The crystal structure of CD8 in complex with YTS156.7.7 Fab and interaction with other CD8 antibodies define the binding mode of CD8 alphabeta to MHC class I

The CD8αβ heterodimer interacts with class I pMHC on antigen-presenting cells as a co-receptor for TCR-mediated activation of cytotoxic T cells. To characterize this immunologically important interaction, we used monoclonal antibodies (mAbs) specific to either CD8α or CD8β to probe the mechanism of CD8αβ binding to pMHCI. The YTS156.7 mAb inhibits this interaction and blocks T cell activation. To elucidate the molecular basis for this inhibition, the crystal structure of the CD8αβ immunoglobulin-like ectodomains were determined in complex with mAb YTS156.7 Fab at 2.7 Å resolution. The YTS156.7 epitope on CD8β was identified and implies that residues in the CDR1 and CDR2-equivalent loops of CD8β are occluded upon binding to class I pMHC. To further characterize the pMHCI/CD8αβ interaction, binding of class I tetramers to CD8αβ on the surface of T cells was assessed in the presence of anti-CD8 mAbs. In contrast to YTS156.7, mAb YTS105.18, which is specific for CD8α, does not inhibit binding of CD8αβ to class I tetramers, indicating the YTS105.18 epitope is not occluded in the pMHCI/CD8αβ complex. Together, these data indicate a model for the pMHCI/CD8αβ interaction similar to that observed for CD8αα in the CD8αα/pMHCI complex, but in which CD8α occupies the lower orientation (membrane proximal to the antigen presenting cell), and CD8β occupies the upper position (membrane distal). The implication of this molecular assembly for the function of CD8αβ in T cell activation is discussed.     

High affinity xenoreactive TCR:MHC interaction recruits CD8 in absence of binding to MHC

The TCR from a xenoreactive murine cytotoxic T lymphocyte clone, AHIII 12.2, recognizes murine H-2D(b) complexed with peptide p1058 (FAPGFFPYL) as well as human HLA-A2.1 complexed with human self-peptide p1049 (ALWGFFPVL). To understand more about T cell biology and cross-reactivity, the ectodomains of the AHIII 12.2 TCR have been produced in E. coli as inclusion bodies and the protein folded to its native conformation. Flow cytometric and surface plasmon resonance analyses indicate that human p1049/A2 has a significantly greater affinity for the murine AHIII 12.2 TCR than does murine p1058/D(b). Yet, T cell binding and cytolytic activity are independent of CD8 when stimulated with human p1049/A2 as demonstrated with anti-CD8 Abs that block CD8 association with MHC. Even in the absence of direct CD8 binding, stimulation of AHIII 12.2 T cells with "CD8-independent" p1049/A2 produces p56(lck) activation and calcium flux. Confocal fluorescence microscopy and fluorescence resonance energy transfer flow cytometry demonstrate CD8 is recruited to the site of TCR:peptide MHC binding. Taken together, these results indicate that there exists another mechanism for recruitment of CD8 during high affinity TCR:peptide MHC engagement.     

Development of antigen-specific CD8+ CTL in MHC class I-deficient mice through CD4 to CD8 conversion

CD8+ CTL are the predominant tumoricidal effector cells. We find, however, that MHC class I-deficient mice depleted of CD8+ T cells are able to mount an effective antitumor immunity after immunization with fused dendritic/tumor cells. Such immunity appears to be mediated by the generation of phenotypic and functional CD8+ CTL through CD4+ to CD8+ conversion, which we have demonstrated at the single cell level. CD4+ to CD8+ conversion depends on effective in vivo activation and is promoted by CD4+ T cell proliferation. The effectiveness of this process is shown by the generation of antitumor immunity through adoptive transfer of primed CD4 T cells to provide protection against tumor cell challenge and to eliminate established pulmonary metastases.     

