Facing two T cell epitopes: a degree of randomness in the primary response is lost upon secondary immunization.

We have analyzed the hierarchy of epitope-specific T cell populations during a primary and a secondary CD8 T cell response. MHC-peptide tetramers were used to track the in vivo kinetics of expansion of T cell populations specific for two Kd-restricted epitopes simultaneously presented by a murine tumor cell following primary or recall immunizations. Individual syngeneic mice generated remarkably different primary CTL responses, as reflected by up to 60-fold differences in the relative contribution of each peptide-specific T cell population to the overall response. In these primary immunizations, the CTL dominance was not dictated by the respective abundance of the presented epitopes. In sharp contrast, the secondary response was systematically associated with a selective expansion of the same epitope-specific population both in vitro and in vivo. In vitro experiments indicated that the extent of expansion of each epitope-specific memory population is modulated by the epitope density. We conclude that, at least for this set of epitopes, the CTL hierarchy is not controlled by the same parameters in a primary vs a secondary response.

Generation of MHC-peptide tetramers: a new opportunity for dissecting T-cell immune responses.

In the past few years, the technical breakthrough in generating MHC-peptide tetramers has revolutionized the analysis of T-cell responses. The major advantage of this technique over currently available methods is the ability of these tetramers to label T lymphocytes according to their antigenic specificity. The present review describes some technical aspects of tetramers generation and discusses some of the numerous possibilities opened up by this new technology.

Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.

Severe combined immunodeficiency-X1 (SCID-X1) is an X-linked inherited disorder characterized by an early block in T and natural killer (NK) lymphocyte differentiation. This block is caused by mutations of the gene encoding the gammac cytokine receptor subunit of interleukin-2, -4, -7, -9, and -15 receptors, which participates in the delivery of growth, survival, and differentiation signals to early lymphoid progenitors. After preclinical studies, a gene therapy trial for SCID-X1 was initiated, based on the use of complementary DNA containing a defective gammac Moloney retrovirus-derived vector and ex vivo infection of CD34+ cells. After a 10-month follow-up period, gammac transgene-expressing T and NK cells were detected in two patients. T, B, and NK cell counts and function, including antigen-specific responses, were comparable to those of age-matched controls. Thus, gene therapy was able to provide full correction of disease phenotype and, hence, clinical benefit.

The peripheral CD8 T cell repertoire is largely independent of the presence of intestinal flora.

While numerous studies have analyzed the shaping of T cell repertoires by self or foreign peptides, little is known on the influence of commensal self peptides derived from the intestinal flora (IF). Here, we have analyzed naive and immune repertoires in mice devoid of IF [germ-free (GF) mice]. First, by means of an extensive CDR3beta sequencing strategy, we show that the naive peripheral CD8 T cell repertoire does not exhibit a major imprint of IF antigens. Second, using MHC-peptide tetramers, CDR3beta length distribution analyses and TCR sequencing, we show that cytotoxic T lymphocyte (CTL) responses specific for two distinct epitopes are quasi-identical in normal and GF mice. Our findings indicate that, in general, peptides derived from the intestinal microflora have little if any influence on CTL responses in the mouse.

Diversity, functionality, and stability of the T cell repertoire derived in vivo from a single human T cell precursor.

In this report, we have analyzed the human T cell repertoire derived in vivo from a single T cell precursor. A unique case of X-linked severe combined immunodeficiency in which a reverse mutation occurred in an early T cell precursor was analyzed to this end. It was determined that at least 1,000 T cell clones with unique T cell receptor-beta sequences were generated from this precursor. This diversity seems to be stable over time and provides protection from infections in vivo. A similar estimation was obtained in an in vitro murine model of T cell generation from a single cell precursor. Overall, our results document the large diversity potential of T cell precursors and provide a rationale for gene therapy of the block of T cell development.

Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion.

How negative selection shapes a polyclonal population of self-reactive T cells has been difficult to address directly because of the lack of means to isolate T cells reactive to a particular self-peptide. Here, using mice transgenic for the TCR-beta chain of a CTL clone directed against a male-specific peptide, we compared the preimmune repertoire reactive to this peptide in male and female animals. Surprisingly, in the presence of the deleting ligand, as many as 25%-40% of reactive T cells escaped clonal deletion. A correlation was found between T cell avidity, TCRalpha structures, and susceptibility to negative selection. These results suggest that numerous low-affinity self-specific T cells persist in the periphery and show that a deleting ligand can specifically narrow the structural diversity of the TCR repertoire.

