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International Immunology Advance Access originally published online on January 13, 2006
International Immunology 2006 18(2):363-374; doi:10.1093/intimm/dxh376
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Mycobacterium tuberculosis in the adjuvant modulates the balance of Th immune response to self-antigen of the CNS without influencing a "core" repertoire of specific T cells

Chiara Nicolò1, Gabriele Di Sante1, Massimiliano Orsini2, Simona Rolla1,3, Sandra Columba-Cabezas4, Vincenzo Romano Spica2,5, Gualtiero Ricciardi2, Bosco Man Chu Chan6 and Francesco Ria1

1 Institute of General Pathology and 2 Institute of Hygiene, Catholic University, L.go F. Vito 1, 00168 Rome, Italy
3 Department of Clinical and Biological Sciences, Ospedale S. L. Gonzaga, University of Turin, 10043 Orbassano, Torino, Italy
4 Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy
5 University of Movement Sciences (IUSM), P.zza L. De Bosis 6, 00194 Rome Italy
6 Biotherapeutics Research Group, Robarts Research Institute and Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada

Correspondence to: F. Ria; E-mail: fria{at}rm.unicatt.it


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
In the present study, we use modified CDR3 beta-chain spectratyping (immunoscope) to dissect the effect of Mycobacterium tuberculosis (MT)-derived proteins on individual PLP139-151-specific cells in the SJL mouse strain. In this model, the immunoscope technique allows the characterization of a public TCR that involves rearrangement of Vbeta10 and Jbeta1.1 and a semi-private TCR characterized by rearrangement of Vbeta4 and Jbeta1.6. Both rearrangements are specific for PLP139-151 and sequences of the CDR3 region of the two beta-chains show a conserved motif for the public rearrangement and related but more variable sequences for the semi-private rearrangement. MT-derived proteins promote increase of IFN-gamma-secreting cells. However, we observe that the presence and amount of MT used during immunization have no effect on the frequency of usage, polarization and in vivo expansion of cells carrying the studied rearrangements. Rather, the strong Th1-promoting effect of adjuvant is possibly due to recruitment toward Th1 of a wider spectrum of TCR repertoires. Therefore, instead of having a comprehensive effect on the entire repertoire, MT modulates the immune response by affecting a subset of antigen-specific T cells whose polarization can be adapted to the environment. This step establishes the final balance between Th1 and Th2 and may be essential for the enhancement or protection of disease.

Keywords: EAE, Mycobacterium tuberculosis, TCR, Th1/Th2 cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Antigen-specific T helper (Th) cells are functionally defined as type 1 and type 2 on the basis of their cytokine secretion (1). Type-1 T cells produce cytokines IFN-gamma, tumor necrosis factor-alpha and IL-2 that mediate the clearance of viruses and intracellular pathogens, promote inflammation and contribute to the development of autoimmune disorders. In comparison, type-2 T cells produce IL-4, IL-5 and IL-10 that regulate isotype switching in the humoral response to parasitic infection and under pathologic condition, the development of allergy. Some T cells display a regulatory phenotype (2) that can be characterized by the secretion of IL-10 or transforming growth factor beta (3). This phenotype can be acquired upon antigenic challenge (4, 5) or established during maturation in the thymus (6).

The development of type-1 and type-2 T cells has been attributed to differential activation of T-bet (7), GATA-3 (8, 9) and STAT 4 and 6 molecules (10). The dominance in the development of either T cell type results in a functional skewing and defines the phenotype of an immune response. It has been shown that at least part of the T cell memory repertoire [the so-called "effector memory T" (EMT) cells (11)] remains polarized. The presence of polarized antigen-specific T cells can in turn influence the priming of new naive T cells (12) and thus, perpetuate the dominant phenotype of the immune response. This is consistent with the view that the phenotype of the immune response is stable through lifetime (13).

In multiple sclerosis (MS), the selection of the myelin-specific TCR repertoire is necessary and occurs early, but it alone is insufficient to cause disease (14). The development of MS requires priming in an appropriate "environment" such that the self-reactive TCR repertoire is polarized and has homing properties for the target tissue. This need of an appropriate environment for the priming of self-reactive T cells is highlighted in the induction of experimental autoimmune encephalitis (EAE), a model system for MS. Immunization of susceptible strains with encephalitogenic proteins or peptides induces a relapsing remitting form of EAE (a model for the same form of MS) when the antigen is emulsified in incomplete Freund's adjuvant (IFA) supplemented with killed and heat-dried Mycobacterium tuberculosis (MT). The induction of EAE is directly related to the amount of MT present during the antigenic challenge. In fact, antigenic challenge in the absence of MT does not only fail to induce EAE but it also confers protection from a later encephalitogenic challenge (15).

MT in Freund's adjuvant shifts T cell activation toward type-1 response [see e.g. (16, 17)]. MT products bind to Toll-like receptors 2 and 4 (18–21), thus activating dendritic cell (DC) to prime T cells and to produce cytokines that promote Th1 response (22, 23). It has been proposed that the polarization toward a type-1 response can be enhanced in two ways, the presence of a pro-inflammatory environment and the selection of T cells that exhibit high affinity for the peptide–MHC complex. In the absence of a pro-inflammatory environment, the recruited repertoire can be polarized to a Th2 or remain at the Th0 phenotype. In vivo expansion of a restricted TCR repertoire can be the result of a decrease in the presentation due to the competition between peptides derived from the antigen and MT (24), as well as between the antigen-reactive and MT-specific naive T cells (25, 26). Together, the presence of a pro-inflammatory environment and competition in antigen presentation favor the selection of a TCR repertoire with high affinity (26, 27).

