Evolution of a Complex T Cell Receptor Repertoire during Primary and Recall Bacterial Infection

By Dirk H. Busch, Ingrid Pilip, and Eric G. Pamer

From the Section of Infectious Diseases and the Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520

    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The mechanisms underlying the genesis and maintenance of T cell memory remain unclear. In this study, we examined the evolution of a complex, antigen-specific T cell population during the transition from primary effector to memory T cells after Listeria monocytogenes infection. T cell populations specific for listeriolysin O (LLO)91-99, the immunodominant epitope recognized by H2-Kd-restricted T lymphocytes, were directly identified in immune spleens using tetrameric H2-Kd-epitope complexes. The T cell receptor (TCR) Vbeta repertoire of specific T cells was determined by direct, ex vivo staining with a panel of mAbs. We demonstrate that LLO91-99-specific, primary effector T cell populations have a diverse TCR Vbeta repertoire. Analyses of memory T cell populations demonstrated similar TCR diversity. Furthermore, experiments with individual mice demonstrated that primary effector and memory T cells have indistinguishable TCR repertoires. Remarkably, after reinfection with L. monocytogenes, LLO91-99-specific T cells have a narrower TCR repertoire than do primary effector or memory T cells. Thus, our studies show that the TCR repertoire of primary effector T lymphocytes is uniformly transmitted to memory T cells, whereas expansion of memory T cells is selective.

Key words: T cell receptor repertoirecytotoxic T lymphocytesListeria monocytogeneseffector/memory T cellsrecall
    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The adaptive immune response to infectious agents is characterized by initial priming and expansion of complex, pathogen-specific T cell populations. The elicited effector T cells participate in the host defense by controlling the infection and eradicating the pathogen. Interestingly, the in vivo dynamics of antigen-specific T cell responses during the course of infection are very similar, even when the pathogens are very different: initial expansion of effector T cells is followed by a rapid contraction phase, leaving a relatively stable pool of memory T cells that provide long-term immunity (1, 2). The mechanisms that determine and regulate this transition from effector to memory T cells are not known. How are memory T cells generated and maintained? When do memory T cells become distinct from effector T cells? Are there qualitative differences between these two populations that might be reflected by differences in their TCR repertoire? These questions are fundamental to our understanding of protective immunity and have important implications for vaccine design and development.

The differences between naive, unprimed T cells and memory T cell populations are dramatic. Memory T cells require less antigen, do not require costimulation for activation, and expand more rapidly than naive T cells (3, 4). Additional phenotypic differences, such as higher surface expression of adhesion molecules, have also been described (2, 5). However, the distinction between effector T cells and memory T cells is less clear. Thus, it is still unknown whether memory T cells are a distinct cell lineage generated during antigen challenge, or if they are directly selected from activated effector T cells (1). If there is selection of memory T cells, the avidity of the TCR-MHC-peptide interaction might be of special importance. Consistent with this notion, recent studies showed that maintenance of naive or memory T cells had distinct requirements, but both required the presence of MHC molecules in the periphery (8).

One approach to determine the differences or similarities between effector and memory T cell populations is to characterize and compare their TCR repertoire. Because of the difficulties identifying small numbers of epitope-specific T cells among much larger populations of nonspecific cells, most of our knowledge of TCR repertoire evolution after immunization comes from systems where a highly restricted T cell population responds to a dominant T cell epitope (6, 9). In these systems, the predominance of a particular TCR Vbeta segment was used to detect antigen-specific T cells for further analyses of TCR Valpha chains and CDR3 sequences. These experiments demonstrated very similar TCR repertoires in effector and memory T cell populations, although in one system some selection for certain CDR3 regions was described (9). However, most effector T cell responses during infectious diseases are highly diverse (13), and it remains unknown whether memory T cell populations maintain this level of TCR repertoire diversity. We have used murine infection with Listeria monocytogenes to study complex T cell responses to infection. Intravenous infection of mice with a sublethal dose of L. monocytogenes causes rapid clearance of the pathogen and the development of very effective, long lasting immunity, which is mainly mediated by MHC class I-restricted CTLs (17, 18). Unlike many viral infections, which cause prolonged or chronic infections, L. monocytogenes is cleared from infected mice (19, 20). Four different Listeria epitopes are presented to CD8+ T lymphocytes by the MHC class I molecule H2-Kd and the in vivo kinetics of T cells responding to these epitopes have been determined (21). The H2-Kd-restricted immunodominant epitope listeriolysin O (LLO)91-991 induces the largest number of CTLs (22). Interestingly, in vitro- expanded, LLO91-99-specific T cells express a highly diverse TCR Vbeta repertoire (25).

In this study we have used tetrameric H2-Kd-LLO91-99 complexes to characterize the TCR Vbeta repertoire of specific effector, memory, and recall T cells after L. monocytogenes infection. Primary effector T lymphocytes specific for the Listeria epitope LLO91-99 are characterized by a diverse TCR Vbeta repertoire. This diversity is maintained in memory T cell populations. Remarkably, rechallenge with L. monocytogenes induces changes in the epitope-specific TCR repertoire, with focus on a narrower range of TCR Vbeta segments. These findings suggest that the breadth of the primary effector TCR repertoire is transmitted to and maintained in the memory compartment. However, expansion of the memory T cell pool narrows the repertoire of recall effector T cells. We propose that contracting or static T cell populations after primary infection maintain TCR diversity, whereas rapidly expanding T cells lose diversity.

    Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mice and Bacteria.

BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME). L. monocytogenes strain 10403s was obtained from Daniel Portnoy (University of California Berkeley, Berkeley, CA) and grown in brain-heart infusion broth.

Immunization with Listeria and Harvesting of Spleen Cells.

Mice were immunized by intravenous injection of 2 × 103 L. monocytogenes 10403s into the tail vein. Spleens were removed 7 d after immunization and splenocytes were harvested by dissociation through a wire mesh and lysis of erythrocytes with ammonium chloride, and subsequently resuspended in RP10+, which consists of RPMI 1640 (GIBCO BRL, Gaithersburg, MD) supplemented with 10% FCS, L-glutamine, Hepes (pH 7.5), beta -mercaptoethanol, penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamicin (50 µg/ml).

Enrichment for CD8+ T Cells.

Splenocytes were enriched for CD8+ T cells by positive separation using the magnetically activated cell separation system (MACS; Miltenyi, Bergisch Gladbach, Germany). Splenocytes were incubated with anti-mouse CD8alpha microbeads for 20 min in separation buffer (PBS, pH 7.45, 0.5% BSA, and 2 mM EDTA), and after two washes cells were applied on a type LS column (Miltenyi) and CD8+ T cells were separated using the MidiMACS (Miltenyi) following the manufacturer's recommendations.

Tetrameric H2-Kd-Peptide Complexes.

MHC-peptide tetramers for staining of epitope-specific T cells were generated as recently described (24, 26). In brief, a specific biotinylation site (27) was added to the COOH terminus of truncated H2-Kd heavy chain (no transmembrane region, truncation after the amino acid in position 284). This fusion protein and beta 2 microglobulin (beta 2m) were expressed in large amounts as recombinant proteins in Escherichia coli using the isopropyl-beta -D-thiogalactopyranoside (IPTG)- inducible pET3a vector system (Novagen, Inc., Madison, WI) and BL21(DE3) as an expression host. Purified heavy chain and beta 2m were dissolved in 8 M of urea and diluted into refolding buffer containing high concentrations of synthetic peptide LLO91-99 (60 µM; Research Genetics Inc., Huntsville, AL) to generate monomeric, soluble H2-Kd-peptide complexes (24, 28). MHC- peptide complexes were purified by gel filtration over a Superdex 200HR column (Pharmacia Biotech AB, Piscataway, NJ), and in vitro biotinylated for 12 h at 20°C in the presence of 15 µg biotin operon repressor protein A (BirA) (AVIDITY, Boulder, CO), 80 µM biotin, 10 mM ATP, 10 mM MgOAc, 20 mM bicine, and 10 mM Tris-HCL (pH 8.3). To remove free biotin, MHC complexes were again purified by gel filtration, and then tetramerized by addition of PE-conjugated streptavidin (Molecular Probes, Eugene, OR) at a molar ratio of 4:1. Tetramers were purified by gel filtration over a Superdex 200HR column and stored at 3-5 mg/ml at 4°C in PBS (pH 8.0) containing 0.02% sodium azide, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 0.5 mM EDTA.

In vitro Peptide Restimulation of LLO91-99-specific T Cell Lines.

T cell lines were established by in vitro peptide restimulation as recently described (25). In brief, 3-4 × 107 spleen cells from immunized mice were incubated in the presence of 3 × 107 irradiated, syngeneic spleen cells that were peptide pulsed for 1 h at 37°C with 10-9 M LLO91-99 peptide. The low peptide concentration is required for optimal in vitro expansion of LLO91-99-specific T cell lines (25). T cell lines were generated by restimulating responder cells every week with 3 × 107 peptide-coated stimulator cells. After the second restimulation, the medium was supplemented with 5% rat Con A supernatant. For short-term in vitro expansion of LLO91-99-specific CD8+ T lymphocytes from peripheral blood, 0.5-0.7-ml blood samples were taken by eye bleeding from mice 7 d after infection with L. monocytogenes. Erythrocytes of heparinized blood samples were lysed with ammonium chloride, and remaining cells were placed in a well of a 24-well plate containing 6 × 106 LLO91-99-coated stimulator cells (see above) and 5% rat Con A supernatant (total volume, 2 ml in RP10+). Cells were analyzed 3 d after a second restimulation.

Staining and Flow Cytometry Analysis.

