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

Philippe Bousso, Fabrice Lemaître, Dhafer Laouini, Jean Kanellopoulos and Philippe Kourilsky

Unité de Biologie Moléculaire du Gène, INSERM U277, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France

Correspondence to: P. Bousso


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
While numerous studies have analyzed the shaping of T cell repertoires by self or foreign peptides, little is known on the influence of commensal self peptides derived from the intestinal flora (IF). Here, we have analyzed naive and immune repertoires in mice devoid of IF [germ-free (GF) mice]. First, by means of an extensive CDR3ß sequencing strategy, we show that the naive peripheral CD8 T cell repertoire does not exhibit a major imprint of IF antigens. Second, using MHC–peptide tetramers, CDR3ß length distribution analyses and TCR sequencing, we show that cytotoxic T lymphocyte (CTL) responses specific for two distinct epitopes are quasi-identical in normal and GF mice. Our findings indicate that, in general, peptides derived from the intestinal microflora have little if any influence on CTL responses in the mouse.

Keywords: cytotoxic T lymphocyte, germ-free mice, intestinal flora, MHC–peptide tetramers, TCR


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
At various stages of their life, the fate of {alpha}ß T lymphocytes is dependent upon interactions with MHC molecules present at the surface of antigen-presenting cells (APC). These interactions occur between the TCR and peptides complexed with self MHC molecules. Self peptides are responsible for the shaping of the T cell repertoire during thymic selection. In addition, it is now clear that the survival of mature T cells requires continuous interactions with MHC molecules in the periphery (1,2). In addition to self peptides, the environment provides a large source of antigens potentially visible to the immune system including food antigens and peptides derived from the intestinal flora (IF). Modulations of systemic immune T cell responses due to soluble or bacterial antigens in the gut have been widely studied. In mice, it has been shown that oral administration of a single dose of antigen induces a rapid activation of antigen-specific T cells in the spleen and that multiple ingestions decrease their number in the periphery (3). Oral administration of antigen does not always result in peripheral tolerance and it has been reported that it can even prime cytotoxic T lymphocyte (CTL) responses under certain conditions (4). Systemic tolerance is not restricted to soluble antigen but has also been observed toward bacterial antigen in rats, mice and humans (58). While peptides derived from the IF can modify the reactivity of the peripheral T cell repertoire, the extent to which it is affected is largely unknown. Due to the large degeneracy of TCR recognition, it is conceivable that IF peptides modulate the response to closely related antigens. Indeed, the numerous aerobic and anaerobic bacteria homed in the intestine might provide multiple mimicry peptides capable of altering particular CTL responses. For example, the work of Loftus et al. has shown that at least two peptides derived from Escherichia coli were cross-recognized by melanoma reactive CTL (9). Using anti-TCRV region antibodies, it has been previously reported that naive germ-free (GF) mice display a TCR Vß usage similar to that observed in conventional mice (10). Yet, whether the presence of the IF affects the fine composition and the complexity of the peripheral TCR chains repertoire is largely unknown.

In order to determine whether the IF has a strong impact on the shaping of T cell repertoires and immune responses, we analyzed TCRß sequences isolated from naive GF or normal [conventional specific pathogen-free (SPF)] mice. We then thoroughly characterized antigen-specific repertoires selected in response to two CD8 epitopes in mice devoid or not of IF. Interestingly, SPF and GF mice mounted extremely similar responses even at the clonal level. Therefore, in general, the shaping of CD8 T cell repertoire specific for a given antigen is independent of the IF.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice and cell line
GF and SPF BALB/c mice were obtained and raised at the CDTA (Orleans, France). GF animals were bred in overpressure isolators, and the absence of bacteria in mice and tumor cells was systematically verified before immunization. In contrast, a complex flora was detected in SPF animals: culture tests revealed the presence of at least Streptococcus, Staphylococcus (lentus, xylosus, scuiri), E. coli and Enterobacter cloacae (CDTA, pers. commun.). Immunization were performed by i.p injection of 107 P815-CW3 tumor cells (11). All mice were maintained in isolators until being sacrificed.

