Janet L. Maryanski

Obligatory Role for Cooperative Signaling by Pre-TCR and Notch during Thymocyte Differentiation

The first checkpoint during T cell development, known as β selection, requires the successful rearrangement of the TCR-β gene locus. Notch signaling has been implicated in various stages during T lymphopoiesis. However, it is unclear whether Notch receptor-ligand interactions are necessary during β selection. Here, we show that pre-TCR signaling concurrent with Notch receptor and Delta-like-1 ligand interactions are required for the survival, proliferation, and differentiation of mouse CD4−CD8− thymocytes to the CD4+CD8+ stage. Furthermore, we address the minimal signaling requirements underlying β selection and show a hierarchical positioning of key proximal signaling molecules. Collectively, our results demonstrate an essential role for Notch receptor-ligand interactions in enabling the autonomous signaling capacity of the pre-TCR complex.

Quantitation and cloning of cytolytic t lymphocytes and their precursors.

The recent identification of soluble growth-promoting factors which may play a role in the regulation of T cell immune responses has led to an explosion of interest in this aspect of cellular immunobiology. While the majority of investigators in this field have concentrated on obtaining detailed information on the molecular species involved in T cell growth and differentiation or on the cellular pathways by which these factors act {this volume), there is also a growing body of information which is based on the utilization of such factors to obtain relatively homogeneous populations of functionally defined T cells that can be subjected to further analysis at the cellular and molecular levels. The aim of the present review is to summarize a series of experiments which fall into this latter category. In particular, we will demonstrate that a soluble factor(s) derived from the supernatant of secondary mixed leukocyte cultures (2° MLC SN) (Ryser et al. 1978) is a potent nonspecific stimulator of cytolytic T lymphocyte (CTL) growth and/or differentiation. Furthermore, by utilizing such 2° MLC SN in a limiting dilution microculture system, we have been able to quantitate individual precursors of CTL (CTL-P) in a variety of experimental situations and to investigate the specificity and function of their clonally derived progeny.

Single-Cell PCR Analysis of TCR Repertoires Selected by Antigen In Vivo: A High Magnitude CD8 Response Is Comprised of Very Few Clones

Taking advantage of a potent MHC class I-restricted response that allows the identification of antigen-selected CD8 T cells directly ex vivo, we characterized the antigen-specific T cell repertoires that develop in individual mice by single-cell PCR analysis. Each of the immune mice displayed distinct yet structurally similar TCR repertoires. The overall repertoire size was estimated to be in the range of 15-20 for most mice. No major differences were observed between primary and secondary responses. Moreover, for a hyperimmunized mouse the antigen-specific TCR repertoire expressed 8 months after the initial immunization was very similar to that found at the peak of the primary response. Our results demonstrate that a high magnitude immune response may be composed of very few clones, and that at least in the system analyzed, the memory response largely reflects the repertoire selected by the peak of the primary response.

Antigen-selected T-cell receptor diversity and self-nonself homology

Recent studies indicate that the T-cell receptor (TCR) repertoire selected by certain antigenic peptide-MHC combinations can be extremely diverse. This is in contrast to earlier studies reporting T-cell responses which were limited in terms of TCR diversity. In this viewpoint, we suggest these variations in TCR diversity may be explained by taking into account the homology between some antigens and self proteins to which T cells are tolerant.

T helper epitopes enhance the cytotoxic response of mice immunized with MHC class I-restricted malaria peptides

We have previously derived MHC class I (H-2Kd) restricted cytotoxic T lymphocytes (CTL) from BALB/c mice immunized with irradiated sporozoites from Plasmodium (P.) berghei and P. yoelii. The CTL recognize synthetic peptides corresponding to a region of the circumsporozoite (CS) protein that is homologous in the two species. In the present study, we have attempted to induce CS-specific CTL by immunization with those peptides in incomplete Freunds adjuvant. Only a low level CTL response was detected in BALB/c mice immunized with synthetic peptides corresponding to the Pb or Py CTL epitopes. In contrast, CS-specific CTL responses could be readily detected in mice injected with mixtures of peptides that combined the P. berghei or P. yoelii CTL epitopes with previously defined T helper epitopes. Several different T helper epitopes were shown to enhance the response when injected as separate peptides in a mixture, or when covalently linked to a CTL epitope. These results may have general implications for the elicitation of CTL responses to defined CTL epitopes and for the design of peptide-based synthetic vaccines.

Competitor analogs for defined T cell antigens: Peptides incorporating a putative binding motif and polyproline or polyglycine spacers

We describe a new approach for modeling antigenic peptides recognized by T cells. Peptide A24 170-182 can compete with other antigenic peptides that are recognized by H-2kd-restricted cytolytic T cells, presumably by binding to the Kd molecule. By comparing substituted A24 peptides as competitors in a functional competition assay, the A24 residues Tyr-171, Thr-178, and Leu-179 were identified as possible contact residues for Kd. A highly active competitor peptide analog was synthesized in which Tyr was separated from the Thr-Leu pair by a pentaproline spacer. The choice of proline allowed the prediction of a probable conformation for the analog when bound to the Kd molecule. The simplest conformation of the A24 peptide that allows the same spacing and orientation of the motif as in the analog would be a nearly extended polypeptide chain incorporating a single 3(10) helical turn or similar structural kink.