For flow cytometry analysis, ~3 × 105 cells were added per staining to a well of a 96-well plate. After incubation at 4°C for 20 min with unconjugated streptavidin (0.5 mg/ml, Molecular Probes) and Fc-block (PharMingen, San Diego, CA) in FACS® staining buffer (SB; PBS, pH 7.45, 0.5% BSA, and 0.02% sodium azide), cells were triple stained with Cy-Chrome-conjugated anti-CD8alpha (clone 53-6.7, PharMingen), PE-conjugated H2-Kd-LLO91-99 tetramers (0.25- 0.5 mg/ml), and FITC-conjugated mAbs specific for TCR-alpha /beta (clone H57-597; PharMingen), or with 13 different TCR Vbeta segments (Vbeta 2, 3, 4, 5.1/2, 6, 7, 8.1/2, 8.1-3(pan), 9, 10, 11, 12, 13, and 14 (all obtained from PharMingen) in SB for 60 min at 4°C. Subsequently, cells were washed three times in SB and then fixed in 1% paraformaldehyde/PBS (pH 7.45). Three-color flow cytometry was performed using a FACSCalibur® flow cytometer and data were further analyzed with CELLQuest software (Becton Dickinson, Mountain View, CA).

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Generation of H2-Kd-LLO91-99 Tetramers.

The interaction of T cell receptors with their cognate MHC-peptide complexes is characterized by relatively low affinity and a high dissociation rate. Therefore, it has not been possible to use monomeric MHC-peptide complexes to identify antigen-specific T cells. However, a recently described approach using tetrameric MHC class I-peptide complexes (26) increases the affinity sufficiently to allow cell staining. We therefore generated tetrameric H2-Kd complexes stabilized with the immunodominant Listeria epitope LLO91-99. Truncated H2-Kd heavy chain (no transmembrane region) containing a genetically engineered biotinylation site at the COOH terminus (Fig. 1 A), and beta 2m were expressed as recombinant proteins in E. coli, and were refolded in the presence of high concentrations of LLO91-99 peptide. Monomeric H2-Kd-peptide complexes were purified by gel filtration (Fig. 1 B) and subsequently enzymatically biotinylated with BirA. H2-Kd-peptide complexes could be immunoprecipitated with the conformation-dependent, H2-Kd- specific mAb SF1-1.1.1 (Fig. 1 C), indicating that complexes were properly refolded, and precipitation experiments with streptavidin agarose beads demonstrated essentially complete biotinylation after treatment with BirA (Fig. 1 C). Since streptavidin has four binding sites for biotin, incubation of the biotinylated complexes in the presence of streptavidin at a molar ratio of 4:1 results in formation of MHC-peptide tetramers (Fig. 1 D). Streptavidin conjugated with PE was used for flow cytometric detection of LLO91-99-specific T lymphocytes.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1.   Folding and biotinylation of soluble H2-Kd and generation of tetramers. (A) The cDNA for murine H2-Kd was mutagenized by PCR to delete the leader sequence (LS), the transmembrane (TM), and the cytosolic domain (CD), and to extend the COOH terminus with a biotinylation sequence (indicated in single letter amino acid code) recognized by the E. coli BirA enzyme. The biotinylated lysine residue is enclosed in a box. (B) Recombinant H2-Kd and human beta 2m were overexpressed in E. coli and inclusion bodies were purified, resolubilized, and folded with LLO91-99 peptide as indicated in Materials and Methods. A typical fast protein liquid chromatography (FPLC) gel filtration absorbance profile demonstrates a large peak consisting of folded H2-Kd, beta 2m (seen in the gel inset), and peptide. HC indicates a small peak of aggregated, unfolded H2-Kd heavy chains. (C) Refolded, FPLC-purified, and biotinylated H2-Kd complexes were either directly subjected to PAGE (first labeled lane) or precipitated with conformation-dependent anti-H2-Kd-specific antibody SF1-1.1.1 (anti-Kd), control mouse IgG (mIgG) or streptavidin-agarose beads (SA). After SA precipitation, only a very small amount of folded H2-Kd could be precipitated with SF1-1.1.1 (anti-Kd, right lane). (D) Biotinylated H2-Kd complexes were mixed with streptavidin and again subjected to FPLC gel chromatography. The absorbance profile demonstrates a high molecular weight complex consisting of tetramerized H2-Kd-beta 2m-peptide complexes (gel inset shows H2-Kd heavy chain, beta 2m, and a faint band of streptavidin). The large peak consists of free streptavidin and carrier BSA (BSA/SA).

Direct Determination of TCR Vbeta Usage Profiles of Complex Epitope-specific T Cell Populations.