Antibodies and flow cytometry
FITC-conjugated anti-BV10 and CyChrome-conjugated anti-CD8 mAb were purchased from PharMingen (San Diego, CA). Biotinylated anti-CD4 was purchased from Caltag (South San Francisco, CA). Phycoerythrin-labeled Kd-CW3 tetrameric complexes were prepared as previously described (12). Cell samples were incubated 1 h with Kd-CW3 tetramers, washed and incubated with the indicated antibodies. Flow cytometry was performed on a FACScan (Becton Dickinson, Mountain View, CA). Data were analyzed using CellQuest software.

Immunoscope analyses
Immunoscope analysis, and BC- and BV-specific primers have been described elsewhere (13). RNA was extracted from indicated cell samples using the Trizol reagent (Gibco/BRL, Gaithersburg, MD) and cDNA was synthesized using MMLV reverse transcriptase (Gibco/BRL).

TCR sequencing of the naive repertoire
Analysis of the diversity of the naive CD8 T cell repertoire was performed essentially as previously described (12). Briefly, splenocytes from either SPF or GF naive mice were prepared as single-cell suspension and depleted of CD4+ cells using streptavidin beads (Dynal, Oslo, Norway). cDNA was prepared and amplified using BV10- and BJ1.2-specific primers. TCRß rearrangements bearing a 6 amino acid long CDR3ß were isolated on a 8% polyacrylamide–7 M urea gel and submitted to a second PCR (20 cycles) using the same primers. The final products were cloned using the Topo TA cloning kit (Invitrogen, Leek, The Netherlands). TCR sequencing was performed using the M13(-20) primer and the ABI PRISM BigDye terminator cycle sequencing ready reaction kit (Perkin-Elmer, Foster City, CA). Samples were run on a 377 DNA sequencer (Perkin-Elmer).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Flow cytometry analysis of thymocytes and splenocytes of GF animals
All mice referred to as GF were tested for the absence of bacteria and sacrificed immediately at arrival. In good agreement with previous reports (10), the serum IgG of these animals were dramatically reduced (50-fold) as compared to SPF animals (data not shown). In all experiments, we also used BALB/c SPF mice which display a complex intestinal microflora as detailed in Methods. Flow cytometry analysis of thymocytes and splenocytes revealed a similar percentage of CD4+ and CD8+ cells in GF and SPF mice (Fig. 1Go), confirming previous studies (10). Absolute numbers of thymocytes were in the normal range in GF mice, whereas a 40% reduction in splenic cells (average 6x107 cells) was observed as compared to age-matched SPF mice (average 108 cells). Taken together, these results ascertain that the SPF and the GF animals used in this study differ by the presence of a complex IF and that the phenotype of the GF animals used here is in good agreement with what has been previously reported.



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Fig. 1. Flow cytometry analysis of thymocytes and splenocytes of SPF and GF mice. Thymocytes or splenocytes of SPF or GF mice were prepared as single-cell suspensions and stained with mAb against CD4 and CD8. Numbers shown are the percentage of cells that fall into indicated quadrant.

