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

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.

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), β-mercaptoethanol, penicillin (100 U/ml), streptomycin (100 μg/ml), and gentamicin (50 μg/ml).

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 CD8α 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.

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-K^d heavy chain (no transmembrane region, truncation after the amino acid in position 284). This fusion protein and β₂ microglobulin (β₂m) were expressed in large amounts as recombinant proteins in Escherichia coli using the isopropyl-β-d-thiogalactopyranoside (IPTG)- inducible pET3a vector system (Novagen, Inc., Madison, WI) and BL21(DE3) as an expression host. Purified heavy chain and β₂m were dissolved in 8 M of urea and diluted into refolding buffer containing high concentrations of synthetic peptide LLO_91–99 (60 μM; Research Genetics Inc., Huntsville, AL) to generate monomeric, soluble H2-K^d–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.

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 LLO_91–99 peptide. The low peptide concentration is required for optimal in vitro expansion of LLO_91–99-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 LLO_91–99-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 LLO_91–99-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.

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-CD8α (clone 53-6.7, PharMingen), PE-conjugated H2-K^d–LLO_91–99 tetramers (0.25– 0.5 mg/ml), and FITC-conjugated mAbs specific for TCR-α/β (clone H57-597; PharMingen), or with 13 different TCR Vβ segments (Vβ 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).

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-K^d complexes stabilized with the immunodominant Listeria epitope LLO_91–99. Truncated H2-K^d heavy chain (no transmembrane region) containing a genetically engineered biotinylation site at the COOH terminus (Fig. 1,A), and β₂m were expressed as recombinant proteins in E. coli, and were refolded in the presence of high concentrations of LLO_91–99 peptide. Monomeric H2-K^d–peptide complexes were purified by gel filtration (Fig. 1,B) and subsequently enzymatically biotinylated with BirA. H2-K^d–peptide complexes could be immunoprecipitated with the conformation-dependent, H2-K^d– 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 LLO_91–99-specific T lymphocytes.

We have recently shown that tetrameric H2-K^d–peptide complexes specifically detect Listeria epitope-specific T cells in vitro and ex vivo (24). LLO_91–99 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). LLO_91–99 tetramers stain essentially all antigen-specific lymphocytes within the complex T cell population (24). Analysis of in vitro expanded LLO_91–99-specific T cell lines revealed a diverse TCR Vβ repertoire (25). To examine whether the TCR Vβ repertoire of LLO_91–99-specific T cell populations can be directly determined by costaining T cells with tetramers and TCR Vβ–specific mAbs, we compared TCR Vβ staining of in vitro expanded LLO_91–99-specific T cell lines in the presence and absence of LLO_91–99–H2-K^d tetramers. First, a T cell line specific for LLO_91–99 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 LLO_91–99 tetramers (Fig. 2,B). Staining with a panel of different TCR Vβ–specific mAbs demonstrated a diverse TCR Vβ profile, which is identical to that obtained when cells were double stained with LLO_91–99 tetramers (Fig. 2 C, gated on CD8⁺, tetramer-positive lymphoblasts). This experiment indicates that double staining with these TCR Vβ mAbs does not interfere with the LLO_91–99 tetramer staining. Similar results were obtained for other LLO_91–99-specific T cell lines (data not shown).

Next, we examined whether double staining with TCR Vβ mAbs and LLO_91–99 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, LLO_91–99 tetramers stain a distinct population of CD8⁺ T cells. Double staining with a TCR-α/β– specific mAb demonstrates high level TCR-α/β surface expression on all tetramer-positive T cells (Fig. 3,A). Double staining with the anti–TCR Vβ 8.1-3 mAb F21.3 identifies a distinct subpopulation within the LLO_91–99-specific T cell population expressing this particular TCR Vβ segment (Fig. 3 B).

We performed direct ex vivo analyses of LLO_91–99-specific, primary effector T cells using 14 different TCR Vβ-specific mAbs, which usually cover >90% of all T cells within this population. Representative histograms of TCR Vβ stainings of CD8⁺/ LLO_91–99 tetramer-positive T cells are shown in Fig. 4. In almost all mice analyzed, substantial subpopulations within the LLO_91–99-specific T cell population could be identified for the TCR Vβ segments Vβ2, 4, 5, 8.1/2, 8.1-3, and 10, whereas for other TCR Vβ segments (Vβ6, 7, 9, 11, or 14) larger subpopulations could only be identified in some individual mice (see also Figs. 6 and 7).