Defective CTLA-4 cycling pathway in Chediak-Higashi syndrome: a possible mechanism for deregulation of T lymphocyte activation.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, also known as CD152) has been shown to play a major role in the regulation of T cell activation. Its membrane expression is highly regulated by endocytosis and trafficking through the secretory lysosome pathway. Chediak-Higashi syndrome (CHS) is an inherited disorder caused by mutations in the lysosomal trafficking regulator gene, LYST. It results in defective membrane targeting of the proteins present in secretory lysosomes, and it is associated with a variety of features, including a lymphoproliferative syndrome with hemophagocytosis. The murine equivalent of CHS, beige mice, present similar characteristics but do not develop the lymphoproliferative syndrome. We show herein that CTLA-4 is present in enlarged, abnormal vesicles in CHS T cells and is not properly expressed at the cell surface after T cell activation, whereas its surface expression is not impaired. It is therefore proposed that the defective surface expression of CTLA-4 by CHS T cells is involved in the generation of lymphoproliferative disease. This observation may provide insight into the role of CTLA-4 in humans.

The composition of a primary T cell response is largely determined by the timing of recruitment of individual T cell clones.

Primary T cell responses rely on the recruitment and proliferation of antigen-specific T cell precursors. The extent of expansion of each individual T cell clone may depend on (a) its frequency before immunization, (b) its proliferative capacity, and (c) the time at which it first encounters its cognate antigen. In this report, we have analyzed the relative contribution of each of these parameters to the shaping of immune repertoires in the T cell response specific for the epitope 170-179 derived from HLA-Cw3 and presented by Kd. By means of hemisplenectomy, we compared immune and naive repertoires in the same animal and found that the frequency of all expanded T cell clones was extremely low before immunization. In particular, the most expanded clones did not derive from high-frequency precursors. In addition, recruited T cells were found to proliferate at the same rate, irrespective of their T cell antigen receptor sequence. Finally, we showed that only T cells that encounter the antigen at early time points account for a significant part of the specific response. Therefore, the contribution of a T cell clone to the immune response is mostly determined by the time of its entry into the immune repertoire, i.e., the time of first cell division after antigen encounter.

Dramatic influence of V beta gene polymorphism on an antigen-specific CD8+ T cell response in vivo.

According to recent crystallographic studies, the TCR-alpha beta contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1 beta (CDR1 beta) and the hypervariable (D)J-encoded CDR3 beta and CDR3 alpha domains. To evaluate directly the relative importance of CDR1 beta polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic V beta a and V beta b mouse strains that differ by a CDR1 polymorphism in the V beta 10 gene segment. The V beta 10-restricted CD8+ T cell response to a defined immunodominant epitope was dramatically reduced in V beta a compared with V beta b mice, as measured either by the expansion of V beta 10+ cells or by the binding of MHC-peptide tetramers. These data indicate that V beta polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1 beta-peptide interactions.

Role of peptide backbone in T cell recognition.

T cells recognize self and nonself peptides presented by molecules of the MHC. Amino acid substitutions in the antigenic peptide showed that T cell specificity is highly degenerate. Recently, determination of the crystal structure of several TCR/MHC-peptide complexes suggested that the peptide backbone may significantly contribute to the interaction with the TCR. To directly investigate the role of the peptide backbone in T cell recognition, we performed a methylene-amino scan on the backbone of an antigenic peptide and measured the capacity of such pseudopeptides to bind their cognate MHC molecule, to sensitize target cells for T cell lysis, and to stimulate IL-2 secretion by two T cell hybridomas. For one of these pseudopeptides, we prepared fluorescent tetramers of MHC molecules and compared the staining of two T cell hybridomas. Our results demonstrate that the peptide backbone has an important contribution to TCR binding and suggest that some interactions between the peptide backbone and the TCR may be partially conserved. We discuss this finding in the perspective of TCR plasticity and T cell function.

A clonal view of alphabeta T cell responses.