Functional characterization of MHC class II-restricted CD8+CD4- and CD8-CD4- T cell responses to infection in CD4-/- mice

Classical CD4(+) and CD8(+) T cells recognize Ag presented by MHC class II (MHCII) and MHC class I (MHCI), respectively. However, our results show that CD4(-/-) mice mount a strong, readily detectable CD8(+) T cell response to MHCII-restricted epitopes after a primary bacterial or viral infection. These MHCII-restricted CD8(+)CD4(-) T cells are more similar to classical CD8(+) T cells than to CD4(+) T cells in their expression of effector functions during a primary infection, yet they also differ from MHCI-restricted CD8(+) T cells by their inability to produce high levels of the cytolytic molecule granzyme B. After resolution of a primary infection, epitope-specific MHCII-restricted T cells in CD4(-/-) mice persist for a long period of time as memory T cells. Surprisingly, upon reinfection the secondary MHCII-restricted response in CD4(-/-) mice consists mainly of CD8(-)CD4(-) T cells. In contrast to CD8(+) T cells, MHCII-restricted CD8(-)CD4(-) T cells are capable of producing IL-2 in addition to IFN-gamma and thus appear to have attributes characteristic of CD4(+) T cells rather than CD8(+) T cells. Therefore, MHCII-restricted T cells in CD4(-/-) mice do not share all phenotypic and functional characteristics with MHCI-restricted CD8(+) T cells or with MHCII-restricted CD4(+) T cells, but, rather, adopt attributes from each of these subsets. These results have implications for understanding thymic T cell selection and for elucidating the mechanisms regulating the peripheral immune response and memory differentiation.     

Evidence for a domain-swapped CD4 dimer as the coreceptor for binding to class II MHC

CD4 is a coreceptor for binding of T cells to APC and the primary receptor for HIV. The disulfide bond in the second extracellular domain (D2) of CD4 is reduced on the cell surface, which leads to formation of disulfide-linked homodimers. A large conformational change must take place in D2 to allow for formation of the disulfide-linked dimer. Domain swapping of D2 is the most likely candidate for the conformational change leading to formation of two disulfide-bonds between Cys130 in one monomer and Cys159 in the other one. Mild reduction of the extracellular part of CD4 resulted in formation of disulfide-linked dimers, which supports the domain-swapped model. The functional significance of dimer formation for coreceptor function was tested using cells expressing wild-type or disulfide-bond mutant CD4. Eliminating the D2 disulfide bond markedly impaired CD4's coreceptor function. Modeling of the complex of the TCR and domain-swapped CD4 dimer bound to class II MHC and Ag supports the domain-swapped dimer as the immune coreceptor. The known involvement of D4 residues Lys318 and Gln344 in dimer formation is also accommodated by this model. These findings imply that disulfide-linked dimeric CD4 is the preferred coreceptor for binding to APC.     

CD1a and MHC class I follow a similar endocytic recycling pathway

CD1 proteins are a family of major histocompatibility complex (MHC) class I-like antigen-presenting molecules that present lipids to T cells. The cytoplasmic tails (CTs) of all human CD1 isoforms, with the exception of CD1a, contain tyrosine-based sorting motifs, responsible for the internalization of proteins by the clathrin-mediated pathway. The role of the CD1a CT, which does not possess any sorting motifs, as well as its mode of internalization are not known. We investigated the internalization and recycling pathways followed by CD1a and the role of its CT. We found that CD1a can be internalized by a clathrin- and dynamin-independent pathway and that it follows a Rab22a- and ADP ribosylation factor (ARF)6-dependent recycling pathway, similar to other cargo internalized independent of clathrin. We also found that the CD1a CT is S-acylated. However, this posttranslational modification does not determine the rate of internalization or recycling of the protein or its localization to detergent-resistant membrane microdomains (DRMs) where we found CD1a to be enriched. We also show that plasma membrane DRMs are essential for efficient CD1a-mediated antigen presentation. These findings place CD1a closer to MHC class I in its trafficking and potential antigen-loading compartments among CD1 isoforms. Furthermore, we identify CD1a as a new marker for the clathrin- and dynamin-independent and DRM-dependent pathway of internalization as well as the Rab22a- and ARF6-dependent recycling pathway.     

Characterizing the impact of CD8 antibodies on class I MHC multimer binding

Many studies have suggested that CD8 Abs affect the binding of class I MHC tetramers/multimers to CD8(+) T cells, which has led to the interpretation that CD8 participates directly in multimer binding. In contrast, a recent publication has argued that CD8 Abs instead cause reorganization of TCR distribution and hence have an indirect effect on multimer binding to the TCR alone. We address these issues by testing the role of CD8 and the impact of CD8 Abs on the binding of normal and mutant multimers to Ag-specific mouse T cells. Our data suggest that, in this system, CD8 Abs act directly on CD8 and only mediate their effects on multimer binding when CD8 is capable of binding to the multimer. These data reinforce the paradigm that CD8 plays an active and direct role in binding of class I MHC multimers.     

