Homeostatic expansion occurs independently of costimulatory signals.

Naive T cells undergo homeostatic proliferation in lymphopenic mice, a process that involves TCR recognition of specific self peptide/MHC complexes. Since costimulation signals regulate the T cell response to foreign Ags, we asked whether they also regulate homeostatic expansion. We report in this study that homeostatic expansion of CD4 and CD8 T cells occurs independently of costimulation signals mediated through CD28/B7, CD40L/CD40, or 4-1BB/4-1BBL interactions. Using DO11.10 TCR transgenic T cells, we confirmed that CD28 expression was dispensable for homeostatic expansion, and showed that the presence of endogenous CD4(+)CD25(+) regulatory cells did not detectably influence homeostatic expansion. The implications of these findings with respect to regulation of T cell homeostasis and autoimmunity are discussed.

The impact of duration versus extent of TCR occupancy on T cell activation: a revision of the kinetic proofreading model.

The widely accepted kinetic proofreading theory proposes that rapid TCR dissociation from a peptide/MHC ligand allows for stimulation of early but not late T cell activation events, explaining why low-affinity TCR ligands are poor agonists. We identified a low-affinity TCR ligand which stimulated late T cell responses but, contrary to predictions from kinetic proofreading, inefficiently induced early activation events. Furthermore, responses induced by this ligand were kinetically delayed compared to its high-affinity counterpart. Using peptide/MHC tetramers, we showed that activation characteristics could be dissociated from TCR occupancy by the peptide/MHC ligands. Our data argue that T cell responses are triggered by a cumulative signal which is reached at different time points for different TCR ligands.

A low affinity TCR ligand restores positive selection of CD8+ T cells in vivo.

The T cell repertoire is shaped by the processes of positive and negative selection. During development, the TCR binds self peptide-MHC complexes in the thymus, and the kinetics of this interaction are thought to determine the thymocyte's fate. For development of CD8(+) T cells, the data supporting such a model have been obtained using fetal thymic organ culture. To confirm the fidelity of this model in vivo, we studied development of OT-I TCR-transgenic mice that expressed different individual K(b) binding peptides in thymic epithelial cells under the control of the human keratin 14 promoter. We used a system that allowed TAP-independent expression of the peptide-MHC complex, such that the ability of given peptides to restore positive selection in TAP(o) mice could be assessed. We found that transgenic expression of a TCR antagonist peptide (E1) in vivo efficiently restored positive selection of OT-I T cells in TAP(o) mice. An unrelated transgenic peptide (SIY) did not restore selection of OT-I T cells, nor did the E1-transgenic peptide restore selection of an unrelated receptor (2C), showing that positive selection is peptide specific in vivo, as observed in organ cultures. Neither E1 nor SIY transgenes increased the polyclonal CD8 T cell repertoire size in non-TCR-transgenic animals, arguing that single class I binding peptides do not detectably affect the size of the CD8 T cell repertoire when expressed at low levels. We also observed that OT-I T cells selected in TAP(o)-E1 mice were functional in their response to Ag; however, there was a lag in this response, suggesting that the affinity of the TCR interaction with MHC-self peptide can result in fine-tuning of the T cell response.

Il-12 enhances CD8 T cell homeostatic expansion.

The size of the T lymphocyte pool is maintained by regulation of T cell production, proliferation, and survival. Under the pressure of a T lymphopenic environment, mature naive T cells begin to proliferate in the absence of Ag, a process called homeostatic expansion. Homeostatic expansion involves TCR recognition of self peptide/MHC ligands, but less is known about the soluble factors that regulate this process. Here we show that IL-12 dramatically enhanced the homeostatic proliferation of CD8 T cells. In contrast, IL-2 had no beneficial effect on homeostatic expansion and, in fact, inhibited T cell expansion induced by IL-12. Using gene-targeted mice, we showed that IL-12 acted directly on the T cells to enhance homeostatic expansion, but that IL-12 cannot override the requirement for TCR interaction with self peptide/MHC ligands in homeostatic expansion. These data indicate that inflammatory cytokines may modulate T cell homeostasis after lymphopenia and have implications for regulation of the T cell repertoire and autoimmunity.

CD8 binding to MHC class I molecules is influenced by T cell maturation and glycosylation.

CD8 serves both as an adhesion molecule for class I MHC molecules and as a coreceptor with the TCR for T cell activation. Here we study the developmental regulation of CD8-mediated binding to noncognate peptide/MHC ligands (i.e., those not bound by the TCR). We show that CD8's ability to bind soluble class I MHC tetramers and to mediate T cell adhesion under shear flow conditions diminishes as double-positive thymocytes mature into CD8(+) T cells. Furthermore, we provide evidence that this decreased CD8 binding results from increased T cell sialylation upon T cell maturation. These data suggest that CD8's ability to interact with class I MHC is not fixed and is developmentally regulated through the T cell's glycosylation state.