The relatively high and reproducible frequencies of T cells specific for the immunodominant Listeria epitope LLO_91–99 (day 7 primary responders: 1.2–1.5%; 5-wk memory cells: 0.4–0.6% within CD8⁺ splenocytes) allowed us to determine TCR Vβ profiles using all 14 different TCR Vβ mAbs among immune splenocytes. In Fig. 5, TCR Vβ profiles of LLO_91–99-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 LLO_91–99-specific effector T cell populations show TCR Vβ diversity, similar to the results obtained with in vitro expanded T cell lines (25, Fig. 2). The predominant Vβ segments that are used are Vβ 2, 4, 8, and 10, whereas other segments are represented at relatively low frequencies. However, there is some variability in the TCR Vβ profiles between individual mice. In the memory pool, LLO_91–99-specific T cell populations are also characterized by diverse TCR Vβ 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 LLO_91–99-specific memory T cells directly reflects the repertoire of primary effector T cells.

Direct ex vivo staining with MHC–peptide tetramers allowed us to compare the TCR Vβ profiles of an epitope-specific T cell population with the overall TCR Vβ usage by CD8⁺ T cells in the same mouse (Fig. 5, black bars). The overall TCR Vβ repertoire of CD8⁺ T cells in the individual mice tested are remarkably similar, suggesting that intermouse variability in LLO_91–99-specific TCRs cannot be attributed to general differences in the frequencies of different TCR Vβ populations. TCR Vβ stainings of naive, BALB/c splenocytes demonstrated very similar overall TCR Vβ repertoires (data not shown). Thus, there is no detectable skewing in the overall TCR Vβ repertoire during primary responses to L. monocytogenes.

Taken together, our data show that LLO_91–99-specific primary effector and memory T cell populations have diverse TCR Vβ repertoires. The prevalence of TCR Vβ chains among LLO_91–99-specific T cells and the general population of CD8⁺ T cells is remarkably similar.

As mentioned above, the mouse to mouse variability in the TCR Vβ profiles does not permit precise correlations between memory and primary effector TCR repertoires. To address this issue, we decided to determine the TCR Vβ repertoire of LLO_91–99-specific effector and memory T cells in the same mouse. Therefore, we expanded LLO_91–99-specific T cell lines from blood samples taken at day 7 during the primary response by short term in vitro peptide stimulation. The TCR Vβ usage of LLO_91–99-specific T cell lines was determined by double staining with LLO_91–99 tetramers and TCR Vβ mAbs, and compared with the TCR Vβ profiles of the LLO_91–99-specific memory T cell population 5 wk after primary infection in the spleen of the same mouse. Fig. 6 shows TCR Vβ profiles of LLO_91–99-specific effector and memory T cell populations determined in individual mice. The TCR Vβ 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., Vβ 7, 9 and 10). T cell populations that are distinct for individual mice, such as the unusual TCR Vβ profile in mouse 386, with an unusually large TCR Vβ9⁺ 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.

We used the same approach to determine the TCR Vβ repertoire of LLO_91–99-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 Vβ repertoires of LLO_91–99-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 Vβ repertoire in the recall population, suggesting a focusing on certain TCR Vβ segments. In particular, less prevalent subpopulations decreased in frequency while the frequency of dominant Vβ 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 Vβ8⁺ subpopulation (in mouse 391, 70% of all recall effector T cells are positive for the anti–TCR Vβ8 panantibody), in mouse 393, TCR Vβ2 becomes the predominant subpopulation. Focusing of the recall TCR repertoire on other TCR Vβ segments was found for Vβ10 in mouse 390 and for Vβ4 in mouse 392. Mouse 390 showed an unusually high TCR Vβ7 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 Vβ7 subpopulation in mouse 390 disappeared almost completely. Taken together, the recall TCR Vβ profiles appear to be more restricted when compared with the corresponding primary effector T cell repertoires.

Our studies comparing the TCR repertoires of effector and memory T cell populations responding to the immunodominant Listeria epitope LLO_91–99 show the following: (a) the memory TCR Vβ repertoire is similar to the repertoire of primary effector cells; and (b) focusing of the repertoire on certain TCR Vβ 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 Vβ 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.

Although there is some mouse to mouse variability in the TCR repertoires of T cell populations selected for the immunodominant Listeria epitope LLO_91–99, 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 Vβ 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 Vβ staining allows us to characterize the majority of T cells within the antigen-specific T cell population (usually >90% of LLO_91–99-specific T cell populations are covered by staining with the 14 different TCR Vβ 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 Vβ segments, indicating that TCR Vβ usage directly correlates with the overall complexity of a given T cell population (29). The complexity of primary effector T cells specific for LLO_91–99 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 LLO_91–99, which may consist of TCRs with different affinities for H2-K^d– LLO_91–99 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 Vβ repertoire of LLO_91–99-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 LLO_91–99-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.

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).

β₂m

β₂ microglobulin

BirA

biotin operon repressor protein A

LLO

listeriolysin O

SB

staining buffer