Mapping the binding site on CD8 beta for MHC class I reveals mutants with enhanced binding

In an effective immune response, CD8+ T cell recognition of virally derived Ag, bound to MHC class I, results in killing of infected cells. The CD8alphabeta heterodimer acts as a coreceptor with the TCR, to enhance sensitivity of the T cells to peptide/MHC class I, and is two orders of magnitude more efficient as a coreceptor than the CD8alphaalpha. To understand the important interaction between CD8alphabeta and MHC class I, we created a panel of CD8beta mutants and identified mutations in the CDR1, CDR2, and CDR3 loops that decreased binding to MHC class I tetramers as well as mutations that enhanced binding. We tested the coreceptor function of a subset of reducing and enhancing mutants using a T cell hybridoma and found similar reducing and enhancing effects. CD8beta-enhancing mutants could be useful for immunotherapy by transduction into T cells to enhance T cell responses against weak Ags such as those expressed by tumors. We also addressed the question of the orientation of CD8alphabeta with MHC class I using CD8alpha mutants expressed as a heterodimer with wild-type CD8alpha or CD8beta. The partial rescuing of binding with wild-type CD8beta compared with wild-type CD8alpha is consistent with models in which either the topology of CD8alphaalpha and CD8alphabeta binding to MHC class I is different or CD8alphabeta is capable of binding in both the T cell membrane proximal and distal positions.     

Social foragers adopt a riskier foraging mode in the centre of their groups

Foraging in groups provides many benefits that are not necessarily experienced the same way by all individuals. I explore the possibility that foraging mode, the way individuals exploit resources, varies as a function of spatial position in the group, reflecting commonly occurring spatial differences in predation risk. I show that semipalmated sandpipers (Calidris pusilla), a social foraging avian species, tended to adopt a riskier foraging mode in the central, more protected areas of their groups. Central birds effectively used the more peripheral group members as sentinels, allowing them to exploit a wider range of resources within the same group at the same time. This finding provides a novel benefit of living in groups, which may have a broad relevance given that social foraging species often exploit a large array of resources.     

Aminoglycoside antibiotics bound to aminoglycoside-detoxifying enzymes and RNA adopt similar conformations

Conformations of ribostamycin and isepamicin, aminoglycoside antibiotics, bound to an aminoglycoside antibiotic, 3′-phosphotransferase, were determined by transferred nuclear Overhauser effect spectroscopy and molecular modeling. Two major conformers of enzyme-bound ribostamycin, a neomycin-group aminoglyeoside were observed. The 3′- and 5″-OH groups (reactive hydroxyl groups) in the conformers are placed in approximate locations. One of the conformers is similar to the structure of paromomycin bound to a 27-nucleotide piece of ribosomal RNA that represents the A-site of the small ribosomal subunit, where rings A and C are in an orthogonal arrangement. Isepamicin, a kanamycin-group aminoglycoside antibiotic, also showed two major enzyme-bound conformations. Both conformations were similar to those observed for bound isepamicin in the active site of an aminoglycoside(6′)-acetyl transferase-Ii. Conformations of other RNA-bound kanamycin-group aminoglycosides were also similar to the enzyme-bound conformations of isepamicin. These observations suggest that aminoglycosides may adopt similar conformations when bound to RNA and protein targets. This may have significant implications in the design of enzyme inhibitors and/or antibiotics.     

Seven GC-rich microbial genomes adopt similar codon usage patterns regardless of their phylogenetic lineages

Seven GC-rich (group I) and three AT-rich (group II) microbial genomes are analyzed in this paper. The seven microbes in group I belong to different phylogenetic lineages, even different domains of life. The common feature is that they are highly GC-rich organisms, with more than 60% genomic GC content. Group II includes three bacteria, which belong to the same subdivision as Pseudomonas aeruginosa in group I. The genomic GC content of the three bacteria is in the range of 26-50%. It is shown that although the phylogenetic lineages of the organisms in group I are remote, the common feature of highly genomic GC content forces them to adopt similar codon usage patterns, which constitutes the basis of an algorithm using a set of universal parameters to recognize known genes in the seven genomes. The common codon usage pattern of function known genes in the seven genomes is GGS type, where G, G, and S are the bases of G, non-G, and G/C, respectively. On the contrary, although the phylogenetic lineages of the three bacteria in group II are quite close, the codon usage patterns of function known genes in these genomes are obviously distinct. There are no universal parameters to identify known genes in the three genomes in group II. It can be deduced that the genomic GC content is more important than phylogenetic lineage in gene recognition programs. We hope that the work might be useful for understanding the common characteristics in the organization of microbial genomes.     