In the present study, we have used the modified CDR3 beta-chain spectratyping (28) to assess how the presence of MT during immunization impacts the usage of two PLP139-151-specific TCR rearrangements in SJL mice. We observed that the presence of MT does not affect the frequency of usage of these repertoires. A provision of a more stringent Th1- or Th2-promoting environment does not modify the polarization of clonotypic cells. Also, MT promotes Th1 polarization by recruitment of a wider spectrum of TCR repertoires that evolve to a Th1 phenotype than that seen in immunization without MT. Thus, some naive T cells are insensitive to the environmental stimuli and constantly differentiate toward the same phenotype. However, the same stimuli can fine-tune differentiation of other naive T cells. We propose that the differentiation properties of the repertoire expanded by MT play an important role in determining the net Th1/Th2 balance in the immune response, which, in turn, could enhance the progression or confer protection from disease development.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice and antigens
Female SJL mice (2 months old, Charles River, Calco, Italy) were used in the experiments reported. PLP139-151 [Ser 140 (29)] was purchased from PRIMM (Milan, Italy) and was >95% pure, as determined by HPLC and mass spectroscopy.

Immunizations and T cell proliferation
Mice were immunized subcutaneously with 10–50 µg per mouse of PLP139-151 in PBS, emulsified 1 : 1 with IFA or CFA (CFA is IFA containing 1 mg ml–1 of killed and heat-dried MT H37RA) or in enriched CFA (IFA containing 4 mg ml–1 of killed and heat-dried MT H37RA) (Sigma–Aldrich, St Louis, MO, USA). Draining lymph nodes (LNs) were harvested 10 days after immunization and LN cells were seeded in 96-well plates (Costar Corp., Cambridge, MA, USA) at 5 x 105 cells per well in the presence of graded concentration of antigen. In all experiments, the culture medium was RPMI 1640 (GIBCO BRL Life Technologies, Basel, Switzerland), supplemented with 2 mM L-glutamine, 50 µM 2-mercaptoethanol, 50 µg ml–1 gentamicin (Sigma–Aldrich) and 10% FCS (GIBCO BRL Life Technologies). When indicated, 72 h later, antigen-specific T cell proliferation was assessed by 3[H]-thymidine incorporation.

TCR repertoire analysis
Repertoire analysis was performed using a modification of a described protocol (13). LN cells were cultured in the presence or absence of PLP139-151 for 3 days in RPMI 1640 medium (Sigma–Aldrich) supplemented as above described. Total RNA was isolated from cell suspensions using RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instruction. cDNA was synthesized using an oligo-dT primer (dT15) (GIBCO BRL Life Technologies). From each cDNA, PCRs were then performed. Sequences of Vbeta-, Cbeta- and Jbeta-specific primers have been deduced from IMGT database (http://imgt.cines.fr) according to the published sequences (28), following the nomenclature of Arden and colleagues (30). In particular, we used a Vbeta10 forward primer 5'-ATCAAGTCTGTAGAGCCGGAGGA-3', or a Vbeta4 forward primer 5'-GCCTCAAGTCGCTTCCAACCTC-3' and a common Cbeta reverse primer 5'-CACTGATGTTCTGTGTGACA-3'. Using 2 µl of this product as a template, run-off reactions were performed with a single internal fluorescent primer for each Jbeta tested. These products were then denatured in formamide and analyzed on an Applied Biosystem 3100 Prism using GeneScan 2.0 software (Applied Biosystem, Foster City, CA, USA). Results are also reported as rate stimulation index (RSI = normalized peak area obtained from cells stimulated with antigen/normalized peak area of non-stimulated cells).

Staining and enrichment of IFN-{gamma}-, IL-4- and IL-10-secreting T cell
PLP139-151-specific T cells secreting IFN-gamma, IL-4 and IL-10 were stained and enriched from LNs of SJL mice (immunized as described above) using MACS® secretion kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instruction, following the protocols for enrichment of low-frequency secreting cells. Briefly, 1 x 107 to 3 x 107 cells obtained from draining LNs were stimulated in the absence (background) or in the presence of 50 µg ml–1 of PLP139-151, in a six-well plate at a concentration of 0.5 x 107 cells ml–1. Three hours later, cells were harvested and submitted to the staining procedure for each cytokine. The enrichment for cytokine-secreting cells was checked by flow cytometry (Coulter Epics FACS, equipped with Lysis software) (Beckman Coulter Inc., Fullerton, CA, USA) analysis. In order to evaluate correctly the number of antigen-specific cells, we examined by FACS 5 x 105 cells both in the background and in positive samples. Total, negatively selected and positively selected cells were collected and prepared for mRNA isolation. In order to prevent uncontrolled loss of mRNA due to scarcity of cells in the positively selected fraction (usually ~104 total cells were recovered in the cytokine-positive fraction), 106 alpha–beta– BW cells were added to the positively selected cells before proceeding with mRNA isolation for the TCR repertoire analysis.

CDR3 sequencing
cDNAs were obtained from antigen-stimulated LN cells or from immunoaffinity-selected cells as described above. Two microliters of each sample were submitted to a first PCR using the mentioned above Vbeta10- or Vbeta4-specific forward primers and the common Cbeta-specific reverse primer. A second nested PCR was then performed using 2 µl of the product of the former reaction as template, the same Vbeta-specific primer and Jbeta1.1- or Jbeta1.6-specific reverse primers. PCR fragments were then cloned by using TOPO TA Cloning® kit (Invitrogen, Carlsbad, CA, USA) according to manufacturer instructions. Transformed Escherichia coli were grown in 5 ml LB medium supplemented with ampicillin, and plasmids were purified by Qiaprep Miniprep columns (Qiagen GmbH), and checked for the presence of the expected inserts by PCR amplification using Vbeta–Jbeta paired primers. Samples that scored positive for the insert were sequenced by M-Medical (Pomezia, Italy) using a M13 forward primer. DNA sequence was translated into protein sequence through the ExPASy Proteomics Server (http://au.expasy.org/).