We have recently shown that tetrameric H2-Kd-peptide complexes specifically detect Listeria epitope-specific T cells in vitro and ex vivo (24). LLO91-99 is an immunodominant epitope inducing relatively high numbers of specific T cells during the course of infection with L. monocytogenes, which can be easily detected by tetramer staining as primary effector T lymphocytes, as well as after establishment of a memory T cell pool (24). LLO91-99 tetramers stain essentially all antigen-specific lymphocytes within the complex T cell population (24). Analysis of in vitro expanded LLO91-99-specific T cell lines revealed a diverse TCR Vbeta repertoire (25). To examine whether the TCR Vbeta repertoire of LLO91-99-specific T cell populations can be directly determined by costaining T cells with tetramers and TCR Vbeta -specific mAbs, we compared TCR Vbeta staining of in vitro expanded LLO91-99-specific T cell lines in the presence and absence of LLO91-99-H2-Kd tetramers. First, a T cell line specific for LLO91-99 was generated from an L. monocytogenes-immunized mouse by in vitro peptide restimulation (Fig. 2 A). Essentially all CD8+ lymphoblasts in the cell culture are stained by LLO91-99 tetramers (Fig. 2 B). Staining with a panel of different TCR Vbeta -specific mAbs demonstrated a diverse TCR Vbeta profile, which is identical to that obtained when cells were double stained with LLO91-99 tetramers (Fig. 2 C, gated on CD8+, tetramer-positive lymphoblasts). This experiment indicates that double staining with these TCR Vbeta mAbs does not interfere with the LLO91-99 tetramer staining. Similar results were obtained for other LLO91-99-specific T cell lines (data not shown).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   Costaining with LLO91-99-H2-Kd tetramers and TCR Vbeta mAbs. A LLO91-99-specific CTL line was generated from an L. monocytogenes-immunized BALB/c mouse by in vitro peptide restimulation. (A) P815 (H2d) target cells were labeled with 51Cr and incubated in the presence (open circles) and absence (closed circles) of 10-6 M LLO91-99 and decreasing numbers of LLO91-99-specific CTL. The percentage of specific lysis and the E/T ratio are indicated. (B) The CTL line was stained for CD8 (anti-CD8alpha Cy-Chrome) and LLO91-99 tetramers (PE-conjugated). Gating for CD8+ blasts revealed that nearly all T cell blasts stained with LLO91-99 tetramers. (C) Lymphoblasts were stained with a panel of FITC-conjugated, Vbeta -specific antibodies in the presence (white bars) and absence (black bars) of LLO91-99 tetramers.

Direct Ex Vivo Determination of TCR Vbeta Usage Profiles.

Next, we examined whether double staining with TCR Vbeta mAbs and LLO91-99 tetramers would allow direct ex vivo TCR repertoire analyses. BALB/c mice were immunized with a sublethal dose of L. monocytogenes, and spleen cells were harvested and enriched for CD8+ T cells 7 d later. As shown in Fig. 3, LLO91-99 tetramers stain a distinct population of CD8+ T cells. Double staining with a TCR-alpha /beta - specific mAb demonstrates high level TCR-alpha /beta surface expression on all tetramer-positive T cells (Fig. 3 A). Double staining with the anti-TCR Vbeta 8.1-3 mAb F21.3 identifies a distinct subpopulation within the LLO91-99-specific T cell population expressing this particular TCR Vbeta segment (Fig. 3 B).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3.   Direct ex vivo TCR staining of LLO91-99-specific T cells. CD8+ T cells from the spleen of a BALB/c mouse immunized 7 d previously with a sublethal dose of L. monocytogenes were stained with LLO91-99 tetramers (PE-conjugated) and FITC-conjugated antibody specific for TCR-alpha /beta (left) and the TCR Vbeta 8 chain (right).

We performed direct ex vivo analyses of LLO91-99-specific, primary effector T cells using 14 different TCR Vbeta -specific mAbs, which usually cover >90% of all T cells within this population. Representative histograms of TCR Vbeta stainings of CD8+/ LLO91-99 tetramer-positive T cells are shown in Fig. 4. In almost all mice analyzed, substantial subpopulations within the LLO91-99-specific T cell population could be identified for the TCR Vbeta segments Vbeta 2, 4, 5, 8.1/2, 8.1-3, and 10, whereas for other TCR Vbeta segments (Vbeta 6, 7, 9, 11, or 14) larger subpopulations could only be identified in some individual mice (see also Figs. 6 and 7).


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 4.   TCR Vbeta staining reveals multiple subpopulations of LLO91- 99-specific T cells. Immune BALB/c CD8+ splenocytes obtained 7 d after L. monocytogenes infection were stained with LLO91-99-specific tetramers and a panel of 14 different FITC-conjugated, Vbeta -specific mAbs. These histograms demonstrate the proportion of cells that are stained with each of the antibodies.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 6.   Primary and memory, LLO91-99-specific T cells from individual mice have indistinguishable ratios of TCR Vbeta chains. (top) Three BALB/c mice were immunized with a sublethal dose of L. monocytogenes and 7 d later peripheral blood lymphocytes were restimulated with LLO91-99-coated splenocytes. 10 d later these T cell lines were stained with LLO91-99 tetramers and the panel of TCR Vbeta -specific antibodies (white bars; primary effector T cells). (bottom) 35 d after infection, these three BALB/c mice were killed and CD8+ T cells were isolated from spleens and stained with LLO91-99 tetramers and the panel of TCR Vbeta -specific antibodies (hatched bars; memory T cells). The percentage of cells stained with each of the TCR antibodies is indicated.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 7.   After a recall response, LLO91-99-specific T cells express a more limited TCR repertoire than do primary effector T cells. Four BALB/c mice were infected with a sublethal dose of L. monocytogenes and 7 d later peripheral blood lymphocytes were used to generate LLO91-99-specific T cell lines, as described for Fig. 6. These T cell lines were stained with LLO91-99 tetramers and the panel of TCR Vbeta -specific antibodies (white bars; primary effector T cells). 35 d after primary infection, these four BALB/c mice were reinfected with a 50-fold higher dose (100,000 bacteria) and 5 d later CD8+ splenocytes were isolated and stained with LLO91-99 tetramers and the TCR Vbeta panel (hatched bars; recall effector T cells). The percentage of cells stained with each of the TCR antibodies is indicated.