 
Complexity of the naive CD8 T cell repertoire in GF animals
In order to determine whether peptides derived from the IF affect the diversity of the naive peripheral repertoire, we examined the complexity of a particular CD8 T cell subset. We have recently described a novel approach to examine the composition of naive repertoires at the amino acid sequence level (12,14). The strategy consists of isolating and sequencing TCRß rearrangements sharing a particular BV–BJ combination and a given CDR3ß size. Using this approach, it is possible to estimate TCRß diversity by counting the number of distinct CDR3ß sequences within the defined subset. We focused our analysis on CD8 T splenocytes displaying the BV10–BJ1.2 combination and bearing a 6 amino acid long CDR3ß, and examined the diversity and the amino acid usage in SPF and GF animals. A total of 252 TCRß sequences were analyzed. Among 126 sequences performed in one SPF naive animal, we identified 81 distinct amino acid sequences. Seventy-three distinct sequences were identified in the GF mouse (among of 126 sequences performed). About 30% of the amino acid sequences were shared by the SPF and the GF mouse. This percentage is similar to that we found previously when analyzing the T cell repertoire of two syngeneic SPF animals (12). To determine whether the IF introduces large biases in the naive peripheral CD8 repertoire, we compared the amino acid usage within the CDR3ß observed in the SPF or the GF mouse. Figure 2Go shows, for each position of the CDR3ß (16), the amino acid usage calculated from the sequences obtained in the SPF or the GF mouse. Few different amino acids were used in positions 1, 4, 5 and 6, which are mostly germline encoded. In contrast, amino acid usage was highly diverse in positions 2 and 3, which are encoded by N additions and/or by the Dß1 segment. At each CDR3ß position, amino acid usage was very similar between SPF and GF mice (Fig. 2Go). These results indicate that even at a high level of resolution, the naive peripheral CD8 T cell repertoire does not appear largely influenced by the IF with regard to its complexity and to amino acid usage within the junctional region.



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Fig. 2. Similar diversity and amino acid usage in the naive peripheral T cell repertoire of SPF and GF mice. TCRß rearrangements corresponding to CD8 splenic T cells displaying BV10–BJ1.2 combination and a 6 amino acid long CDR3ß were isolated and sequenced (as described in Methods). Based on the 126 sequences performed in both SPF and GF animals, we calculated the amino acid occurrence for each CDR3ß position.

 
Analysis of antigen-selected repertoires in GF animals
We then investigated whether peptides derived from the resident flora had any influence on the shaping of T cell responses. For that purpose, we characterized antigen-selected T cells in response to two CTL epitopes in SPF or GF animals. It has been shown previously that DBA/2 mice, when injected i.p with P815-CW3 tumor cells, mount a strong CD8 T cell response against the Kd epitope 170–179 derived from the HLA-Cw3 molecule (15). This response is highly restricted in terms of TCR usage: all expanded T cell clones display BV10 and essentially BJ1.2 usage, a 6 amino acid long CDR3ß, a serine and a glycine at positions 1 and 3 of the CDR3ß (16). In preliminary experiments, we observed a similar response in BALB/c mice, although the number of CW3-specific T cells was lower compared to DBA/2 mice (P. Bousso, unpublished). We have shown recently that Kd-CW3 tetramers allow the identification of all CW3-specific T cells (12). Therefore, 2 weeks after i.p injection of P815-CW3 tumor cells, splenocytes were triple stained with Kd-CW3 tetramers, anti-CD8 and anti-BV10 antibodies. Figure 3Go shows a representative staining obtained on a naive mouse, an immune SPF and an immune GF mice. Both SPF and GF mice mounted a response against the CW3 epitope as revealed by a significant staining of CD8 T cells with Kd-CW3 tetramers (usually from 5 to 15%). In addition, the vast majority (if not all) of CW3-specific T cells used the BV10 segment. Therefore, GF mice can respond against the CW3 epitope and display a BV restriction similar to that observed in conventional and SPF mice.



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Fig. 3. The response of GF mice against the CW3 epitope is restricted to BV10+ cells. BALB/c mice (SPF or GF) were immunized i.p with 107 P815-CW3 tumor cells. Two weeks later, splenocytes were stained with Kd-CW3 tetramers, and anti-CD8 and anti-BV10 antibodies. Profiles are gated on CD8+ cells and show a representative staining of a naive BALB/c (left), an immune SPF mouse (middle) and an immune GF mouse (right).