Modeling of CDR3-beta region carrying the Vbeta10–Jbeta1.1 (97 bp) rearrangement
CDR3-beta chains sequences characterised by PGT or SGS (Fig. 3A and B) NDN region were modeled into a .pdb file using the Geno3D modeling service (http://geno3d-pbil.ibcp.fr) (31), and analyzed using the Swiss-Pdb Viewer 3.7 software. The structure published in (32) was used as template.


Figure 3
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  		Fig. 3. Modeling of CDR3-beta region carrying the Vbeta10–Jbeta1.1 (97 bp) rearrangement. CDR3-beta chains sequences characterised by SGS (A) or PGT (B) NDN region were modeled into a .pdb file using the Geno3D modeling service, and analyzed using the Swiss-Pdb Viewer 3.7 software, as described in Methods. Blue (A) and yellow (B) indicate the side chains of Ser and Pro at position 1 of the NDN-encoded region.

 
Assessment of the number of clonotypic cells
In order to assess the number of clonotypic cells among the positively selected IFN-gamma+, a volume of the fraction collected after immunoaffinity magnetic sorting containing 100 antigen-specific CD4+ IFN-gamma-secreting cells was aliquoted at a final concentration of three of these cells per vial. RNA was prepared as described above and cDNA was synthesized. Samples were then submitted to immunoscope analysis. The number of samples positive for the expected clonotypic peak divided by the total number of cells tested (i.e. 100) expresses the frequency of T cells bearing the Vbeta10–Jbeta1.1 rearrangement. Finally, multiplication of this value by the number of CD4+ IFN-gamma-secreting cells per 106 LN cells gives an estimated number of antigen-specific Vbeta10–Jbeta1.1 (97bp)+, CD4+ and IFN-gamma-secreting cells per 106 total LN cells.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
PLP139-151-specific TCR repertoire comprises public and semi-private rearrangements
According to immunoscope analysis of other antigen-specific immune responses (28), Vbeta CDR3-length fragment analysis of individual response to PLP139-151 in SJL strain yields three distinct types of distribution of Vbeta–Jbeta fragments length, as shown in Fig. 1(A). The large majority of recombinations (~75%) maintains a Gaussian distribution, as for instances the reported Vbeta2–Jbeta 2.5. Such Gaussian distribution is perturbed in a second group of recombinations, although not in an antigen-dependent fashion (see, e.g. the spectra obtained for Vbeta15–Jbeta 2.1). In this case, the antigen-dependent RSI of a candidate peak is <2. In a third group of recombinations (exemplified by rearrangement Vbeta 19–Jbeta 2.7 in Fig. 1A) however, the RSI is >2. Therefore, we consider that perturbation of the Gaussian depends on the presence of the peptide antigen during in vitro culture in this group of recombinations (~20% of total spectra examined). When a complete analysis of CDR3 length distribution is performed in individual mice, several Vbeta–Jbeta rearrangements appear expanded in vitro in this antigen-driven manner (Fig. 1B). As shown in Fig. 1(B), this analysis confirms that the response to PLP139-151 in SJL mice is characterized by a spread usage of TCRs (33).


Figure 1
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  		Fig. 1. TCR repertoire usage in SJL mice in the immune response to PLP139-151. SJL mice were immunized subcutaneously with 50 µg per mouse PLP139-151 emulsified in CFA. After 10 days, cells from draining LNs were collected and cultured at 5 x 106 cells ml–1 in the presence or absence of 20 µg ml–1 PLP139-151. After 3 days, cells from individual mice were harvested and modified CDR3 beta-chain spectratyping was performed as described in Methods. (A) Representative Vbeta–Jbeta CDR3 length spectra of T cells obtained from one representative mouse for three rearrangements, in the absence and in the presence of antigenic peptide. The peaks that disturb Gaussian distribution of CDR3 length (in antigen-dependent or antigen-independent manner) are shaded in gray and the RSI (normalized peak area obtained from cells stimulated with antigen/normalized peak area of non-stimulated cells) are reported. (B) Complete immunoscope analysis of the individual response of a SJL mouse to PLP139-151. Filled squares indicate Vbeta–Jbeta rearrangements showing antigen-driven expansion of one peak.

 
Comparison of TCR usage in response to PLP139-151 among several mice shows that antigen-driven expansions of rearrangements are (to a large extent) private for each mouse. Nevertheless, this comparison also shows that SJL mice immunized with PLP139-151 constantly display the antigen-driven expansion of a TCR carrying a Vbeta10–Jbeta1.1 rearrangement, characterized by a 97-bp length. In addition, half of immunized mice also use a PLP139-151-specific TCR rearrangement, defined by a Vbeta4–Jbeta1.6 recombination resulting in a product of 162 bp length. Results are shown in Fig. 2(A). The two rearrangements differ in their ability to proliferate in vitro in response to PLP139-151 (Fig. 2A). Thus, in seven out of eight cases, T cells bearing the Vbeta10–Jbeta1.1 (97 bp) rearrangement exhibited maximal proliferative expansion at a concentration (0.5 µg ml–1) of PLP139-151 that is less than half-maximal in the standard LN proliferation assay (data not shown). In comparison, a 10-fold increase in the peptide concentration (5–50 µg ml–1) was required for maximal stimulation of T cells bearing Vbeta4–Jbeta1.6 (162 bp) rearrangement.


