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

Abstract

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.

Surface Expression of MPT64 as a Fusion with the PE Domain of PE_PGRS33 EnhancesMycobacterium bovisBCG Protective Activity againstMycobacterium tuberculosisin Mice

“…In four distinct experiments, spleen cells obtained from mice vaccinated with H PE-⌬MPT64-BCG were stimulated with recombinant MPT64, and IFN-␥-secreting cells were enriched as described previously (37). In two experiments, cells from three mice were pooled and examined; in the other two experiments, cells from a total of 6 mice were examined individually.…”

“…Results are also reported as relative stimulation indices (RSI; calculated as normalized peak area obtained from cells stimulated with Ag/normalized peak area of nonstimulated cells). According to our experience with other model antigens (37,47), T cells carrying a T cell receptor (TCR) rearrangement can be considered expanded in a peptide-driven manner when the RSI is Ն2.…”

Mycobacterium smegmatisExpressing a Chimeric Protein MPT64-Proteolipid Protein (PLP) 139–151 Reorganizes the PLP-Specific T Cell Repertoire Favoring a CD8-Mediated Response and Induces a Relapsing Experimental Autoimmune Encephalomyelitis

“…Percoll gradient was performed, and mononuclear cells were extracted at the 30-70% interface. Collected cells were washed twice mixed with 10 6 ab-BW cells (as previously described for analysis of low numbers of T cells [28]) and resuspended in RLT buffer for RNA extraction.…”

“…cDNA was synthesized using an oligo-dT primer (dT15) (Life Technologies). For complete "Immunoscope" analysis (28), cDNA was subjected to PCR amplification using a common Constant (C) b primer (CACT-GATGTTCTGTGTGACA) in combination with the variable b (BV) primers previously described (32), following the nomenclature of Arden et al (33). Using 2 ml of this product as a template, run-off reactions were performed with a single internal fluorescent primer for each BJ tested (described previously [32]).…”

“…The plasmid vector was constructed using a pair of complementary oligonucleotides, synthesized to assemble the double-stranded mini-fragments of the previously published BV10 CDR3 "Y L C A S S P G T N T E V F F G" (28), using the following primers: sense, 59-CTAGCTAGCGCCACCA-TGTATCTCTGTGCCAGCAGCCCGGGGACAAACACAGAAGTCTTCT-TTGGTTAAGCGGCCGCTAAACTAT-39; and antisense, 59-ATAGTTT AGCGGCCGCTTAACCAAAGAAGACTTCTGTGTTTGTCCCCGGGCT-GCTGGCACAGAGATACATGGTGGCGCTAGCTAG-39.…”