 
In order to refine our repertoire analysis, we examined the CDR3ß size distribution (Immunoscope) of immune splenocytes isolated from two SPF or GF animals. The upper panels of Fig. 4Go show BV10–BC size distribution profiles. The Gaussian-like profile obtained in naive animals is altered in immune SPF mice which display a clonal expansion corresponding to T cells bearing a 6 amino acid long CDR3ß. The same clonal expansions are detected in immune GF mice indicating a similar responding T cell repertoire. These expansions correspond in both cases at least in part of T cells bearing the BJ1.2 segment since a clonal expansion corresponding to 6 amino acid long CDR3ß is detected on BV10–BJ1.2 profiles (Fig. 4Go, middle panels).



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Fig. 4. The presence of the IF does not modify CW3- and P1E-specific T cell repertoires. BALB/c mice (SPF or GF) were immunized i.p with P815-CW3. Two weeks later, splenocytes were depleted of CD4+ cells and cDNA was prepared. Immunoscope analyses were performed on these samples. The CW3-specific response was monitored by analyzing BV10–BC (upper panels) and BV10–BJ1.2 (middle panels) profiles. The P1E-specific response was analyzed on BV1–BJ2.5 profiles (lower panels). Clonal expansions corresponding to CW3- or P1E-specific T cells are indicated by arrows.

 
Finally, using the same approach, we examined T cell responses against another antigen derived from P815-CW3 cells. It has been shown previously that DBA/2 mice, when immunized with P815 tumor cells, display clonal expansions corresponding to T cells bearing the BV1–BJ2.5 combination with a 9 amino acid long CDR3ß. In addition, these cells correspond to CTL specific for the P1E antigen (17). Accordingly, we examined the P1E-specific response in SPF or GF animals immunized with the P815-CW3 tumor cell line. As shown in Fig. 4Go(lower panels), GF animals do respond against the P1E epitope as detected by a specific clonal expansion. In summary, GF mice were able to mount responses against both epitopes. Most interestingly, similar features were observed in the selected T cells whether mice were lacking or not the resident flora.

Analysis of the TCRß chains specific for the CW3 and P1E epitopes in immune SPF or GF mice
To characterize the clonal composition of antigen-selected repertoires in GF animals, we sequenced the TCRß of either P1E- or CW3-specific T cells. From the data presented in Fig. 4Go, it can be noted that, in immune animals, the BV10–BJ1.2 (respectively BV1–BJ2.5) profile is largely dominated by the peak corresponding to 6 (respectively 9) amino acid long CDR3. This observation indicates that almost all T cells displaying BV10–BJ1.2 (respectively BV1–BJ2.5) combination are CW3- (respectively P1E-) specific. For that reason, we cloned and sequenced the PCR products corresponding to these combinations. Shown in Table 1Go are the TCRß chains corresponding to the CW3-specific T cells and derived from one immune SPF or GF mouse. All these sequences bear the SXG motif within the CDR3ß and are identical or highly homologous to those of previously reported CTL clones derived from DBA/2 mice (16,18). TCRß chains corresponding to P1E-specific T cells (bearing BV1 and BJ2.5 segments with a 9 amino acid long CDR3ß) derived from either immune SPF or GF animals are shown in Table 2Go and were also similar to the previously characterized public response (17). Taken together, our results demonstrate that antigen-selected repertoires characterized for two CTL responses are largely independent of the IF.


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Table 1. CDR3ß sequences of CW3-specific T cells
 

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Table 2. CDR3ß sequences of P1E-specific T cells
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
This study was undertaken to investigate whether the large source of antigens derived from the IF has any impact on the shaping of peripheral CTL responses. Both naive and immune repertoires were analyzed in SPF or GF BALB/c. Our results demonstrate that, by and large, CD8 T cell repertoires are mostly independent of the resident flora.