Figure 2
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  		Fig. 2. SJL mice use a public Vbeta10–Jbeta1.1 rearrangement and a semi-private Vbeta4–Jbeta1.6 rearrangement specific for PLP139-151. (A) Eight SJL mice were immunized with 50 µg PLP139-151 emulsified in CFA as described in the legend to Fig. 1 and CDR3 beta-chain spectratyping was performed after stimulation at the indicated concentrations of antigen. Dose responses for the public Vbeta10–Jbeta1.1 (97 bp) (left panel) and semi-private Vbeta4–Jbeta1.6 (162 bp) (right panel) rearrangements are shown. Results are reported as RSI for each mouse. Dashed line represents the significance threshold (i.e. 2). Closed symbols indicate mice showing significant antigen-driven expansion, while open symbols indicate mice that did not show antigen-driven expansion. (B) SJL mice were immunized with a mixture containing 50 µg PLP139-151 and 100 µg HEL, emulsified in CFA as described in the legend to Fig. 1. CDR3 beta-chain spectratyping for Vbeta10–Jbeta1.1 and Vbeta4–Jbeta1.6 rearrangements was performed after 3 days culture in the absence of antigen or in the presence of 5 µg ml–1 PLP139-151 or of 10 µM HEL. Columns at the bottom of the panel show average and standard deviation of triplicate proliferation (as counts per minute) of LNs cells cultured in the three conditions.

 
Proliferation in response to antigen stimulation can be the result of specific cognate interaction of T cells with antigen-loaded antigen-presenting cell (APC) or of bystander proliferation, linked to IL-2 secretion by specific cells. Therefore, to asses if proliferation of the two rearrangements is peptide specific, we challenged SJL mice with a mixture containing PLP139-151 and hen egg lysozyme (HEL) in CFA. Cells from draining LNs were then cultured for 3 days without antigen, in the presence of PLP139-151 or of HEL, and immunoscope analysis was performed for the public and the semi-private rearrangements. Results are shown in Fig. 2(B). Expansion of the two rearrangements is achieved only when proliferation is driven by PLP139-151 and not when HEL induces similar level of proliferation. These data, together with data obtained in the evaluation of cytokine secretion and by immunization in the absence of MT (see below) indicate that expansion of both T cell rearrangements is due to cognate interaction with APC presenting the PLP139-151 peptide, rather than to IL-2-mediated bystander proliferation.

Though not a direct measurement, the sensitivity to antigen stimulation in vitro has been shown to reflect the antigen avidity of a T cell [see, e.g. (34)]. Thus, taken together, results reported in Fig. 2 suggest that the Vbeta10–Jbeta1.1 (97 bp) cells represent a high antigen avidity repertoire, whereas Vbeta4–Jbeta1.6 (162 bp) behaves as a repertoire characterized by lower avidity.

PLP139-151 has been shown to elicit a class II-restricted T cell response. As a further step to characterize the public and semi-private repertoires, we examined their expression of co-receptor molecules. Thus, LN cells obtained from mice immunized with PLP139-151/CFA were stimulated in vitro with peptide antigen for 3 days. CD4+ and CD4– populations were fractionated by immunoaffinity magnetic sorting. As expected, both rearrangements were segregated with CD4+ cells (data not shown).

Usage of conserved CDR3 sequence characterizes the public repertoire, while the less conserved motif characterizes semi-private repertoire
Public rearrangements are expected to display a conserved amino acid sequence. We therefore examined the possibility that the two PLP139-151-specific rearrangements studied show homologies in their CDR3-beta sequences among mice. Thus, we compared the CDR3 region of the two Vbeta–Jbeta in the antigen-stimulated samples from several individual mice. As a first hint into the possibility that some sequences actually represent the CDR3-beta regions of PLP139-151-specific TCRs, we expected that CDR3 encoded by fragments of the 97 bp (for Vbeta10–Jbeta1.1) and 162 bp (for Vbeta4–Jbeta1.6) should be enriched in conserved sequences among individual mice. In contrast, CDR3 encoded by fragments of different length should present more variable sequences.

Table 1 shows the sequences of 23 CDR3 Vb10–Jbeta1.1 obtained from five individual mice in three independent experiments. The majority of fragments sequenced (15/23) was of 97 bases yielding a NDN region of three amino acid residues (hereafter named position 1, 2 and 3 starting from the first residue after the CASS stretch). CDR3s of this length from all five mice present a Gly residue at position 2, in a total of 12 out of 15 sequences obtained, and a Thr at position 3. However, CDR3 from two mice also shows Ser at this position, that represents a conservative change for Thr. Focusing on the CDR3s that have this G-T/S motif (that is present in all of the five mice), the residue at position 1 is Ser in three mice and Pro in two. One CDR3 also shows a Tyr residue at this position. Although Ser and Pro have distinct biochemical properties, that do not allow to consider Ser to Pro a conservative change, both amino acids are most frequently involved in beta turns of the secondary structure of proteins. In order to examine if the two sequences (SGS and PGT) preserve the tertiary structure of the antigen-recognition site, we modeled the beta-chain using the Geno3D modeling service, as described in Methods. Results (reported in Fig. 3) show that the side chain of residue in position 1 points away from the putative antigen-binding site. This residue appears to be mainly involved in the establishment of a bow that allows formation of a polar surface created by side chains of the two S of CASS (position –1 and –2), of residue in position 3 and by residues N and T belonging to J (+1 and +2). This surface possibly contacts the antigen complex. Thus, position 1 can accommodate either P or S since its side chain would not contact the antigen. Comparison of the modeling of tertiary structure of TCR beta-chains containing the two CDR3 regions is shown in Fig. 3. It is noticeable that any combination between SGS and PGT in this CDR3 creates the same tertiary structure.


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  		Table 1. Sequences of Vß10–Jß1.1 CDR3 public clonotypea

 
We therefore consider as candidate CDR-beta motif for the PLP139-151-specific Vbeta10–Jbeta1.1 (97 bp) rearrangement a sequence S/P-G-T/S. This motif is found in 10 out of the 15 sequences that exhibited the characteristic 97 bp product. In comparison, CDR3s of other lengths use this motif at a frequency of only one out of eight, indicating that it is used almost exclusively by T cells belonging to the public rearrangement. This sequence would play an important role in providing direct interactions with the antigenic complex (position 3) and facilitating other residues (–2, –1, +1 and +2) to gain contact with the antigenic complex. A strong relevance for Gly residues in the CDR3 region of myelin-specific T cell has also been observed in the MBP Ac1-9/B10 Pl model (35).