Immunogenic (tum−) variants obtained by mutagenesis of mouse mastocytoma P815

Mutagen treatment of mouse mastocytoma P815 produces highly inununogenic “tum−” variants. Most of these variants express potent transplantation antigens which are not present on the original P815 tumor cells. These tum− antigens, which appear to be specific for each variant, elicit a strong cytolytic T lymphocyte (CTL) response, but do not seem to induce a specific antibody response. As a first step in the isolation of the gene of a tum− antigen, we attempted DNA-mediated gene transfer. As a DNA recipient cell we used P1.HTR, a highly transfectable P815 cell line, whose selection has been previously described. For the detection of antigen-expressing cells in transfected populations we developed a procedure that relies on the ability of these cells to stimulate the proliferation of the relevant CTL. Using DNA from tum− variant P91 mixed with a plasmid carrying an antibiotic resistance gene, we obtained several independent transfectants expressing a tum− antigen, at a frequency of approximately 1 in 13 000 antibiotic-resistant transfectants. These transfectants express only one of the two tum− antigens that were identified on P91, suggesting that these tum− antigens correspond to different genes. We expect that the detection procedure described here will be-suitable for the identification of transfectants for any gene that determines the expression of an antigen recognized by CTL.

Isolation and characterization of protective cytolytic T cells in a rodent malaria model system

Protective immunity against malaria is induced by immunization with irradiation-attenuated sporozoites. Here we report the isolation of cytolytic T-cell (CTL) clones from BALB/c (H-2d) mice immunized with either Plasmodium berghei or Plasmodium yoelii sporozoites. The epitopes recognized by these CTL can be mimicked by synthetic peptides corresponding to a homologous region in the CS proteins of both rodent malaria species. Both peptides are recognized by the CTL in the context of the same MHC class I molecule, H-2 Kd. In vivo adoptive transfer of the CTL clones into non-immune syngeneic mice protected them from a lethal challenge of infectious sporozoites.

T Cell Receptor Gene Rearrangement Lineage Analysis Reveals Clues for the Origin of Highly Restricted Antigen-specific Repertoires

Due to ordered, stage-specific T cell receptor (TCR)-β and -α locus gene rearrangements and cell division during T cell development, a given, ancestral TCR-β locus VDJ rearrangement might be selected into the mature T cell repertoire as a small cohort of “half-sibling” progeny expressing identical TCR-β chains paired with different TCR-α chains. The low frequency of such a cohort relative to the total αβ TCR repertoire precludes their direct identification and characterization in normal mice. We considered it possible that positive selection constraints might limit the diversity of TCR-α chains selected to pair with β chains encoded by an ancestral VDJ-β rearrangement. If so, half-sibling T cells expressing structurally similar, but different TCR-α chains might recognize the same foreign antigen. By single cell polymerase chain reaction analysis of antigen-specific TCRs selected during a model anti-tumor response, we were able to identify clusters of T cells sharing identical VDJ-β rearrangements but expressing different TCR-α chains. The amplification of residual DJ-β rearrangements as clonal markers allowed us to track T cells expressing different TCR-α chains back to a common ancestral VDJ-β rearrangement. Thus, the diversity of TCR-αs selected as partners for a given VDJ-β rearrangement into the mature TCR repertoire may indeed be very limited.

A quantitative, single-cell PCR analysis of an antigen-specific TCR repertoire selected during an in vivo CD8 response: direct evidence for a wide range of clone sizes with uniform tissue distribution

The development of T cell effector and memory responses against foreign antigens (Ags) involves the activation, differentiation and proliferation of naive T cells expressing distinct Ag-specific TCRs. Understanding the complexity of Ag-selected TCR repertoires in individual responders in terms of the sequences selected and their relative frequencies may provide indications about how a repertoire is established and suggest ways to influence the outcome of an immune response. Most methods of repertoire analysis are unsuitable for calculating the relative in vivo frequencies of Ag-specific clones (expressing distinct TCRs) selected during an immune response, whereas sequence data obtained by single-cell PCR analysis directly reflect cell frequencies if a sufficiently large number of cells is sampled. Using a CD8 T cell response in normal mice in which Ag-selected cells are identified by cell surface phenotype and rearranged TCRBV sequences are determined by PCR amplification of genomic DNA directly from single cells, we have analyzed a large number (>200 per animal) of structurally-related Ag-specific TCRs to calculate the frequencies of distinct TCRs selected by individual mice. We found that each responder selects a unique Ag-specific TCR repertoire in which the various TCRBV sequences are present in a wide range of frequencies. However, the overall distribution of sequences is quite similar for different responder animals. Moreover, an individuals selected TCR repertoire is uniformly represented among Ag-specific CD8 cells circulating in the blood or localized in the spleen or liver. Relatively few sequences make up the bulk of the repertoire and account for the oligoclonality observed in earlier studies. We discuss various models that could account for this skewed distribution of an Ag-selected TCR repertoire.