TCR Vbeta Profiles of Primary Effector and Memory T Cells.

The relatively high and reproducible frequencies of T cells specific for the immunodominant Listeria epitope LLO91-99 (day 7 primary responders: 1.2-1.5%; 5-wk memory cells: 0.4-0.6% within CD8+ splenocytes) allowed us to determine TCR Vbeta profiles using all 14 different TCR Vbeta mAbs among immune splenocytes. In Fig. 5, TCR Vbeta profiles of LLO91-99-specific T cell populations are shown for six individual mice, three analyzed at the peak of the primary response and three analyzed 5 wk after primary infection with L. monocytogenes (memory phase). Primary LLO91-99-specific effector T cell populations show TCR Vbeta diversity, similar to the results obtained with in vitro expanded T cell lines (25, Fig. 2). The predominant Vbeta segments that are used are Vbeta 2, 4, 8, and 10, whereas other segments are represented at relatively low frequencies. However, there is some variability in the TCR Vbeta profiles between individual mice. In the memory pool, LLO91-99-specific T cell populations are also characterized by diverse TCR Vbeta repertoires, overall showing an extent of diversity similar to that of primary effector T cell populations. However, there is variability between individual mice, making it difficult to determine if the TCR repertoire of LLO91-99-specific memory T cells directly reflects the repertoire of primary effector T cells.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 5.   LLO91-99-specific primary and memory T cell repertoires closely reflect the general TCR repertoire of BALB/c CD8+ T cells. Six BALB/c mice were infected with a sublethal dose of L. monocytogenes, and CD8+ T cells from three mice were stained for TCR Vbeta expression 7 d after infection (top, primary effector T cells) and from the remaining three mice 35 d after infection (bottom, memory T cells). White bars indicate the percentage of LLO91-99 tetramer-positive cells that stain with the individual TCR Vbeta specific antibodies. Black bars indicate the percentage of overall CD8+ T cells that stain with the TCR Vbeta -specific antibodies. Minimum number of gated CD8+ and tetramer-positive T cells for each TCR Vbeta staining was 2,000 for primary effector T cells and 1,000 for memory T cells, respectively. n.d. = not done.

Direct ex vivo staining with MHC-peptide tetramers allowed us to compare the TCR Vbeta profiles of an epitope-specific T cell population with the overall TCR Vbeta usage by CD8+ T cells in the same mouse (Fig. 5, black bars). The overall TCR Vbeta repertoire of CD8+ T cells in the individual mice tested are remarkably similar, suggesting that intermouse variability in LLO91-99-specific TCRs cannot be attributed to general differences in the frequencies of different TCR Vbeta populations. TCR Vbeta stainings of naive, BALB/c splenocytes demonstrated very similar overall TCR Vbeta repertoires (data not shown). Thus, there is no detectable skewing in the overall TCR Vbeta repertoire during primary responses to L. monocytogenes.

Taken together, our data show that LLO91-99-specific primary effector and memory T cell populations have diverse TCR Vbeta repertoires. The prevalence of TCR Vbeta chains among LLO91-99-specific T cells and the general population of CD8+ T cells is remarkably similar.

Effector and Memory TCR Vbeta Repertoire Analyses in Individual Mice.

As mentioned above, the mouse to mouse variability in the TCR Vbeta profiles does not permit precise correlations between memory and primary effector TCR repertoires. To address this issue, we decided to determine the TCR Vbeta repertoire of LLO91-99-specific effector and memory T cells in the same mouse. Therefore, we expanded LLO91-99-specific T cell lines from blood samples taken at day 7 during the primary response by short term in vitro peptide stimulation. The TCR Vbeta usage of LLO91-99-specific T cell lines was determined by double staining with LLO91-99 tetramers and TCR Vbeta mAbs, and compared with the TCR Vbeta profiles of the LLO91-99-specific memory T cell population 5 wk after primary infection in the spleen of the same mouse. Fig. 6 shows TCR Vbeta profiles of LLO91-99-specific effector and memory T cell populations determined in individual mice. The TCR Vbeta repertoires determined at the two time points are very similar, if not identical. In particular, even relatively small T cell subpopulations persist among memory T cells (e.g., Vbeta 7, 9 and 10). T cell populations that are distinct for individual mice, such as the unusual TCR Vbeta profile in mouse 386, with an unusually large TCR Vbeta 9+ subpopulation, is maintained in the memory pool and underlines the importance of performing serial repertoire analyses in individual mice. These data indicate that the TCR repertoire of acute effector T cells is transmitted to the memory T cell compartment.