The sequences of the CDR3 characterizing the semi-private Vbeta4–Jbeta1.6 (162 bp) rearrangement show distinct structural properties. Table 2 reports the CDR3-beta chain residues of 35 TCR Vbeta4–Jbeta1.6 rearrangements in three individual mice from distinct experiments. Residue at position 1 (Gln) is coded by the germ line Vbeta4 (36). Frequency of usage of this amino acid is the same between the recombination of 162 bp and all of the other recombinations. Asp at position 2 represents also a germ line residue and it is used at a frequency similar to that seen in other unrelated recombinations. However, two out of three mice use also Gly in this position, a residue that is not used in this position by any other of the unrelated sequences. The selective antigen-driven pressure on the Vbeta4–Jbeta1.6 162-bp rearrangement was more evident at position 3 showing a preference for residues Pro and Thr that create a bow in the structure while Gly was selectively diminished. Position 4 was occupied either by hydrophobic residues (Phe and Val) or by Gly. Residue at position 5 uses either Tyr or Trp in ten 162 bp clonotypic sequences. Therefore, the semi-private clonotypic cells have a consensus sequence CASS (1)Q-(2)D/G-(3)P/T-(4)F/V/G-(5)Y/W NSP. The bend created by residues in position 3 may play a role in the recognition of PLP139-151.


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  		Table 2. Sequences of Vß4–Jß1.6 CDR3 regions obtained from LNC after 3 days stimulation with PLP139-151a

 
Finally, a homogeneous CDR3 sequence was obtained in one mouse for a 159-bp sequence (PTVY). Since we did not find a corresponding peak that expanded upon stimulation with PLP139-151, we suggest that cells bearing this CDR3 region may belong to a private repertoire that is expanded in vivo in response to MT.

T cells bearing the public TCR co-purify with antigen-specific, IFN-{gamma}-secreting cells
To study the polarization of clonotypic cells, we first enriched LN cells for IFN-gamma-, IL-10- and IL-4-secreting cells and then analyzed the individual populations by CDR3 beta-chain spectratyping. It has to be pointed out that we could not count the antigen-specific IL-4-secreting cells using this method (see below), even after prolonged antigen stimulation (up to 16 h). Nevertheless, we performed the enrichment and carried out the analysis of IL-4-secreting cells. Thus, SJL mice were immunized with PLP139-151 re-suspended in CFA. Eight days later, IFN-gamma+, IL-10+ or IL-4+ enrichment of cells from draining LNs was performed and the presence of Vbeta10–Jbeta1.1 (97 bp) cells was examined in total, positively selected and negatively selected cells. Figure 4 shows the result of a representative experiment out of the four performed. For the positively selected fraction, both IFN-gamma- and IL-10-secreting cells achieved two-log fold enrichment. The peak corresponding to the public Vbeta10–Jbeta1.1 rearrangement co-segregated with the IFN-gamma-producing cells. Of a total of four independent experiments performed, this public rearrangement was never detected among the IL-10- or IL-4-producing cells. Together, our results show that T cells of the public rearrangement Vbeta10–Jbeta1.1 (97 bp) are characterized by high antigen avidity and IFN-{gamma} producing Th1 phenotype.


Figure 4
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  		Fig. 4. Vbeta10–Jbeta1.1 clonotypic cells are enriched among antigen-specific, IFN-gamma-secreting T cells. SJL mice were immunized as described in the legend of Fig. 2. After 8 days, 3 x 107 cells obtained from draining LNs were cultured in the absence or in the presence of 50 µg of the antigen peptide at the concentration of 5 x 106 cells ml–1 in six-wells plates. After 3 h, PLP139-151-specific T cells secreting IFN-gamma and IL-10 were stained and enriched using MACS® secretion kit. The enrichment for cytokine-secreting cells was checked by FACS (Coulter). CDR3 length distribution for the Vbeta10–Jbeta1.1 recombination in total, positively or negatively selected fractions of IL-10- and IFN-gamma-secreting populations are shown. Side by side, the staining for CD4–FITC/IL-10–PE and CD4–FITC/IFN-gamma–PE is reported, for each fraction. The peak corresponding to the public rearrangement (Vbeta10–Jbeta1.1, 97 bp) is indicated by an arrow. The results are from one representative experiment out of four performed.

 
To further confirm that cells polarized toward Th1 actually belong to the public rearrangement, we cloned and sequenced the Vbeta10–Jbeta1.1 rearrangement in the IFN-gamma+ population from three pooled mice challenged with IFA or CFA. Results show that cells enriched for PLP139-151-driven IFN-gamma secretion shared the public Vbeta10–Jbeta1.1 sequence that was previously identified as candidate motif for PLP139-15-specific public TCR rearrangement (Table 3). However, it is notable that position 3 holds more frequently Ser than Thr, opposite to what is observed in the total antigen-stimulated samples (see Table 1), and that we also find Thr in position 1 of the CDR3 (a conservative substitution for Ser). Using the modeling strategy described above, also TGT sequence appears to maintain the same relationship between residues at position –1, –2, 3, +1 and +2 of the CDR3 shown in Fig. 3. While cloning of TCR from total LN cells stimulated in vitro with the antigen results in 10/23 of plasmids containing the canonical sequence (i.e. 40%, Table 1), enrichment for IFN-gamma secretion results in 11 canonical sequences out of 16 plasmids (69%, Table 3). These observations strengthen the hypothesis that the public TCR sequences identified in the antigen-stimulated samples are specific for PLP139-151. Confirming this hypothesis, both SGS and PGT sequences were found in the CNS of SJL mice during acute EAE (C. Nicolò, in preparation). These results also demonstrate that carrying out immunoscope analysis after the purification for Th1 cells yields a more precise picture about the utilization of studied rearrangement.