CD5 costimulation up-regulates the signaling to extracellular signal-regulated kinase activation in CD4+CD8+ thymocytes and supports their differentiation to the CD4 lineage

CD5 positively costimulates TCR-stimulated mature T cells, whereas this molecule has been suggested to negatively regulate the activation of TCR-triggered thymocytes. We investigated the effect of CD5 costimulation on the differentiation of CD4+CD8+ thymocytes. Coligation of thymocytes with anti-CD3 and anti-CD5 induced enhanced tyrosine phosphorylation of LAT (linker for activation of T cells) and phospholipase C-gamma (PLC-gamma) compared with ligation with anti-CD3 alone. Despite increased phosphorylation of PLC-gamma, this treatment down-regulated Ca2+ influx. In contrast, the phosphorylation of LAT and enhanced association with Grb2 led to activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase. When CD3 and CD5 on CD4+CD8+ thymocytes in culture were coligated, they lost CD8, down-regulated CD4 expression, and induced CD69 expression, yielding a CD4+(dull)CD8-CD69+ population. An ERK inhibitor, PD98059, inhibited the generation of this population. The reduction of generation of CD4+CD8- cells resulted from decreased survival of these differentiating thymocytes. Consistent with this, PD98059 inhibited the anti-CD3/CD5-mediated Bcl-2 induction. These results indicate that CD5 down-regulates a branch of TCR signaling, whereas this molecule functions to support the differentiation of CD4+CD8+ thymocytes by up-regulating another branch of TCR signaling that leads to ERK activation.     

Interleukin 4 induces CD4+/CD8- to CD8+/CD4- transformation of human neonatal T cells by way of a double positive intermediate

We have determined that IL-4 induces the generation of CD4+/CD8+ T cells in cultures of neonatal lymphocytes. Sorting, positive, and negative selection experiments indicate that these cells arise from a subpopulation of CD4+/CD8- cells present in the neonate but not in the adult. We have further determined that these IL-4 generated "double positive" cells further differentiate to express only the CD8 marker. Our findings suggest an undescribed and dramatic role for IL-4 in T cell differentiation.     

Patterns of costimulation of T cell clones by cross-linking CD3, CD4/CD8, and class I MHC molecules

The ability of mAb to class I MHC molecules, CD3, or CD4/CD8 to stimulate human T cell clones alone or in combination was examined. Cross-linking each of these surface Ag with appropriate mAb and goat anti-mouse Ig (GaMIg) resulted in a unique pattern of increase in intracellular free calcium ([Ca2+]i) and different degrees of functional activation. Cross-linking class I MHC molecules provided the most effective stimulus of IL-2 production and proliferation. Cross-linking more than one surface Ag induced a compound calcium signal with characteristics of each individual response. Cross-linking CD3 + HLA-A,B,C caused a rapid and prolonged increase in [Ca2+]i and synergistically increased IL-2 production and proliferation of all clones. Cross-linking CD3 + CD4/CD8 also generated a compound calcium signal and increased IL-2 production and DNA synthesis. Purposeful inclusion of CD3 was not required for costimulation as cross-linking HLA-A,B,C + CD4/CD8 also increased [Ca2+]i, IL-2 production, and proliferation. Cross-linking three surface Ag, CD3 + HLA-A,B,C + CD4/CD8, resulted in the greatest initial and sustained [Ca2+]i, IL-2 production, and DNA synthesis. Although there was a tendency for the various stimuli to increase both [Ca2+]i and functional responsiveness, neither the magnitude nor duration of the increased [Ca2+]i correlated with the amount of IL-2 produced or the ultimate proliferative response. To determine whether costimulation required that the various surface molecules were cross-linked together, experiments were carried out using isotype specific secondary antibodies. Augmentation of [Ca2+]i and costimulation of functional responses were noted when class I MHC molecules were cross-linked and CD3 was bound, but not cross-linked. Similarly, costimulation through CD3 and CD4/CD8 was observed when CD4/CD8 was cross-linked and the CD3 complex was engaged by an anti-CD3 mAb which was not further cross-linked. In contrast, costimulation by class I MHC molecules and CD4/CD8 was only observed when these molecules were cross-linked together. These data demonstrate that cross-linking class I MHC determinants or CD4/CD8 provides a direct signal to T cell clones that can be enhanced when CD3 is independently engaged. The results also indicate that T cell clones can be stimulated without engaging CD3 by the combination of signals delivered via class I MHC molecules and CD4/CD8, but only when these determinants were cross-linked together. These studies have demonstrated that these cell surface molecules differ in their capacity to deliver activation signals to T cell clones and also exhibit unique patterns of positive cooperativity in signaling potential.     

