-tyrosine kinase whose activity can in turn be regulated by the membrane-bound protein-tyrosine-phosphatase CD45. Here we show that antibody engagement of CD45 external domains enhances Lck tyrosine kinase activity in CD4+CD8+ thymocytes, inhibits TCR expression, and inhibits differentiation of immature CD4+CD8+ thymocytes into mature T cells. Thus, engagement of the external domains of CD45 tyrosine phosphatase can regulate the ability of immature CD4+CD8+ thymocytes to undergo positive selection, suggesting an important regulatory role for intrathymic ligands that are capable of engaging CD45 within the thymus.     

Instability of assembled T-cell receptor complex that is associated with rapid degradation of zeta chains in immature CD4+CD8+ thymocytes.

The intracellular fate of newly synthesized T-cell receptor (TCR) chains was compared in CD4+CD8+ (double positive; DP) thymocytes and in CD4+CD8- or CD4-CD8+ (single positive; SP) thymocytes. Purified DP and SP thymocytes from normal adult mice were analyzed by pulse-chase metabolic labeling and immunoprecipitation with specific anti-TCR antibodies. Biosynthesis of invariant chains (CD3 gamma, -delta, -epsilon, and zeta) was comparable between DP and SP thymocytes, whereas DP thymocytes synthesized TCR alpha and TCR beta chains at lower and higher levels than SP thymocytes, respectively. These newly synthesized TCR chains were degraded at different rates in SP thymocytes based on their sensitivities for degradation as previously reported: TCR alpha, TCR beta, CD3 gamma, and CD3 delta chains were rapidly degraded and CD3 epsilon and zeta chains were stable. Although the degradation rates of clonotypic and invariant CD3 chains were similar in DP and SP thymocytes, the zeta subunit was rapidly degraded in DP thymocytes (t1/2, approximately 1.5 hr). Degradation of zeta was inhibited by NH4Cl, implicating lysosomes as the site of degradation. Comparison of TCR subunit assembly in DP and SP thymocytes demonstrated that, despite the same relative rate of formation of TCR complexes in a pulse period (30 min), complete complexes were unstable and degraded during the subsequent 6 hr of chase in DP thymocytes. This contrasted with the stability and a progressive increase in the levels of completely assembled complexes in SP thymocytes. Thus, these results demonstrate that a unique posttranslational regulation operates in the formation of TCR complexes in DP thymocytes and that lack of stability of complete TCR complexes is a crucial mechanism that may account for the limited surface TCR expression on this thymocyte subset.     

p56lck-independent activation and tyrosine phosphorylation of p72syk by T-cell antigen receptor/CD3 stimulation.

Activation of resting T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex is initiated by rapid tyrosine phosphorylation of cellular proteins. Protein-tyrosine kinases (PTKs) of the src family are known to be important, but the mechanism of their recruitment and their interactions with PTKs of other families are incompletely understood. We show that a member of another family of PTKs, the p72syk kinase, is constitutively bound to the TCR/CD3 complex and becomes tyrosine phosphorylated and activated within 1 min after TCR/CD3 stimulation. This activation did not depend on the presence of p56lck in T cells and in transfected COS cells. In both cases, however, the phosphorylation of cellular substrates was augmented by src family PTKs. We propose that p72syk may act as an immediate receptor-activated kinase upstream of the related p70zap PTK and the src family PTKs p56lck and p59fyn in T cells and that these src family PTKs act as signal amplifiers.     

The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex.

Many mammalian receptors have been found to regulate cell growth by virtue of a protein-tyrosine kinase domain in their cytoplasmic tail. We recently described an association of the CD4 antigen with a T-cell-specific protein-tyrosine kinase (p56lck; formerly termed pp58lck; EC 2.7.1.112). This interaction represents a potential mechanism by which T-cell growth may be regulated and offers a model by which other members of the src family (products of c-src, c-yes, c-fgr, etc.) may interact with mammalian growth factor receptors. As in the case of the CD4 antigen, the CD8 antigen appears to serve as a receptor for nonpolymorphic regions of products of the major histocompatibility complex and has been implicated in the regulation of T-cell growth. In this study, we reveal that the human CD8 antigen is also associated with the T-cell-specific protein-tyrosine kinase (p56lck). The associated p56lck kinase was detected by use of both in vitro and in vivo labeling regimes using an antiserum to the C terminus of p56lck. Two-dimensional nonequilibrium pH-gradient gel electrophoresis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated the similarity of p56lck to the protein-tyrosine kinase associated with the CD4 antigen. The catalytic activity of p56lck was revealed by the autophosphorylation of the 55- to 60-kDa kinase and the occasional labeling of a 35-kDa protein. Last, we demonstrate directly that members of the CD3 complex, including the gamma, delta, and epsilon chains, as well as a putative zeta subunit, can be phosphorylated at tyrosine residues by the CD4/CD8.p56lck complex.     