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  		Table 3. Sequences of Vß10–Jß1.1 CDR3 region obtained from LN cells after selection of IFN-{gamma}+ PLP139-151-specific T cellsa

 
Effect of presence and amount of MT on recruitment and polarization of public and semi-private, PLP139-151-specific TCR repertoires
To assess the role that competition plays in the shaping of the TCR repertoire, we compared the frequency of recruitment for the two PLP139-151-specific TCRs during the response to PLP139-151. Thus, mice were immunized with 50 µg of PLP139-151 using as adjuvant IFA, IFA containing 1 mg ml–1 of MT (CFA) or IFA containing 4 mg ml–1 of MT. As shown in Table 4, the frequency of usage of either rearrangement remained the same in all of the conditions tested. The public rearrangement was being used even in mice challenged in the presence of large amounts of the competing MT. Also, the usage of the semi-private repertoire did not occur more frequently when mice were challenged in the absence of MT. Thus, at a qualitative level, the recruitment of T cell repertoire is not dramatically influenced by the amounts of competing antigenic determinants.


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  		Table 4. Frequency of usage of Vß10–Jß1.1 (97 bp) public and Vß4–Jß1.6 (162 bp) semi-private clonotypic cells in mice immunized with PLP139-151 in the presence of different amounts of MTa

 
We next assessed the effect of MT on the polarization of the public Vbeta10–Jbeta1.1 (97 bp) rearrangement. We analyzed by CDR3 beta-chain spectratyping of LN cells enriched for IFN-gamma, IL-10 and IL-4 secretion, after challenge with PLP139-151 in IFA (n = 3 experiments), CFA (n = 4) or IFA/MT (4 mg ml–1) (n = 3). Thus, IFN-gamma+, IL-10+ or IL-4+ LN cells were stained and sorted. The presence of clonotypic cells was then examined in total, positively selected and negatively selected cells. As summarized in Table 5, independent of the presence or the amounts of MT used, cells carrying the public Vbeta10–Jbeta1.1 rearrangement polarized exclusively toward Th1 by segregating with the IFN-gamma, but not the IL-10- or IL-4-producing cells.


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  		Table 5. Polarization of Vß10–Jß1.1 (97 bp) and Vß4–Jß1.6 (162 bp) clonotypic cells in mice immunized with PLP139-151 in the presence of different amounts of MTa

 
We subsequently examined the cytokine-secreting profile of the T cells carrying the semi-private rearrangement Vbeta4–Jbeta1.6 (162 bp), and tested the ability of adjuvant to modulate its differentiation toward IFN-gamma, IL-10 and IL-4 secretion. In order to account for the low frequency of usage, each experiment was performed using LN cells pooled from a minimum of three mice in mice challenged with IFA (n = 2 independent experiments), CFA (n = 3 independent experiments) and in five individual mice challenged with antigen in IFA and in three individual mice challenged with IFA + 4 mg MT. In all of the experiments, the semi-private rearrangement Vbeta4–Jbeta1.6 (162 bp) showed no co-segregation with IFN-gamma-, IL-10- or IL-4-secreting cells. At present, we cannot formally discriminate between the possibilities that cells carrying this rearrangement secrete other cytokines, or that they represent CMT cells, characterized by the ability to proliferate but fail to secrete effector cytokines. Together, our results show that the presence of MT in the adjuvant during priming fails to impact the polarization of either group of T cells.

The presence of MT increases the number of IFN-{gamma}-secreting CD4+ T cells but does not promote expansion in vivo of Th1 Vbeta10–Jbeta1.1 cells
We next examined if priming in the pro-inflammatory environment triggered by co-injection of antigen with MT promotes a more robust expansion in vivo of the same clonotypic cells that differentiate to Th1 after priming in IFA. To measure the Th1/Th2-modulating effect of MT, we first assessed the number of antigen-specific, cytokine-producing CD4+ cells by cytofluorimeter. LN cells were obtained by pooling cells from groups of three mice immunized with peptide antigen re-suspended in IFA, CFA or in IFA supplemented with 4 mg ml–1 of MT (Fig. 5A). Draining LNs were collected 10 days after immunization and cells were re-stimulated for 3 h in vitro in the presence of PLP139-151. Cells were stained and analyzed by flow cytometry to quantify IFN-gamma+ CD4+ and IL-10+ CD4+ cells. Antigen-specific, cytokine-secreting cells were estimated as the number of CD4+ cytokine+ cells in the sample stimulated by antigen peptide after a background subtraction of the cell number obtained in the absence of the 3 h stimulation in vitro with the peptide.


Figure 5
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  		Fig. 5. MT increases in vivo the number of IFN-{gamma}-secreting CD4+ T cells, by promoting polarization toward Th1 of a wider TCR repertoire. (A) Three mice per group were immunized with 50 µg PLP139-151 emulsified with IFA, CFA or IFA with 4 mg ml–1 of MT. After 10 days, cells from draining LNs were pooled and stimulated in vitro with 50 µg ml–1 PLP139-151. After 3 h of stimulation, cells were stained for the CD4/IFN-gamma (black columns) and CD4/IL-10 (white columns) and checked by FACS (Coulter) analysis. Data are the average + SD of four experiments for IFN-gamma secretion and two experiments for IL-10 secretion. (B) Two groups of three mice were immunized with 50 µg PLP139-151 per mouse emulsified in IFA or CFA and, after 10 days, LN cells were stained and enriched for antigen-driven IFN-gamma production as described above and in the legend of Fig. 3. To establish the number of Th1 cells carrying clonotypic Vbeta10–Jbeta1.1, a total of 100 antigen-specific CD4/IFN-gamma-secreting cells were divided into 33 aliquots at a final concentration of three cells per vial. All the aliquots were then analyzed for the presence of the expected 97-bp peak of the public rearrangement (Vbeta10–Jbeta1.1) by modified CDR3 beta-chain spectratyping. The frequency of T cells bearing the Vbeta10–Jbeta1.1 rearrangement (dashed columns) per 106 LN cells was determined by dividing the number of samples that were positive for the expected clonotypic peak by the total number of cells tested (i.e. 100), and then multiplying this value for the total number of specific CD4/IFN-gamma-secreting cells per 106. Black columns indicate the number of antigen-specific, IFN-gamma-secreting CD4+ cells per 106 total LN cells in each sample.