Recall TCR Repertoires Differ from the Primary Effector T Cell Population.

We used the same approach to determine the TCR Vbeta repertoire of LLO91-99-specific, primary effector T cells from peripheral blood samples and compared this to the repertoire in mice rechallenged with L. monocytogenes. As shown in Fig. 7, the recall TCR Vbeta repertoires of LLO91-99-specific T cell populations differ substantially from the repertoires of primary effector T cell populations. For all four mice, we found a more restricted TCR Vbeta repertoire in the recall population, suggesting a focusing on certain TCR Vbeta segments. In particular, less prevalent subpopulations decreased in frequency while the frequency of dominant Vbeta populations generally increased. Although focusing could be detected in all four mice, specific changes were not uniform and differed from mouse to mouse. Whereas mice 390, 391, and 392 show focusing mostly onto the TCR Vbeta 8+ subpopulation (in mouse 391, 70% of all recall effector T cells are positive for the anti-TCR Vbeta 8 panantibody), in mouse 393, TCR Vbeta 2 becomes the predominant subpopulation. Focusing of the recall TCR repertoire on other TCR Vbeta segments was found for Vbeta 10 in mouse 390 and for Vbeta 4 in mouse 392. Mouse 390 showed an unusually high TCR Vbeta 7 subpopulation in the effector T cell population. Unlike mouse 386 in Fig. 6, where the "fingerprint" was also found in the memory pool, after reimmunization the TCR Vbeta 7 subpopulation in mouse 390 disappeared almost completely. Taken together, the recall TCR Vbeta profiles appear to be more restricted when compared with the corresponding primary effector T cell repertoires.

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our studies comparing the TCR repertoires of effector and memory T cell populations responding to the immunodominant Listeria epitope LLO91-99 show the following: (a) the memory TCR Vbeta repertoire is similar to the repertoire of primary effector cells; and (b) focusing of the repertoire on certain TCR Vbeta segments occurs during rechallenge with the antigen. Furthermore, our studies demonstrate the use of tetrameric MHC class I-peptide complexes for direct ex vivo TCR repertoire analyses, and for the first time show the TCR repertoire evolution of a complex T cell population responding to bacterial infection.

Previous studies investigating the evolution of TCR repertoires during in vivo T cell responses have resulted in the following two models. In the first model, memory T cell receptor repertoires directly reflect those selected during the primary response, remain stable over time, and are not influenced by repetitive antigen exposure (11, 12). The second involves selection for particular T cell receptors during the transition from effector to memory T cells, which results in a more restricted memory TCR repertoire (9). Both models are based on findings in experimental systems where T cell responses are directed at single, highly dominant epitopes, expanding relatively uniform T cell populations that express a predominant TCR Vbeta chain. With our studies we wanted to investigate whether the diversity of a highly complex effector T cell population responding to an infectious agent is maintained in the memory pool. Our findings support a third model, which combines aspects from both of the above-mentioned models (Fig. 8). Although the TCR repertoire of memory T cells appears to directly reflect the repertoire selected during the primary response, the composition of the repertoire can be substantially modified during reexposure with the antigen.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 8.   Model for TCR repertoire evolution during primary and recall infection with L. monocytogenes. The diversity of a pathogen-specific T cell population expanded during primary infection (arrows indicate time points of infection) is maintained in the memory pool. After rechallenge with the pathogen, the recall TCR repertoire is more restricted compared with the primary effector and memory T cell populations. These differences might be due to different in vivo expansion rates of T cells within the epitope-specific population.

Although there is some mouse to mouse variability in the TCR repertoires of T cell populations selected for the immunodominant Listeria epitope LLO91-99, the TCR profile elicited by primary infection is highly conserved in the memory T cell population of the same individual mouse. This is consistent with observations in other experimental systems (11). In our studies, we determined the TCR Vbeta repertoire of epitope-specific T cell populations, which provides a general picture of the overall diversity of the epitope-specific response. Although this approach does not determine the complete diversity of the T cell response, its advantage is that TCR Vbeta staining allows us to characterize the majority of T cells within the antigen-specific T cell population (usually >90% of LLO91-99-specific T cell populations are covered by staining with the 14 different TCR Vbeta mAbs) directly ex vivo without the need of further in vitro propagation. The validity of this approach is supported by the recent finding that the degree of TCR repertoire diversity of EBV-specific T cell populations is maintained on the level of the TCR Vbeta segments, indicating that TCR Vbeta usage directly correlates with the overall complexity of a given T cell population (29). The complexity of primary effector T cells specific for LLO91-99 is precisely maintained in the memory T cell population. This finding suggests that selection of memory T cells is either very similar to or completely overlaps the selection of primary effector T cells during priming. Our results are interesting in the context of recent findings, which show that the maintenance of memory T cells is far less dependent on the presence of the restricting MHC molecule than is the maintenance of naive, unprimed T cells (8). Thus, individual T cell subpopulations within the broad repertoire of the T cells specific for LLO91-99, which may consist of TCRs with different affinities for H2-Kd- LLO91-99 complexes, are treated equivalently in the memory compartment. However, our experiments do not rule out the possibility that further maturation and qualitative changes of memory T cell populations occur very slowly over prolonged periods of time. Thus, it will be of particular interest to monitor TCR repertoires of complex memory T cell populations over longer periods of time.