Beta-chain broadens range of CD8 recognition for MHC class I molecule.

It is known that the alpha-chain of CD8 binds to a negatively charged loop composed of residues 223 to 229 on MHC class I Ag and that binding of CD8 alpha enhances Ag recognition of T cells. We have recently shown that the mouse CD8 alpha homodimer does not bind to either the HLA class I alpha 3 domain or a mutant of H-2Kb Ag containing a substitution of glutamine for methionine at residue 224, which brings this residue toward the human consensus. Here we report a complementary study of the CD8 beta-chain. The functional role of the CD8 beta-chain was analyzed by using four T cell hybridoma lines expressing mouse CD8 alpha and transfected with the mouse CD8 beta gene. As compared with the lines expressing only CD8 alpha, allorecognition of the chimeric H-2Kb Ag that contains the HLA class I alpha 3 domain was enhanced in lines expressing both CD8 alpha and -beta. This enhancement was blocked by either anti-CD8 mAb or anti-HLA class I alpha 3 domain mAb. In addition, we show that CD8 alpha beta binds the H-2Kb mutant Ag at residue 224. These results suggest that the beta-chain allows the CD8 alpha beta heterodimer to recognize the chimeric H-2Kb Ag. A model for the role of the beta-chain is presented.     

The role of CD8 alpha' in the CD4 versus CD8 lineage choice.

During thymic development the recognition of MHC proteins by developing thymocytes influences their lineage commitment, such that recognition of class I MHC leads to CD8 T cell development, whereas recognition of class II MHC leads to CD4 T cell development. The coreceptors CD8 and CD4 may contribute to these different outcomes through interactions with class I and class II MHC, respectively, and through interactions with the tyrosine kinase p56lck (Lck) via their cytoplasmic domains. In this paper we provide evidence that an alternatively spliced form of CD8 that cannot interact with Lck (CD8 alpha') can influence the CD4 vs CD8 lineage decision. Constitutive expression of a CD8 minigene transgene that encodes both CD8 alpha and CD8 alpha' restores CD8 T cell development in CD8 alpha mutant mice, but fails to permit the development of mismatched CD4 T cells bearing class I-specific TCRs. These results indicate that CD8 alpha' favors the development of CD8-lineage T cells, perhaps by reducing Lck activity upon class I MHC recognition in the thymus.     

Examination of CD8+ T cell function in humans using MHC class I tetramers: similar cytotoxicity but variable proliferation and cytokine production among different clonal CD8+ T cells specific to a single viral epitope

Following infection by human T cell lymphotrophic virus-I (HTLV-I), high frequencies of polyclonal Tax11-19-reactive CD8(+) T cells can be detected in the peripheral blood. To investigate whether there are differences in the effector functions of these cells, we generated a panel of Tax11-19-reactive T cell clones by single cell sorting of HLA-A2/Tax11-19 tetramer binding CD8(+) T cells followed by repeated stimulation with PHA and IL-2. Examination of the TCRs revealed 17 different T cell clones with unique clonal origins. Nine representative CD8(+) T cell clones showed a similar cytotoxic dose-response activity against Ag-pulsed target cells, even though they express different TCRs. This cytotoxic effector function was not influenced by the engagement of either CD28 or CD2 costimulatory molecules. In contrast to the cytotoxic activity, qualitatively different degrees of proliferative response and cytokine secretion were observed among T cell clones of different clonal origin. The induction of proliferation and cytokine secretion required the engagement of costimulatory molecules, particularly CD2-LFA-3 interaction. These results indicate that functionally diverse, polyclonal CTL populations can be activated specific to a single immunodominant viral epitope; they can manifest virtually identical cytotoxic effector function but have marked differences in proliferation and cytokine secretion.     

