Natural killer T cells reactive to a single glycolipid exhibit a highly diverse T cell receptor beta repertoire and small clone size.

CD1d-restricted natural killer (NK) T cells reactive with the glycolipid alpha-galactosylceramide (alpha-GalCer) are a distinct lymphocyte sublineage. They express an invariant Valpha14-Jalpha18 T cell receptor (TcR), but the role of the beta chain has been controversial. Here, we have used CD1d tetramers to identify and isolate NK T cells based on their antigen specificity. In mice lacking germline Vbeta8, most of the alpha-GalCer-reactive T cells express either Vbeta2 or Vbeta7, strong Vbeta selection being revealed by the lack of an increase in other Vbeta regions. By contrast to the selection for complementarity determining region (CDR) 3beta sequences in some anti-peptide responses, alpha-GalCer-reactive T cells have polyclonal CDR3beta sequences. There is little CDR3beta sequence redundancy between organs or individual mice, and, surprisingly, there also is no evidence for organ-specific CDR3beta sequence motifs. These data argue against a T cell receptor-mediated self-reactivity for tissue-specific CD1d-bound ligands. Each NKT clone is represented by only 5-10 cells. This clone size is similar to naive conventional T cells, and much lower than that reported for memory T cells, although NK T cells have an activated/memory phenotype.

NKT cells derive from double-positive thymocytes that are positively selected by CD1d.

CD1d-reactive NKT cells are a separate T cell sublineage. Instructive models propose that NKT cells branch off the mainstream developmental pathway because of their T cell antigen receptor specificity, whereas stochastic models would propose that they develop from precursor cells committed to this sublineage before variable-gene rearrangement. We show here that immature double-positive (DP) thymocytes form the canonical rearranged Valpha gene of NKT cells at nearly equivalent frequencies in the presence or absence of CD1d expression. After interacting with CD1d in the thymus, these cells give rise to expanded populations of NKT cells-including both CD4+ and double-negative lymphocytes in the thymus and periphery-that express this alpha chain. These results confirm the existence of a DP intermediate for CD1d-reactive NKT cells. They also show that the early developmental stages of these T cells are not governed by a distinct mechanism, which is consistent with the TCR-instructive model of differentiation.

Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers.

A major group of natural killer (NK) T cells express an invariant Valpha14(+) T cell receptor (TCR) specific for the lipoglycan alpha-galactosylceramide (alpha-GalCer), which is presented by CD1d. These cells may have an important immune regulatory function, but an understanding of their biology has been hampered by the lack of suitable reagents for tracking them in vivo. Here we show that tetramers of mouse CD1d loaded with alpha-GalCer are a sensitive and highly specific reagent for identifying Valpha14(+) NK T cells. Using these tetramers, we find that alpha-GalCer-specific T lymphocytes are more widely distributed than was previously appreciated, with populations of largely NK1.1(-) but tetramer-binding T cells present in the lymph nodes and the intestine. Injection of alpha-GalCer leads to the production of both interferon gamma and interleukin 4 by nearly all NK T cells in the liver and the majority of the spleen within 2 h. These cells mostly disappear by 5 h, and they do not reappear after 1 wk. Curiously, tetramer-positive thymocytes do not rapidly synthesize cytokines, nor do they undergo decreases in cell number after lipid antigen stimulation, although they express equivalent TCR levels. In summary, the data presented here demonstrate that alpha-GalCer-specific NK T cells undergo a unique and highly compartmentalized response to antigenic stimulation.

Systemic activation and antigen-driven oligoclonal expansion of T cells in a mouse model of colitis.

Transfer of CD4+CD45RBhigh T cells into immunodeficient mice results in both the expansion of the transferred T cells and colitis. Here we show that colitis pathogenesis requires expression of MHC class II molecules by the immune-deficient host. Analysis of the TCRbeta repertoire of the cells found in the large intestine of diseased mice revealed a population with restricted TCR diversity. Furthermore, nucleotide sequence analysis demonstrated the selection for particular CDR3beta amino acid sequence motifs. Collectively, these data indicate that the expansion of T cells in the intestine and colitis pathogenesis are likely to require the activation of Ag-specific T cells, as opposed to nonspecific or superantigen-mediated events. There is relatively little overlap, however, when the TCR repertoires of different individuals are compared, suggesting that a number of Ags can contribute to T cell expansion and the generation of a T cell population in the intestine. Surprisingly, many of the expanded clones found in the large intestine also were found in the spleen and elsewhere, although inflammation is localized to the colon. Additionally, donor-derived T cells appear to be activated in both the intestine and the spleen at early time points after cell transfer. Together, these results strongly suggest that disease induction in this model involves either the early and systemic activation of antigen-specific T cells or the rapid dispersal of T cells activated at a particular site.

Interleukin 4-producing CD4 T cells arise from different precursors depending on the conditions of antigen exposure in vivo.