Cutting edge: In situ tetramer staining of antigen-specific T cells in tissues.

Staining Ag-specific T cells with fluorescently labeled tetrameric MHC/peptide complexes has provided a powerful experimental approach to characterizing the immune response. In this report, we describe an extension of this method to directly visualize Ag-specific T cells in tissues. We successfully stained transgenic T cells with MHC tetramers in spleen sections from both 2C and OT-1 TCR transgenic mice. In addition, with the in situ tetramer staining technique, we detected a very small population of Ag-specific T cells in tissue after adoptive transfer of transgenic TCR T cells to a syngeneic nontransgenic mouse. We also show that the in situ tetramer technique can be applied to lightly fixed as well as frozen tissue, thus extending the method to archived tissue collections. This in situ tetramer staining technique offers a general approach to tracking the Ag-specific T cells in tissues.

Role of 2CT cell receptor residues in the binding of self- and allo-major histocompatibility complexes.

T cell clone 2C recognizes the alloantigen L(d) and the positive selecting major histocompatibility complex (MHC), K(b). To explore the molecular basis of T cell antigen receptor (TCR) binding to different peptide/MHC (pMHC) complexes, we performed alanine scanning mutagenesis of the 2C TCR. The TCR energy maps for QL9/L(d) and SIYR/K(b) were remarkably similar, in that 16 of 41 Valpha and Vbeta alanine mutants showed reduced binding to both ligands. Several TCR residues varied in the magnitude of energy contributed to binding the two ligands, indicating that there are also unique interactions. Residues in complementarity determining region 3alpha showed the most notable differences in binding energetics among the ligands QL9/L(d), SIYR/K(b), and the clonotypic antibody 1B2. Various lines of evidence suggest that these differences relate to the mobility of this loop and point to the key role of conformational dynamics in pMHC recognition.

Positive selection is limited by available peptide-dependent MHC conformations.

Recent data suggest that the diversity of self peptides presented in the thymus during development contributes to positive selection of a diverse T cell repertoire. We sought to determine whether a previously defined "hole in the immunological repertoire" could be explained by the absence of an appropriate selecting self peptide. The repertoire defect in question is the inability of bm8 mice to make an H-2K-restricted response to OVA. Like other OVA-specific, H-2K-restricted receptors, OT-I-transgenic T cells are not positively selected in bm8 mice. Using criteria we had previously established for identifying positive selection ligands, we found peptides that could restore positive selection of OT-I thymocytes in bm8 mice. Thus, the T cell repertoire can be limited by a requirement for specific self peptides during development. Data with MHC-specific Abs suggested that peptides might be able to force MHC residues to adopt different conformations in Kb vs Kbm8. This shows that peptides can potentially contribute to ligand diversity both directly (via variability in the solvent-exposed side chains) and indirectly (through their effect on the MHC conformation). Our data support a model where self peptide diversity allows selection of T cells specific for a broad range of MHC conformations.

Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers.

Recent data using MHC/peptide tetramers and dimers suggests that the T cell coreceptors, CD4 and CD8, although important for T cell activation, do not play a direct role in facilitating T cell receptor (TCR) binding to multivalent MHC/peptide ligands. Instead, a current model proposes that coreceptors are recruited only after a stable TCR-MHC/peptide complex has already formed and signaled. In contrast, we show using multimeric class I MHC/peptide ligands that CD8 plays a critical (in some cases obligatory) role in antigen-specific TCR binding. T cell activation, measured by calcium mobilization, was induced by multimeric but not monomeric ligands and also showed CD8 dependency. Our analysis using anti-CD8 antibodies revealed that binding to different epitopes of CD8 can either block or augment TCR-MHC/peptide interaction. These effects on TCR binding to high-affinity agonist ligands were even more pronounced when binding to multimeric low-affinity ligands, including TCR antagonists, was studied. Our data have important implications for the role of CD8 in TCR binding to MHC/peptide ligands and in T cell activation. In addition, our results argue against the view that multimeric MHC/peptide ligands bind directly and solely to the TCR; rather, our data highlight a pivotal contribution of CD8 for this association.

Interleukin-7 mediates the homeostasis of naïve and memory CD8 T cells in vivo.