Quantitative analyses of antigen (Ag)-specific alphabeta T cell populations have provided a large body of information on the natural course of T cell immune responses. New tools are now available to determine the clonal composition of Ag-specific pools in individual responders, an approach which offers direct insights into the generation of T cell immune responses and establishment of protective immunity. The present review discusses the parameters that determine the composition of Ag-specific T cell responses. Emphasis is placed on the role of the naive alphabeta T cell repertoire and on the dynamics of individual Ag-specific T cell clones during the successive phases of an immune response.

The Qa-1b molecule binds to a large subpopulation of murine NK cells.

Recent studies on human NK cells have demonstrated that the NK cell CD94/NKG2 receptors bind to the nonclassical MHC class I molecule HLA-E. A functional CD94/NKG2 complex has not yet been identified in rodents, but cDNA encoding rat and mouse CD94 and NKG2 have recently been cloned, suggesting that CD94/NKG2 receptors may exist in species other than man. The mouse nonclassical MHC class I molecule Qa-1 shares several features with HLA-E. This suggests that Qa-1 may be similarly recognized by murine NK cells. To study the ability of Qa-1 to bind to murine NK cells, we have produced a soluble tetrameric form of Qa-1b. In the present study, we demonstrate that Qa-1b tetramers distinctly bind to a large subset of fresh or IL-2-activated NK1.1+/CD3- splenocytes independently of the expression of Ly49 inhibitory receptors. Binding occurs whether NK cells have evolved in an MHC class I-expressing or in an MHC class I-deficient environment. Our data suggest the existence of a Qa-1-recognizing structure on a large subpopulation of murine NK cells that may be similar to the human CD94/NKG2 heterodimeric complex.

Immunological issues in vaccine trials: T-cell responses.

T-cell responses are both extremely diverse and dependent on the MHC of the immunized (or infected) individual. Apart from T-cell proliferation assays, the most informative functional T-cell assays are still difficult to perform. Antibody measurements provide a very indirect assessment of the helper arm of the T-cell response. On the other hand, measuring cytolytic T cells (CTL) remains a difficult task, which has precluded the evaluation of CTL responses in vaccine efficacy trials. Accordingly, even though there are reasons to suspect that CTL are important to clear certain infections and to vaccinate against certain diseases, particularly chronic viral infections such as that caused infection by HIV, the data to support these claims are largely missing in humans. Improving and automating CTL assays would have a significant impact on vaccine design. The Immunoscope technology is a PCR based approach which describes the T cell repertoire by several thousands of measurements. This allows the detection of clonal expansions and to evaluate the oligoclonality of pathological T cell infiltrates in humans. In the mouse, it has allowed us to establish the concept of public T-cell responses which are recurrent in individual animals sharing the same MHC. This concept can occasionally apply to humans since we found a public T-cell expansion in DR2a patients at the onset of multiple sclerosis. Single chain class I MHC molecules have been produced, purified, homogeneously loaded with an antigenic peptide, and coated on to beads. This formulation appears to be efficient for induction of primary CTL in vitro . A similar approach can be used to purify peptide specific T cells, and its coupling with the Immunoscope technology is being considered. The potential of these new approaches for T-cell analyses will be discussed.

Individual variations in the murine T cell response to a specific peptide reflect variability in naive repertoires.

Previous studies have analyzed the diversity of T cell responses upon immunization. Little is known, however, about the individual variability of naive repertoires and its influence on immune responses. In the present study, T cells specific for a Kd-restricted epitope derived from HLA-A2 were purified from individual immunized mice using tetramers of MHC-peptide. Their TCRbeta chains were sequenced revealing strong biases but large variations in BJ usage and clonal composition. Most importantly, sequence analysis from nonimmunized mice demonstrated the preexistence of a small set of splenic precursors, distinct in each mouse and comprising less than 200 cells. Therefore, differences in precursor pools appear to be the major source of individual variability in antigen-selected repertoires.

Enrichment of antigen-specific T lymphocytes by panning on immobilized MHC-peptide complexes.

Numerous studies have focused on characterizing and monitoring antigen-specific T cells during the course of an immune response. Mostly indirect methods were used to circumvent the low frequency of T cell precursors and the inherent complexity of T cell receptor (TcR)-MHC-peptide interactions. Here, we took advantage of peptide-specific adhesion induced by immobilized MHC-peptide complexes. We describe a simple technique which allows enrichment in antigen-specific T lymphocytes among a heterogeneous CD8+ T cell population. Enrichment of T cells according to their specificity should facilitate their characterization and provide an attractive tool for immunotherapy.