The precursor origin of T helper (Th) cell subsets in vivo has been difficult to study and remains poorly investigated. We have previously shown that chronic administration of soluble protein antigen induces selective development of antigen-specific CD4 Th2 cells in genetically predisposed mouse strains. To analyze the origin of effector T cells in this model, we designed a competitive polymerase chain reaction-based approach to track public BV-J rearrangement expressed by CD4 T cells specific for hen egg white lysozyme (HEL) in BALB/c mice. We show that public T cell clones are predominantly associated with type 1 or 2 effector Th cells recovered after primary immunization in complete or incomplete Freund's adjuvant, respectively. Conversely, continuous administration of soluble antigen, which induces strong memory Th2 response, is associated with a dose-dependent reduction of public clone size by a mechanism resembling clonal anergy. Thus, soluble HEL-induced Th2 cells do not express the public complementarity determining region 3 motifs characteristic of immunogenic challenge in the presence of adjuvant. These results demonstrate that there are multiple pathways of induction of Th2 responses depending on the condition of antigen exposure in vivo, i.e., clonal immune deviation versus recruitment of a different pool of precursor cells.

Cutting edge: TCR alpha beta+ CD8 alpha alpha+ T cells are found in intestinal intraepithelial lymphocytes of mice that lack classical MHC class I molecules.

TCR alpha beta+ intestinal intraepithelial lymphocytes (IEL) can express either the typical CD8 alpha beta heterodimer or an unusual CD8 alpha alpha homodimer. Both types of CD8+ IEL require class I molecules for their differentiation, since they are absent in beta2m-/- mice. To gain insight into the role of class I molecules in forming TCR alpha beta+ CD8+ IEL populations, we have analyzed the IEL in mice deficient for either TAP, beta 2m, CD1, or K and D. We find that K-/-D-/- mice have TCR alpha beta+ CD8 alpha alpha+ IEL, although they are deficient for TCR alpha beta+ CD8 alpha beta+ cells. This indicates that at least some TCR alpha beta+ CD8 alpha alpha+ IEL require only nonclassical class I molecules for their development. Surprisingly, the TCR alpha beta+ CD8 alpha alpha+ IEL are significantly increased in K-/-D-/- mice, suggesting a complex interaction between CD8+ IEL and class I molecules that might include direct or indirect negative regulation by K and D, as well as positive effects mediated by nonclassical class I molecules.

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.

Quantitative analysis of the T cell repertoire selected by a single peptide-major histocompatibility complex.

The positive selection of CD4+ T cells requires the expression of major histocompatibility complex (MHC) class II molecules in the thymus, but the role of self-peptides complexed to class II molecules is still a matter of debate. Recently, it was observed that transgenic mice expressing a single peptide-MHC class II complex positively select significant numbers of diverse CD4+ T cells in the thymus. However, the number of selected T cell specificities has not been evaluated so far. Here, we have sequenced 700 junctional complementarity determining regions 3 (CDR3) from T cell receptors (TCRs) carrying Vbeta11-Jbeta1.1 or Vbeta12-Jbeta1.1 rearrangements. We found that a single peptide-MHC class II complex positively selects at least 10(5) different Vbeta rearrangements. Our data yield a first evaluation of the size of the T cell repertoire. In addition, they provide evidence that the single Ealpha52-68-I-Ab complex skews the amino acid frequency in the TCR CDR3 loop of positively selected T cells. A detailed analysis of CDR3 sequences indicates that a fraction of the beta chain repertoire bears the imprint of the selecting self-peptide.

Type II and III receptors for immunoglobulin G (IgG) control the presentation of different T cell epitopes from single IgG-complexed antigens.

T cell receptors on CD4(+) lymphocytes recognize antigen-derived peptides presented by major histocompatibility complex (MHC) class II molecules. A very limited set of peptides among those that may potentially bind MHC class II is actually presented to T lymphocytes. We here examine the role of two receptors mediating antigen internalization by antigen presenting cells, type IIb2 and type III receptors for IgG (FcgammaRIIb2 and FcgammaRIII, respectively), in the selection of peptides for presentation to T lymphocytes. B lymphoma cells expressing recombinant FcgammaRIIb2 or FcgammaRIII were used to assess the presentation of several epitopes from two different antigens. 4 out of the 11 epitopes tested were efficiently presented after antigen internalization through FcgammaRIIb2 and FcgammaRIII. In contrast, the 7 other epitopes were efficiently presented only when antigens were internalized through FcgammaRIII, but not through FcgammaRIIb2. The capacity to present these latter epitopes was transferred to a tail-less FcgammaRIIb2 by addition of the FcgammaRIII-associated gamma chain cytoplasmic tail. Mutation of a single leucine residue at position 35 of the gamma chain cytoplasmic tail resulted in the selective loss of presentation of these epitopes. Therefore, the nature of the receptor that mediates internalization determines the selection of epitopes presented to T lymphocytes within single protein antigens.