The naïve and memory T lymphocyte pools are maintained through poorly understood homeostatic mechanisms that may include signaling via cytokine receptors. We show that interleukin-7 (IL-7) plays multiple roles in regulating homeostasis of CD8+ T cells. We found that IL-7 was required for homeostatic expansion of naïve CD8+ and CD4+ T cells in lymphopenic hosts and for CD8+ T cell survival in normal hosts. In contrast, IL-7 was not necessary for growth of CD8+ T cells in response to a virus infection but was critical for generating T cell memory. Up-regulation of Bcl-2 in the absence of IL-7 signaling was impaired after activation in vivo. Homeostatic proliferation of memory cells was also partially dependent on IL-7. These results point to IL-7 as a pivotal cytokine in T cell homeostasis.

Homeostatic expansion and phenotypic conversion of naïve T cells in response to self peptide/MHC ligands.

Recent data suggest that survival of resting, naïve T cells requires an interaction with self MHC molecules. From analysis of the class I MHC-restricted T cell receptor transgenic strain OT-I, we report a different response. Rather than merely surviving, these T cells proliferated slowly after transfer into T-depleted syngeneic hosts. This expansion required both T cell "space" and expression of normal levels of self class I MHC molecules. Furthermore, we demonstrate that during homeostatic expansion in a suitable environment, naïve phenotype (CD44(low)) OT-I T cells converted to memory phenotype (CD44(med/high)), despite the absence of foreign antigenic stimulation. On the other hand, cells undergoing homeostatic expansion did not acquire cytolytic effector function. The significance of these data for reactivity of T cells with self peptide/MHC ligands and the implications for normal and abnormal T cell homeostasis are discussed.

Expression of the transcription factor lung Krüppel-like factor is regulated by cytokines and correlates with survival of memory T cells in vitro and in vivo.

The transcription factor lung Krüppel-like factor (LKLF) is involved in naive T cell survival. Expression of LKLF is rapidly down-regulated upon T cell stimulation, raising the question of whether LKLF is reexpressed after activation, and what factors are required for such reexpression. Furthermore, the expression of LKLF in resting memory cells has not been determined. Here, we use the OT-I TCR transgenic mouse system to address these issues. LKLF was found to be reexpressed following culture of activated CD8 T cells in certain cytokines (IL-2, IL-7) but not others (IL-12) known to influence CTL development. Interestingly, induction of LKLF reexpression corresponded with long-term T cell survival and development of memory T cell phenotype. Furthermore, using OT-I cells stimulated in vivo, we demonstrated that Ag induced rapid LKLF down-regulation and that the factor is expressed by in vivo-derived memory T cells.

Cutting edge: a test of the dominant negative signal model for TCR antagonism.

The mechanism by which TCR antagonists interfere with T cell activation is unclear. One popular hypothesis is that incomplete early signaling events induced by these ligands dominantly inhibit the T cell's ability to respond to a copresented agonist ligand. Here we test this "dominant negative" signal hypothesis by studying T cells expressing two distinct MHC class I-restricted TCRs (2C and OT-I). Although responses through each TCR can be efficiently inhibited by their specific antagonists, we found no evidence for "cross-antagonism" in which an antagonist for receptor "A" blocks responses through receptor "B." Such inhibition would have been expected were the dominant negative signaling hypothesis correct, and alternative models for TCR antagonism are discussed.

Qualitative and quantitative differences in T cell receptor binding of agonist and antagonist ligands.

The kinetics of interaction between TCR and MHC-peptide show a general relationship between affinity and the biological response, but the reported kinetic differences between antigenic and antagonistic peptides are very small. Here, we show a remarkable difference in the kinetics of TCR interactions with strong agonist ligands at 37 degrees C compared to 25 degrees C. This difference is not seen with antagonist/positive selecting ligands. The interaction at 37 degrees C shows biphasic binding kinetics best described by a model of TCR dimerization. The altered kinetics greatly increase the stability of complexes with agonist ligands, accounting for the large differences in biological response compared to other ligands. Thus, there may be an allosteric, as well as a kinetic, component to the discrimination between agonists and antagonists.

A divalent major histocompatibility complex/IgG1 fusion protein induces antigen-specific T cell activation in vitro and in vivo.

Activation of antigen-specific T cell clones in vivo might be possible by generating soluble MHC molecules; however, such molecules do not induce effective T cell responses unless cross-linked. As a first step in generating a soluble MHC molecule that could function as an antigen-specific immunostimulant, the extracellular domains of the murine H-2Kb MHC class I molecule were fused to the constant domains of a murine IgG1 heavy chain, resulting in a divalent molecule with both a TCR-reactive and an Fc receptor (FcR)-reactive moiety. The fusion protein can be loaded with peptide and can induce T cell activation in a peptide-specific, MHC-restricted manner following immobilization on plastic wells or following cross-linking by FcR+ spleen cells. The fusion protein induces partial T cell activation in vivo in a mouse transgenic for a TCR restricted to H-2Kb. This fusion protein molecule may be useful to study peptide-MHC interactions and may provide a strategy for boosting in vivo antigen-specific T cell responses, such as to viral or tumor antigens.