 
In three independent experiments, mice were challenged by antigen in IFA. The number of antigen-specific, CD4+ IFN-gamma-secreting cells ranged from 100 to 250 per 106 total LN cells. This number of cells was tripled by the presence of 1 mg ml–1 MT during immunization, reaching a number of IFN-gamma-secreting CD4+ cells ranging from 400 to 1000 per 106 total LN cells (four independent experiments). This difference is statistically significant (P = 0.029). Using MT at 4 mg ml–1 during immunization, however, does not further enhance the number of antigen-specific IFN-gamma-secreting CD4+ cells.

In contrast, we observed a decrease of IL-10-secreting T cells during the rise in Th1 cells. Immunization with PLP139-151 in IFA yielded 140–200 antigen-specific CD4+ IL-10-secreting cells per 106 total LN cells. When 1 mg ml–1 of MT was added to the adjuvant, this figure fell to 90 and then to 70 when 4 mg MT ml–1 was used. We could not determine the number of antigen-specific IL-4-secreting cells in three independent experiments by using the described assay, also after longer time period of culture in the presence of peptide antigen.

To establish the number of Th1 cells carrying the public Vbeta10–Jbeta1.1 TCRs, two groups of mice were challenged with antigen in IFA or CFA. PLP139-151-specific IFN-gamma-secreting CD4+ cells were counted and enriched. As described in Methods, a large number of samples each containing on average three of such cells were prepared. After the first round of Vbeta10–Cbeta PCR amplification was performed, samples were then subjected to immunoscope analysis using the Jbeta1.1-specific primer (Fig. 5B). In the LN of mice immunized in the presence of IFA CD4+ IFN-gamma-secreting T cells were found at 200 out of 106 LN cells. This figure increased to ~450 per 106 LN cells when immunization was performed in CFA. However, the numbers of T cells defined by the Vbeta10–Jbeta1.1 (97 bp) rearrangement were 20 and 16 per 106 LN cells for immunization in the presence of IFA and CFA, respectively. While the presence of 1 mg ml–1 MT during immunization increased the number of CD4+ IFN-gamma-secreting T cells by ~2-fold, it had no effect on the number of T cells with the Vbeta10–Jbeta1.1 (97 bp) rearrangement within this group.

Taking together the results reported we show that the presence of MT in the adjuvant does not influence recruitment, polarization and in vivo expansion of studied groups of T cells. Therefore, the effect of MT on the increase of Th1 cells is not due to a more robust expansion in vivo of the repertoires that were in common with that recruited by using IFA; rather, MT possibly increases the number of Th1 cells by inducing differentiation toward Th1 of a wider spectrum of TCR rearrangements. The studied rearrangements will therefore represent a core TCR repertoire that is insensitive (within the tested range) to environment.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In the present study, we use modified CDR3 beta-chain spectratyping to dissect the effect of MT-derived proteins on individual PLP139-151-specific cells. We examined the behavior of a public and a semi-private rearrangement. As expected, MT in the adjuvant strongly promoted the overall polarization toward Th1 of PLP139-151-specific immune response. However, when we assessed polarization at the level of the selected individual rearrangements, we failed to observe a shift in differentiation of either type of cell as a direct effect of MT. The overall polarization of the immune response is most likely achieved by recruitment of a wider TCR repertoire to differentiate into Th1 cells. We therefore conclude that naive T cells specific for PLP139-151 can be divided into two groups. One group is characterized by a stable pre-disposition to differentiate toward a fixed phenotype while the second has differentiation properties that are flexible and amenable to modulation by the environment where priming occurs.

MS and its disease model EAE represent a group of human and experimental pathologies of complex etiology. In both cases, genetic and environmental factors converge to determine the onset, evolution and outcome of the disease (37–39). In this study, MT represents the "environment dependent" element that controls EAE induction or protection in SJL and other experimental models. By monitoring the behavior of clonotypic cells, we examined how the presence of MT affects the recruitment and polarization of T cells during disease induction.

During the immune response, the recruitment of antigen receptor repertoire is critically determined by competition and precursor frequency. Competition occurs among antigens competing for the presenting element (40–43) and among T cells to access the APC (25, 44). The presence of multiple antigenic determinants during immunization will give rise to a more limited antigen receptor repertoire that is characterized by a greater affinity for each determinant (26). Therefore, the presence of other antigenic determinants in the "antigenic" environment at the time of priming can impact the recruitment of self-reactive repertoire. The frequency of precursors has been found to also determine the repertoire usage for T cell responses (45, 46). It has also been suggested that competition among T cells actually occurs only in recall responses, where the number of antigen-specific T cells is high enough to interfere with the access to APC (47).

Here we show that the presence of MT does not restrict the TCR repertoire by competition. On the contrary, our data indicate an enlargement of the Th1 repertoire. This suggests that the precursor frequency is a more relevant factor in determining TCR usage than competition in this experimental model. The amounts of MT used in this study encompass the biologically active spectrum, spanning from the dose that enhances "disease induction" to the one that confers "disease protection." This is consistent with reports showing that genetic factors contribute to skew the T cell repertoire that pre-disposes to the development of MS (14).