The TCR Vbeta repertoire of LLO91-99-specific T cells becomes narrower in immune mice after rechallenge with L. monocytogenes. This observation differs from findings in other experimental systems, where antigen reexposure had no influence on the TCR repertoire (11). However, those experiments were characterized by a highly restricted primary T cell response, a factor that may limit further restriction during a recall response. The basis for repertoire focusing in our system is not known. Although the mice were rechallenged with a much larger infecting dose of L. monocytogenes (100,000 bacteria for recall infection compared with 2,000 bacteria for primary infections), bacterial clearance occurs much more rapidly in immune mice than in naive mice (48-72 h compared with 1 wk). It is possible that the kinetics of bacterial clearance and, consequently, the differences in the overall antigen quantity may account for qualitative changes in the expanded T cell population. Thus, T cell clones that are capable of responding to lower amounts of epitope, perhaps on the basis of a higher avidity for the cognate MHC-peptide complexes, might have a selective advantage. However, preliminary experiments analyzing recall TCR repertoires in response to 20-fold lower infecting doses of L. monocytogenes demonstrated similar expansion of LLO91-99-specific T cells with an identical extent of TCR repertoire focusing (Busch, D.H., and E.G. Pamer, unpublished data).

Determining the cellular and molecular basis for repertoire focusing after reexposure to antigen will require further investigation. Studies of the relative affinities of more focused T cell populations for their cognate peptide-MHC complex may be particularly informative.

    Footnotes

Address correspondence to Eric G. Pamer, Sections of Infectious Diseases and Immunobiology, Yale University, New Haven, CT 06520. Phone: 203-785-3561; Fax: 203-785-3864; E-mail: eric.pamer{at}yale.edu

Received for publication 17 February 1998 and in revised form 8 April 1998.

This work was supported by National Institutes of Health grants AI-33143 and AI-39031. E.G. Pamer is a Pew Scholar in the Biomedical Sciences, and D.H. Busch is a research fellow of the Deutsche Forschungsgemeinschaft (DFG).

Abbreviations used in this paper beta 2m, beta 2 microglobulin; BirA, biotin operon repressor protein A; LLO, listeriolysin O; SB, staining buffer.