Preselection thymocytes are more sensitive to T cell receptor stimulation than mature T cells.

During T cell development, thymocytes which are tolerant to self-peptides but reactive to foreign peptides are selected. The current model for thymocyte selection proposes that self-peptide-major histocompatibility complex (MHC) complexes that bind the T cell receptor with low affinity will promote positive selection while those with high affinity will result in negative selection. Upon thymocyte maturation, such low affinity self-peptide-MHC ligands no longer provoke a response, but foreign peptides can incidentally be high affinity ligands and can therefore stimulate T cells. For this model to work, thymocytes must be more sensitive to ligand than mature T cells. Contrary to this expectation, several groups have shown that thymocytes are less responsive than mature T cells to anti-T cell receptor for antigen (TCR)/CD3 mAb stimulation. Additionally, the lower TCR levels on thymocytes, compared with T cells, would potentially correlate with decreased thymocyte sensitivity. Here we compared preselection thymocytes and mature T cells for early activation events in response to peptide-MHC ligands. Remarkably, the preselection thymocytes were more responsive than mature T cells when stimulated with low affinity peptide variants, while both populations responded equally well to the antigenic peptide. This directly demonstrates the increased sensitivity of thymocytes compared with T cells for TCR engagement by peptide-MHC complexes.

Enhanced sensitivity for sequence determination of major histocompatibility complex class I peptides by membrane preconcentration-capillary electrophoresis-microspray-tandem mass spectrometry.

Sequence analysis of antigenic major histocompatibility complex (MHC) class I peptides requires minimizing sample loss and enhancing mass spectrometric sensitivity. In order to facilitate such analyses, we have coupled on-line membrane preconcentration-capillary electrophoresis (mPC-CE) with microspray mass spectrometry (mPC-CE-microMS) and tandem mass spectrometry (mPC-CE-microMS/MS). Specifically, cell lysate from approximately 10(9) EG-7 mouse tumor cells was immunoprecipitated and the released MHC class I peptides were subjected to reverse-phase HPLC. An HPLC fraction containing antigenic peptide(s) shown to induce T-cell stimulation was subjected to mPC-CE-microMS. Approximately 10 microL (from 100 microL) of the fraction was pressure-injected and concentrated on a styrenedivinylbenzene (SDB) impregnated membrane. The peptides were eluted from the membrane with approximately 100 nL of 80% methanol, sandwiched between a leading stacking buffer (LSB, also serving as CE separation medium) of approximately 110 nL of 0.1% acetic acid in 10% methanol, and a trailing stacking buffer (TSB) of approximately 110 nL of 0.1% NH4OH. On application of the CE voltage the peptides are subjected to moving boundary transient isotachophoresis and focused. The peptides were separated in a Polybrene-coated capillary with application of -20 kV in reverse polarity mode and subsequently sprayed via an emitter coupled to the CE capillary by a liquid junction containing a platinum wire. An ion at m/z 482.3 was detected and subjected to mPC-CE-microMS/MS and determined to be SIINFEKL, a peptide (OVA) known to be antigenic in the mouse model system. Sensitivity enhancement over conventional mPC-CE-MS and MS/MS was approximately 100-fold.

T-cell selection.

Major histocompatibility complex (MHC) molecules in the thymus select from the repertoire of germline receptors those that will be most useful in mounting responses to antigen in the periphery. Recent data has shed light on the involvement of self peptides presented by the MHC in this process, and has indicated that a requirement for the constant recognition of 'self' may be essential for T-cell longevity in the periphery.

Functional mapping of the orientation for TCR recognition of an H2-Kb-restricted ovalbumin peptide suggests that the beta-chain subunit can dominate the determination of peptide side chain specificity.

T cells recognize a complex of antigenic peptide bound to the class I or class II products of the MHC. Crystallographic analysis of the interaction between MHC class I-bound peptide fragments and specific TCR have recently been described and highlight the importance of the CDR3 in determining peptide specificity. The results presented here show functional data for TCR recognition of the H2-Kb class-I restricted determinant derived from OVA (SIINFEKL) that are consistent with the TCR orientation defined by these crystal structures. In addition, we also found that the beta-chain CDR3 dominates side chain specificity for the most exposed regions within this peptide. The data also suggest that this orientation and pattern of beta-chain dominance may extend to the recognition of a second H2-Kb-restricted peptide from the herpes simplex virus type 1 glycoprotein B (SSIEFARL), which shares a common alpha-chain contact with the OVA peptide. These results are discussed in terms of a common orientation for TCR-ligand interaction and the greater potential for TCR beta-chain CDR3 diversity in determining peptide side chain specificity.