The observation that the presence of multiple antigenic determinants during immunization does not limit the repertoire selection has relevance for rationale design of vaccines. It implies in fact that vaccine composed by several antigens may be as effective as the administration of the individual antigens in terms of repertoire selection. In cancer vaccines, the spectrum of antigenic determinants and TCR repertoires may be extremely restricted because of self-tolerance. The observations reported show that simultaneous vaccination with several candidate antigenic determinants will not impair activation of the suitable TCR repertoires. At the same time, multiple vaccines may offer advantage by spreading the effect on Th polarization of some compounds (such as, in our case, MT) present in the mixture to other antigens.

The overall Th1/Th2 balance can be determined by immunological history (12), genetic background (48) and type and activation status of APC (49, 50), mostly (but not exclusively) by influencing the cytokine and chemokine milieu in which priming occurs (51, 52). Other regulatory factors include the amount of antigen used for challenge (53), co-stimulatory molecules involved (54), as well as the affinity/avidity of the TCR that together determine the strength and duration of the interaction of the trimolecular complex TCR–peptide antigen–MHC (55). Several longstanding observations have shown that MT promotes polarization of the immune response toward Th1 (16, 17). Here, we observed that in the presence of MT, the increase in the number of Th1 cells were accompanied by a decrease of the potentially protective IL-10-producing cells. Instead of having a comprehensive effect on the entire repertoire, our results show that MT modulates the immune response by affecting a restricted T cell population whose Th1/Th2 polarization is susceptible to influence by the environment such as DC secretion of cytokines.

The role of TCR in determining the polarization of T cells is still unclear. Observations in several models support the hypothesis that T cells can differentiate toward Th1 or Th2, depending on the cytokine milieu in which priming occurs (56, 57); in these models the TCR itself does not have a major role. Studies of the recruitment of public rearrangements in polyclonal models indicate that some cells appear able to differentiate only into one phenotype, when activated by a fixed antigen (58–60). Also, several observations indicate that the alpha-chain of the TCR plays a relevant role in determining the fate of T cells. Thus, a modification of a single amino acid residue of the CDR3 of the alpha-chain can modify the Th1/Th2 fate of T cells bearing the receptor (61). In the non-obese diabetic mouse it appears that the length of the CDR3 region of the alpha-chain influences polarization of T cells (62). In this model, a short CDR3 region appears to favor Th1 differentiation by increasing the affinity of the TCR for its ligand, whereas a longer CDR3 region decreases the TCR–peptide-MHC affinity and promotes Th2 development. Taken together, these data indicate that characteristics intrinsic to the TCR bias the evolvement of Th1 and Th2 phenotype.

In the present study, we observe two populations of T cells based on their polarization properties. One appears as a "core" repertoire that is relatively insensitive to the environmental condition and consistently drifts toward the same phenotype. In comparison, the polarization of another population is flexible and can be fine-tuned depending on the environmental conditions. We propose that together, the net balance in the activation between these two populations defines the Th1/Th2 phenotype of an immune response.

We observe a group of T cell characterized by usage of a specific rearrangement that apparently fails to acquire the secretory effector phenotype during the primary response. It has been reported that memory T cells can be divided into EMT or CMT cells (11). While both types of cell produce IL-2 and quickly proliferate in response to antigen in vitro, CMT cells fail to secrete type-1 or type-2 cytokines, home exclusively to lymphoid organs and need further encounter with the antigen before they can acquire full effector phenotype (63). These cells may represent a reservoir for further differentiation during memory response. It has been suggested that antigen affinity and avidity differ between the initial primary activation and the subsequent memory response (64). This can be related to changes in the requirement of co-stimulatory interactions and raft association of the signal machinery (65). Several observations indicate that the repertoire used by memory or effector CD8+ cells does not change, while a paper by Baron and co-workers provides evidences of a large distinction within the memory compartment between CMT and EMT in their TCR usage (66). We show here that the semi-private clonotypic repertoire proliferates in response to the antigen, but it fails to produce any of the tested cytokines. In addition, cells carrying this rearrangement fail to home to the CNS during the acute phase of EAE (C. Nicolò, in preparation). Taken together, these observations suggest that this rearrangement may actually identify a group of CMT. Our data suggest that the CDR3 TCR repertoires of CD4+ EMT and CMT cells may not completely overlap. It will be of the outmost relevance to assess the relative contribution of either type of cells to the natural history of immune response, when dealing with diseases characterized by relapsing events such as MS. In addition to T cells that differentiated to effectors, the TCR repertoires used by CMT cells should also be considered in the design of diagnostic and therapeutic trials centered on TCR, such as antagonist peptide or vaccination protocols.

In conclusion, we show that simultaneous immunization with several independent antigenic determinants does not limit the spectrum of the TCR repertoire for each of the determinants, and the antigenic environment can fine-tune the immune response by affecting the polarization of a restricted T cell population. Regulation of the width of the activated TCR repertoire specific for a self-antigen and of its polarization to Th1 represents a crucial constraint for the development of organ-specific autoimmune diseases.


   Acknowledgements
 
This work was supported by Grant RG-3339-A-1 from the National Multiple Sclerosis Society to F.R., and by grant from Canadian Institutes of Health Research/Natural Sciences and Engineering Research Council of Canada to B.M.C.C. The authors thank B. Giardina for insightful discussion.


   Abbreviations
 
APC   antigen-presenting cell
CFA   complete Freund's adjuvant
CMT   central memory T cell
DC   dendritic cell
EAE   experimental autoimmune encephalitis
EMT   effector memory T cell
HEL   hen egg lysozyme
IFA   incomplete Freund's adjuvant
LN   lymph node
MS   multiple sclerosis
MT   Mycobacterium tuberculosis
RSI   rate stimulation index

   Notes
 
Transmitting editor: S. Romagnani

Received 30 May 2005, accepted 25 November 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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