Contributions by ionic and steric features of ligands to their binding with phosphorylcholine-specific immunoglobulin IgA H-8 as determined by fluorescence spectroscopy.

The murine myeloma IgA H-8 Fab' fragment which exhibits a binding specificity for phosphorylcholine was assayed for its ability to bind with a number of charged ligands. Monitoring of the ligand-induced changes of protein fluorescence provided a fast and accurate method of determining the equilibrium binding constants. The binding data along with fluorescence spectral properties of the protein permitted an assessment of the relative importance of some binding parameters as well as an evaluation of certain ionic and steric contributions made by ligands exhibiting significant binding affinity for the antibody fragment. Among the conclusions reached is that the dielectric of the binding site microenvironment is important in determining the strength of binding and that hydrophobic groups surrounding a quaternary cationic ligand are important in creating an appropriate binding site of low dielectric value.     

Whole protein and defined CD8(+) and CD4(+) peptides linked to penetratin targets both MHC class I and II antigen presentation pathways

Cytoplasmic delivery and cross-presentation of proteins and peptides is necessary for processing and presentation of antigens for the generation of cytotoxic T cells. We previously described the use of the 16 amino acid peptide penetratin from the Drosophila Antennapedia homeodomain (penetratin, Antp) to transport cytotoxic T lymphocyte epitopes derived from ovalbumin (OVA) or the Mucin-1 tumor-associated antigen into cells. We have now shown that penetratin covalently conjugated to OVA protein and linked in tandem to CD4(+) and/or CD8(+) T-cell epitopes from OVA-stimulated T cells in vitro (B3Z T-cell hybridoma and OT-I and OT-II T cells). The induction of these responses was directly mediated by the penetratin peptide as linking a nonspecific 16-mer peptide to OVA or mixing did not induce CD8(+) or CD4(+) T-cell responses in vitro. Furthermore, interferon (IFN)-γ-secreting CD4(+) and CD8(+) T cells were induced which suppressed B16.OVA tumor growth in C57BL/6 mice. Tumor protection was mediated by a CD8(+) T-cell-dependent mechanism and did not require CD4(+) help to protect mice 7 days after a boost immunization. Alternatively, 40 days after a boost immunization, the presence of CD4(+) help enhanced antigen-specific IFN-γ-secreting CD8(+) T cells and tumor protection in mice challenged with B16.OVA. Long-term CD8 responses were equally enhanced by antigen-specific and universal CD4 help. In addition, immunization with AntpOVA significantly delayed growth of B16.OVA tumors in mice in a tumor therapy model.     

CD8alpha+ and CD11b+ dendritic cell-restricted MHC class II controls Th1 CD4+ T cell immunity

The activation, proliferation, differentiation, and trafficking of CD4 T cells is central to the development of type I immune responses. MHC class II (MHCII)-bearing dendritic cells (DCs) initiate CD4(+) T cell priming, but the relative contributions of other MHCII(+) APCs to the complete Th1 immune response is less clear. To address this question, we examined Th1 immunity in a mouse model in which I-A(beta)(b) expression was targeted specifically to the DCs of I-A(beta)b-/- mice. MHCII expression is reconstituted in CD11b(+) and CD8alpha(+) DCs, but other DC subtypes, macrophages, B cells, and parenchymal cells lack of expression of the I-A(beta)(b) chain. Presentation of both peptide and protein Ags by these DC subsets is sufficient for Th1 differentiation of Ag-specific CD4(+) T cells in vivo. Thus, Ag-specific CD4(+) T cells are primed to produce Th1 cytokines IL-2 and IFN-gamma. Additionally, proliferation, migration out of lymphoid organs, and the number of effector CD4(+) T cells are appropriately regulated. However, class II-negative B cells cannot receive help and Ag-specific IgG is not produced, confirming the critical MHCII requirement at this stage. These findings indicate that DCs are not only key initiators of the primary response, but provide all of the necessary cognate interactions to control CD4(+) T cell fate during the primary immune response.