LFA-1 signaling through p44/42 is coupled to perforin degranulation in CD56+CD8+ natural killer cells

Leukocyte function antigen 1 (LFA-1) is essential for the formation of immune cell synapses and plays a role in the pathophysiology of various autoimmune diseases. We investigated the molecular details of LFA-1 activation during adhesion between cytotoxic cells and a target model leukemia cell. The cytolytic activity of a CD3-CD8+CD56+ natural killer (NK) subset was enhanced when LFA-1 was activated. In a comparison of LFA-1 ligands, intercellular adhesion molecule 2 (ICAM-2) and ICAM-3 promoted LFA-1-directed perforin release, whereas ICAM-1 had little effect. Ligand-induced LFA-1 clustering facilitated perforin release, demonstrating LFA-1 could regulate degranulation mechanisms. LFA-1 induced the activation of src family kinases, Vav1 and p44/42 mitogen-activated protein kinase (MAPK), in human CD56+ NK cells as evidenced by intracellular phospho-epitope measurements that correlated with effector-target cell binding and perforin-granzyme A-mediated cytolytic activity. These results identify novel, specific functional consequence of LFA-1-mediated cytolytic activity in perforin-containing human NK subsets.     

CD4+ T lymphocytes mediate in vivo clearance of plasmid DNA vaccine antigen expression and potentiate CD8+ T-cell immune responses

There is evidence that the limited immunogenicity of plasmid DNA vaccines is the result, at least in part, of the rapid clearance of vaccine antigen expression by antigen-specific immune responses. However, the cell types responsible for the clearance of plasmid DNA vaccine antigens are not known. Here we demonstrate that macrophages, NK cells, and CD8(+) T cells did not significantly contribute to the DNA antigen clearance but CD4(+) T cells played the crucial role in attenuating plasmid DNA vaccine antigen expression. Adoptive transfer experiments demonstrate that CD4(+) T cells facilitated DNA vaccine antigen clearance in a Fas/FasL-dependent manner. Furthermore, we show that depletion of CD4(+) T cells prevented the clearance of vaccine antigen and the appearance of a CD8(+) T-cell immune response. Inoculation of major histocompatibility complex class II KO mice with the plasmid DNA led to persistent antigen expression and abolition of a CD8(+) T-cell immune response. Importantly, the prolongation of antigen expression by disrupting the CD4(+) T-cell Fas/FasL myocytes signaling led to a 3- to 5-fold increase of antigen-specific CD8(+) T-cell responses. These data demonstrate a dominant role of CD4(+) T cell-mediated cytotoxicity in plasmid DNA vaccine antigen clearance.     

An NK1.1+ CD4+8- single-positive thymocyte subpopulation that expresses a highly skewed T-cell antigen receptor V beta family.

In the present report we describe a CD4+8- heat stable antigen-negative (HSA-) thymocyte subpopulation that expresses a distinguishably low density of alpha beta T-cell antigen receptors (TCRlo) from the majority of CD4+8- high-density TCR (TCRhi) mature-type thymocytes. This subpopulation appears relatively late in life. Analysis of MEL-14, Pgp-1 (CD44), ICAM-1 (CD54), and NK1.1 expression on this subpopulation revealed that the CD4+8- TCRlo population was a population having unique characteristics (MEL-14-, CD44+, ICAM-1+, and NK1.1+) compared to the CD4+8- TCRhi thymocytes, most of which are MEL-14+, CD44-, ICAM-1-, and NK1.1-. When TCR beta-chain variable region (V beta) usage was analyzed, this thymic population expressed predominantly products of V beta 7 and V beta 8.2 TCR gene families. Interestingly, cells with V beta 8.1 TCRs, which are reactive to Mls-1a antigens, were not eliminated from the CD4+8- HSA- TCRlo subpopulation but had been eliminated from the major CD4+8- HSA- TCRhi subpopulation in Mls-1a strains. A subset with a phenotype similar to the CD4+8- HSA- TCRlo thymocytes was also identified primarily in bone marrow, and this subset constituted approximately half of the CD4+ T cells in the bone marrow. The CD4+8- HSA- TCRlo cells showed extremely high proliferative responses to immobilized anti-TCR antibody but generated negligible responses to allogeneic H-2 antigens compared to the responses generated by the major CD4+8- HSA- CD3hi cells. However, the CD4+8- HSA- TCRlo cells in Mls-1b mice mounted vigorous proliferative responses to Mls-1a antigens but not in Mls-1a mice. The properties of this T-cell subset suggest that these cells belong to a lineage distinct from the major T-cell population.     