The resident flora colonizes the intestinal tract shortly after birth. Concomitantly, T lymphocytes originating from the thymus populate the Peyer's patches (19), which are lymphoid structures endowed with the ability to process luminal antigens (20). IF peptides may affect immunological responses by at least two mechanisms. On the one hand, IF peptides could create holes in the T cell repertoires by deleting numerous T lymphocytes. Alternatively, cross-recognition with related (mimicry) peptides derived from the IF could have pre-selected T cell clones with particular TCR rearrangements that would be favored upon antigenic stimulation.

We tested the first hypothesis by carefully comparing the complexity of the CD8 repertoire in GF or SPF mice. Previous works have demonstrated that individuals are tolerant to autologous (but not to heterologous) IF (7). This tolerance is both local and systemic, and could therefore contribute significantly to the shaping of T cell repertoires by altering T cell responses that could cross-react with an intestinal antigen. Our analysis of the naive CD8 repertoire allowed us to compare its diversity in GF or SPF animals. The comparison of the number of distinct TCRß sequences (in a defined T cell subset) revealed a similar TCR diversity in SPF and GF animals. Therefore, the presence of the IF does not induce a significant decrease in TCR diversity. In addition, amino acid usage within the CDR3ß was extremely similar in SPF and GF mice. While other studies have documented biases in the TCR repertoire due to self peptides in the thymus (21), we demonstrate here that, at a high level of resolution, the peripheral CD8 T cell repertoire does not bear a major imprint of the IF. However, it remained possible that changes in T cell repertoires could occur without being detected by TCRß sequencing. Therefore, we characterized antigen-selected repertoires in response to two different CTL epitopes. Interestingly, we found that mice mounted similar responses whether or not they lack the IF. Close similarities of the T cell repertoires were observed at three levels of resolution: (i) identical BV restriction was observed after co-staining with MHC–peptide tetramers and anti–BV10 antibody, (ii) similar expansions were detected by Immunoscope analyses and (iii) similar TCRß rearrangements were revealed by TCR sequencing. Thus, IF peptides do not modify these two oligoclonal CTL responses. Although it remains possible that CTL cell responses displaying a more heterogeneous TCR repertoire could be affected by the IF, our results overall suggest that IF peptides have little (if any) influence on the complexity and the reactivity of the CTL precursors.

The other hypothesis would be that, among the T cell specific for a given antigen, a fraction has already reacted with IF peptides and would be favored upon the antigenic stimulation. Such a phenomenon could explain why many antigen-specific responses (including response against the CW3 epitope) are associated with a very limited TCR usage. (22,23). In such a case, one would expect the expansion of a much more diverse T cell repertoire in response to the CW3 (or the P1E) epitope in GF mice. Clearly, our results rule out this hypothesis, since similar restriction in TCR usage was observed in GF and SPF mice in response to the CW3 and P1E epitopes. Thus, in general, IF peptides do not narrow CD8+ antigen-specific repertoires.

We focused our analysis on CTL response and CD8 repertoires but it should be noted that the T cell response against the CW3 epitope is CD4 dependent (24). Therefore, in this system, the CD4 help is provided also under GF conditions. While it remains possible that some CD8 antigen-specific responses may be affected by the IF, our results show that it is not a general rule. Thus, in contrast to self peptides, peptides derived from the IF do not contribute significantly to the shaping of CTL repertoires.


    Acknowledgments
 
The authors wish to thank A. Bandeira for helpful discussion, and D. Guy-Grand and P. Arstila for critical review of the manuscript. This work was supported by grants from l'Association pour la Recherche contre le Cancer, La Ligue Nationale contre le Cancer (subvention: axe immunologie des tumeurs) and from the European Community. P. B. was a recipient of a fellowship from the Délégation Générale de l'Armement.


    Abbreviations
 
APC antigen-presenting cell
CTL cytotoxic T lymphocyte
GF germ-free
IF intestinal flora
SPF conventional specific-pathogen free

    Notes
 
Transmitting editor: A. Fischer

Received 16 August 1999, accepted 30 November 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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