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1. Ahmed, R., and D. Gray. 1996. Immunological memory and protective immunity: understanding their relation. Science. 272: 54-60 [Abstract].
2. Sprent, J.. 1997. Immunological memory. Curr. Opin. Immunol. 9: 371-379 [Medline].
3. Pihlgren, M., P.M. Dubois, M. Tomkowiak, T. Sjogren, and J. Marvel. 1996. Resting memory CD8+ T cells are hyperreactive to antigenic challenge in vitro. J. Exp. Med. 184: 2141-2151 [Abstract/Free Full Text].
4. Bruno, L., J. Kirberg, and H. von Boehmer. 1995. On the cellular basis of immunological memory. Immunity. 2: 37-43 [Medline].
5. Pihlgren, M., L. Lightstone, C. Mamalaki, G. Rimon, and D. Kioussis. 1995. Expression in vivo of CD45RA, CD45RB and CD44 on T cell receptor-transgenic CD8+ T cells following immunization. Eur. J. Immunol. 25: 1755-1759 [Medline].
6. Walker, P.R., T. Ohteki, J.A. Lopez, H.R. MacDonald, and J.L. Maryanski. 1995. Distinct phenotypes of antigen-selected CD8 T cells emerge at different stages of an in vivo immune response. J. Immunol. 155: 3443-3452 [Abstract].
7. Zimmerman, C., K. Brduscha-Riem, C. Blaser, R.M. Zinkernagel, and H. Pircher. 1996. Visualization, characterization, and turnover of CD8+ memory T cells in virus-infected hosts. J. Exp. Med. 183: 1367-1375 [Abstract].
8. Tanchot, C., F.A. Lemonnier, B. Perarnau, A.A. Freitas, and B. Rocha. 1997. Differential requirements for survival and proliferation of CD8 naive and memory T cells. Science. 276: 2057-2062 [Abstract/Free Full Text].
9. McHeyzer-Williams, M.G., and M.M. Davis. 1995. Antigen-specific development of primary and memory T cells in vivo. Science. 268: 106-111 [Medline].
10. McHeyzer-Williams, M.G., J.D. Altman, and M.M. Davis. 1996. Tracking antigen-specific helper T cell responses. Curr. Opin. Immunol. 8: 278-284 [Medline].
11. Walker, P.R., A. Wilson, P. Bucher, and J.L. Maryanski. 1996. Memory TCR repertoires analyzed long-term reflect those selected during the primary response. Int. Immunol. 3: 1131-1138 .
12. Maryanski, J.L., C.V. Jongeneel, P. Bucher, J.L. Casanova, and P.R. Walker. 1996. Single-cell PCR analysis of TCR repertoires selected by antigen in vivo: a high magnitude CD8 response is comprised of very few clones. Immunity. 4: 47-56 [Medline].
13. Casanova, J.L., P. Romero, C. Widmann, P. Kourilsky, and J.L. Maryanski. 1991. T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire. J. Exp. Med. 174: 1371-1383 [Abstract].
14. Cole, G.A., T.L. Hogg, and D.L. Woodland. 1994. The MHC class I-restricted T cell response to Sendai virus infection in C57BL/6 mice: a single immunodominant epitope elicits an extremely diverse repertoire of T cells. Int. Immunol. 6: 1767-1775 [Abstract].
15. Horwitz, M.S., Y. Yanagi, and M.B. Oldstone. 1994. T-cell receptors from virus-specific cytotoxic T lymphocytes recognizing a single immunodominant nine-amino-acid viral epitope show marked diversity. J. Virol. 68: 352-357 [Abstract].
16. Cose, S.C., J.M. Kelly, and F.R. Carbone. 1995. Characterization of diverse primary herpes simplex virus type 1 gB-specific cytotoxic T-cell response showing a preferential V beta bias. J. Virol. 69: 5849-5852 [Abstract].
17. Kaufmann, S.H.E., H.R. Rodewald, E. Hug, and G.D. Libero. 1988. Cloned Listeria monocytogenes specific non-MHC-restricted Lyt-2+ T cells with cytolytic and protective activity. J. Immunol. 140: 3173-3179 [Abstract/Free Full Text].
18. Pamer, E.G. 1997. Immune response to Listeria monocytogenes. In Host Response to Intracellular pathogens. S.H.E. Kaufmann, editor. R.G. Landes, Austin, TX. 131-142.
19. van der Most, R.G., A. Sette, C. Oseroff, J. Alexander, K. Murali-Krishna, L.L. Lau, S. Southwood, J. Sidney, R.W. Chesnut, M. Matloubian, and R. Ahmed. 1996. Analysis of cytotoxic T cell responses to dominant and subdominant epitopes during acute and chronic lymphocytic choriomeningitis virus infection. J. Immunol. 157: 5543-5554 [Abstract].
20. Steven, N.M., A.M. Leese, N.E. Annels, S.P. Lee, and A.B. Rickinson. 1996. Epitope focusing in the primary cytotoxic T cell response to Epstein-Barr virus and its relationship to T cell memory. J. Exp. Med. 184: 1801-1813 [Abstract].
21. Pamer, E.G., A.J. Sijts, M.S. Villanueva, D.H. Busch, and S. Vijh. 1997. MHC class I antigen processing of Listeria monocytogenes proteins: implications for dominant and subdominant CTL responses. Immunol. Rev. 158: 129-136 [Medline].
22. Vijh, S., and E.G. Pamer. 1997. Immunodominant and subdominant CTL responses to Listeria monocytogenes infection. J. Immunol. 158: 3366-3371 [Abstract].
23. Busch, D.H., H.G.A. Bouwer, D. Hinrichs, and E.G. Pamer. 1997. A nonamer peptide derived from Listeria monocytogenes metalloprotease is presented to cytotoxic T lymphocytes. Infect. Immun. 65: 5326-5329 [Abstract].
24. Busch, D.H., I.M. Pilip, S. Vijh, and E.G. Pamer. 1998. Coordinate regulation of complex T cell populations responding to bacterial infection. Immunity. 8: 353-362 [Medline].
25. Busch, D.H., I.M. Pilip, and E.G. Pamer. 1998. MHC class I/peptide stability: implications for immunodominance, in-vitro proliferation and diversity of responding CTL. J. Immunol. 160: 4441-4448 [Abstract/Free Full Text].
26. Altman, J.D., P.A.H. Moss, P.J.R. Goulder, D.H. Barouch, M.G. McHeyzer-Williams, J.I. Bell, A.J. McMichael, and M.M. Davis. 1996. Phenotypic analysis of antigen specific T lymphocytes. Science. 274: 94-96 [Abstract/Free Full Text].
27. Schatz, P.J.. 1993. Use of peptide libraries to map the substrate specificity of a peptide modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Biotechnology. 11: 1138-1143 [Medline].
28. Garboczi, D.N., U. Utz, P. Ghosh, A. Seth, J. Kim, E.A. VanTienhoven, W.E. Biddison, and D.C. Wiley. 1996. Assembly, specific binding, and crystallization of a human TCR-alpha beta with an antigenic Tax peptide from human T lymphotropic virus type 1 and the class I MHC molecule HLA-A2. J. Immunol. 157: 5403-5410 [Abstract].
29. de Campos-Lima, P.O., V. Levitsky, M.P. Imreh, R. Gavioli, and M.G. Masucci. 1997. Epitope-dependent selection of highly restricted or diverse T cell receptor repertoires in response to persistent infection by Epstein-Barr virus. J. Exp. Med. 186: 83-89 [Abstract/Free Full Text].

Copyright © 1998 by The Rockefeller University Press.
0022-1007/98/07/61/10 $2.00