Negative selection of semimature CD4(+)8(-)HSA+ thymocytes requires the BH3-only protein Bim but is independent of death receptor signaling

T cell receptor/CD3 ligation induces apoptosis in semimature CD4(+)8(-)HSA+ thymocytes, and this helps establish immunological tolerance and constitutes one of the safeguards against autoimmune disease. We analyzed several knockout and transgenic mouse lines and found that T cell receptor/CD3-ligation-induced killing of semimature thymocytes occurred independently of Fas and "death receptor" signaling in general but required the proapoptotic BH3-only protein Bim and could be inhibited by Bcl-2. Loss of Apaf-1 or caspase-9, which act downstream of the Bcl-2 family protein family, provided only minor protection, indicating that the "apoptosome" functions as an amplifier rather than as an essential initiator of this death program. These results reveal the mechanisms of apoptosis in negative selection of semimature thymocytes and have implications for immunological tolerance and autoimmunity.     

Molecular modification of idiotypes from B-cell lymphomas for expression in mature dendritic cells as a strategy to induce tumor-reactive CD4+ and CD8+ T-cell responses

Most non-Hodgkin B-cell lymphomas (NHLs) are characterized by the clonal expansion of a single cell expressing a unique rearranged immunoglobulin gene. This idiotype (Id) is a tumor-specific antigen that can be immunologically targeted. The therapeutic efficacy of Id-based vaccines correlates best with detection of cellular immune responses, although these have not been as well characterized as the humoral responses. This study exploited a molecular approach to modify the Id of 38C13 lymphoma for processing via class I and II antigen-processing pathways and evaluated protein expression in dendritic cells (DCs) to simultaneously stimulate tumor reactive CD8(+) and CD4(+) lymphocytes. Recombinant vaccinia viruses (rVVs) were constructed, coding for Id fused with the targeting signal of the lysosomal-associated membrane protein1 (Id-LAMP1) to promote antigen presentation in the context of major histocompatibility complex (MHC) class II. Mature DCs infected with rVV/Id-LAMP1 elicited both CD4(+) and CD8(+) Id-specific T cells and protected animals from tumor challenge. Id-specific CD8(+) cells were required to mediate the effector phase of a therapeutic response, and CD4(+) cells were beneficial in the induction phase of the response. These results demonstrate that fusing Id to LAMP1 enhances CD8(+) and CD4(+) Id-specific responses for NHLs and may be useful therapeutically.     

p56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: implications for T-cell costimulation.

T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.     

Interleukin 2 induces CD8+ T cell-mediated suppression of human immunodeficiency virus replication in CD4+ T cells and this effect overrides its ability to stimulate virus expression.

The nonlytic suppression of human immunodeficiency virus (HIV) production from infected CD4+ T cells by CD8+ lymphocytes from HIV-infected individuals is one of the most potent host-mediated antiviral activities observed in vitro. We demonstrate that the pleiotropic cytokine interleukin 2 (IL-2), but not IL-12, is a potent inducer of the CD8+ HIV suppressor phenomenon. IL-2 induces HIV expression in peripheral blood or lymph node mononuclear cells from HIV-infected individuals in the absence of CD8+ T cells. However, IL-2 induces CD8+ T cells to suppress HIV expression when added back to these cultures, and this effect dramatically supersedes the ability to IL-2 to induce HIV expression. Five to 25 times fewer CD8+ cells were required to obtain comparable levels of inhibition of viral production if they were activated in the presence of IL-2 as compared with IL-12 or no exogenous cytokine. Furthermore, IL-2 appeared either to induce a qualitative increase in HIV suppressor cell activity or to increase the relative frequency of suppressor cells in the activated (CD25+) CD8+ populations. Analyses of proviral levels in peripheral blood mononuclear cells suggest that CD8+ T cell-mediated lysis of in vivo infected cells is not induced by IL-2. These results have implications for our understanding of the effects of impaired IL-2 production during HIV disease as well as the overall effects of IL-2-based immunotherapy on HIV replication in vivo.