Antigen presentation by dendritic cells focuses T cell responses against immunodominant peptides: studies in the hen egg-white lysozyme (HEL) model.

T lymphocyte responses to a protein Ag are restricted to a limited number of determinants and not to all peptides capable of binding to MHC class II molecules. This focusing of the immune response is defined as immunodominance and has been observed with numerous protein Ags. In the H-2d haplotype, hen egg-white lysozyme (HEL)-specific T lymphocytes react with I-Ed-restricted peptides derived from a single immunodominant (ID) region (HEL 103-117). Moreover, we have recently found that another region of HEL (HEL 7-31) binds to I-Ad molecules and is efficiently processed and presented by splenocytes. HEL7-31 is as tolerogenic as the ID region in HEL transgenic mice. The present report demonstrates that the subdominance of the HEL 7-31 region is not due to a defect in the T cell repertoire, since specific TCRs can be found in all BALB/c mice. We show that normal and lymphoma B cells present efficiently HEL regions 103-117 and 7-31, whereas dendritic cells favor the ID region only. These results suggest that dendritic cells play a major role in the focusing of the immune response against a few antigenic determinants, while B lymphocytes may diversify the T cell response by presenting a more heterogeneous set of peptide-MHC complexes.

Determinant selection for T-cell tolerance in HEL-transgenic mice: dissociation between immunogenicity and tolerogenicity.

The induction of T-cell tolerance to self-antigens has been extensively characterized for immunodominant (ID) regions. However, tolerance toward other minor self-determinants has received less attention. In the H-2(d) haplotype, HEL contains a single ID determinant (region 102-120) presented by I-E(d) MHC class II molecules. The present study evaluates the role of subdominant and cryptic HEL regions in maintaining tolerance. We have generated a mutated HEL antigen, HEL mu, whose ID region does not bind to I-E(d). Lymph node cells from HEL-immunized mice proliferated strongly to HEL mu in vitro. Two new stimulatory regions common to HEL and HEL mu were uncovered. They are produced during antigen processing and prime specific T lymphocytes. HEL-Tg mice were tolerant to these determinants, thus confirming their in vivo presentation. These HEL regions were as tolerogenic as the HEL ID determinant, despite their poor immunogenicity. These results demonstrate that there is not always a correlation between tolerogenicity and immunogenicity, a finding that may be critical for understanding T-cell tolerance.

Is antigen processing guided by major histocompatibility complex molecules?

Major histocompatibility complex class I (MHC-1) molecules bind peptide fragments derived from cytosolic antigens, and class II (MHC-2) molecules bind fragments of proteins that enter the endocytic pathway. How peptides of the right affinity and size are generated in vivo is still the focus of intense research. Current data are consistent with the view that precursor peptides of varying length are produced in the cytosol and transported into the endoplasmic reticulum lumen where nascent MHC-1 could sample the peptides for their affinity. High-affinity peptides would form stable complexes with MHC-1, which are resistant to proteolysis by luminal enzymes; peptides unable to bind to MHC-1 presumably undergo proteolysis in the lumen. On the other hand, multiple mechanisms are probably used to load MHC-2. Some proteins denatured in the acidic and reducing environment of the endosomes most likely bind to MHC-2 through the antigen's immunodominant region, and the exposed portions of the antigen are degraded by endosomal proteases. Other antigens must first be proteolysed into peptide fragments, which compete among themselves for binding to MHC-2, whereas heat shock proteins could also contribute peptides for MHC-2 loading. Because of their respective loading modes, there is a partial correlation between the MHC-2 binding affinity of the protein fragments and their in vivo immunodominance, which may not necessarily be the case for MHC-1.

Dissociation of the peptide/MHC class I complex: pH dependence and effect of endogenous peptides on the activation energy.

Dansylated peptides were used to characterize the dissociation of peptides from a recombinant class I major histocompatibility complex protein. Dissociation of endogenous, low-affinity peptides from the class I molecule Kd had an activation energy of 6.78 +/- 0.64 kcal/mol in the 14 to 26 degrees C temperature range, but there was a break in the Arrhenius plot between 12 and 14 degrees C. Dissociation of a dansylated, high-affinity peptide had an activation energy of 20.24 +/- 1.69 kcal/mol, and there was similarly a break in the plot. Both direct interactions between peptide and the class I heavy chain and indirect effects of the peptide affinity on the extent of light chain association with heavy chain may contribute to the difference in activation energies, while the break in the Arrhenius plots implies a temperature-dependent conformational change. Dissociation was also slowest at neutral pH, but the peptide/class I complex dissociated rapidly at pH greater than 9 and less than 5, suggesting that endocytosed class I proteins would most likely lose their bound peptides at the acidic pH of endosomes.