Chiara Nicolò

Distinct and Non-Overlapping T Cell Receptor Repertoires Expanded by DNA Vaccination in Wild-Type and HER-2 Transgenic BALB/c Mice

Central tolerance to tumor-associated Ags is an immune-escape mechanism that significantly limits the TCR repertoires available for tumor eradication. The repertoires expanded in wild-type BALB/c and rat-HER-2/neu (rHER-2) transgenic BALB-neuT mice following DNA immunization against rHER-2 were compared by spectratyping the variable (V)β and the joining (J)β CDR 3. Following immunization, BALB/c mice raised a strong response. Every mouse used one or more CD8+ T cell rearrangements of the Vβ9-Jβ1.2 segments characterized by distinct length of the CDR3 and specific for 63-71 or 1206-1214 rHER-2 peptides. In addition, two CD4+ T cell rearrangements recurred in >50% of mice. Instead, BALB-neuT mice displayed a limited response to rHER-2. Their repertoire is smaller and uses different rearrangements confined to CD4+ T cells. Thus, central tolerance in BALB-neuT mice acts by silencing the BALB/c mice self-reactive repertoire and reducing the size of the CD8+ T cell component. CD8+ and CD4+ T cells from both wild-type and transgenic mice home to tumors. This definition of the T cell repertoires available is critical to the designing of immunological maneuvers able to elicit an effective immune reaction against HER-2-driven carcinogenesis.

Collagen-specific T-cell repertoire in blood and synovial fluid varies with disease activity in early rheumatoid arthritis

IntroductionType II collagen is a DR4/DR1 restricted target of self-reactive T cells that sustain rheumatoid arthritis. The aim of the present study was to analyze the T-cell receptor repertoire at the onset of and at different phases in rheumatoid arthritis.MethodsWe used the CDR3 BV-BJ spectratyping to study the response to human collagen peptide 261–273 in 12 patients with DR4+ rheumatoid arthritis (six at the onset of disease and six during the course of disease) and in five healthy DR4+ relatives.ResultsThe collagen-specific T-cell repertoire is quite restricted at the onset of disease, involving approximately 10 rearrangements. Within the studied collagen-specific rearrangements, nearly 75% is shared among patients. Although the size of the repertoire used by control individuals is comparable to that of patients, it is characterized by different T-cell receptors. Part of the antigen-specific T-cell repertoire is spontaneously enriched in synovial fluid. The specific T-cell repertoire in the periphery was modulated by therapy and decreased with the remission of the disease. Failure of immunoscopy to detect this repertoire was not due to suppression of collagen-driven proliferation in vitro by CD4+ CD25+ T cells. Clinical relapse of the disease was associated with the appearance of the original collagen-specific T cells.ConclusionsThe collagen-specific T-cell receptor repertoire in peripheral blood and synovial fluid is restricted to a limited number of rearrangements in rheumatoid arthritis. The majority of the repertoire is shared between patients with early rheumatoid arthritis and it is modulated by therapy.

TLR2: a crossroads between infections and autoimmunity?

Environment has both pathogenic and protective roles in the determination of autoimmune disease development, possibly through infectious agents. TLR2 has the capability to recognize the widest range of PAMPs, and it is important for the recognition of mycobacteria and gram-positive bacteria. Here we review recent information showing that TLR2 ligands, its signaling machinery and the effects of its engagement on T cell polarization and differentiation, all play a decisive role in experimental models of autoimmunity. Thus, we propose that engagement of TLR2 is an important crossroads between encounter with bacteria and development of self-reactive diseases.

Erbb2 DNA Vaccine Combined with Regulatory T Cell Deletion Enhances Antibody Response and Reveals Latent Low-Avidity T Cells: Potential and Limits of Its Therapeutic Efficacy

Rat (r)Erbb2 transgenic BALB-neuT mice genetically predestined to develop multiple invasive carcinomas allow an assessment of the potential of a vaccine against the stages of cancer progression. Because of rErbb2 expression in the thymus and its overexpression in the mammary gland, CD8+ T cell clones reacting at high avidity with dominant rErbb2 epitopes are deleted in these mice. In BALB-neuT mice with diffuse and invasive in situ lesions and almost palpable carcinomas, a temporary regulatory T cells depletion combined with anti-rErbb2 vaccine markedly enhanced the anti-rErbb2 Ab response and allowed the expansion of latent pools of low-avidity CD8+ T cells bearing TCRs repertoire reacting with the rErbb2 dominant peptide. This combination of a higher Ab response and activation of a low-avidity cytotoxic response persistently blocked tumor progression at stages in which the vaccine alone was ineffective. However, when diffuse and invasive microscopic cancers become almost palpable, this combination was no longer able to secure a significant extension of mice survival.

Surface expression of MPT64 as a fusion with the PE domain of PE_PGRS33 enhances Mycobacterium bovis BCG protective activity against Mycobacterium tuberculosis in mice.

ABSTRACT To improve the current vaccine against tuberculosis, a recombinant strain of Mycobacterium bovis bacillus Calmette-Guérin (rBCG) expressing a Mycobacterium tuberculosis vaccine candidate antigen (MPT64) in strong association with the mycobacterial cell wall was developed. To deliver the candidate antigen on the surface, we fused the mpt64 gene to the sequence encoding the PE domain of the PE_PGRS33 protein of M. tuberculosis (to create strain HPE-ΔMPT64-BCG), which we have previously shown to transport proteins to the bacterial surface. In a series of protection experiments in the mouse model of tuberculosis, we showed that (i) immunization of mice with HPE-ΔMPT64-BCG provides levels of protection significantly higher than those afforded by the parental BCG strain, as assessed by bacterial colonization in lungs and spleens and by lung involvement (at both 28 and 70 days postchallenge), (ii) rBCG strains expressing MPT64 provide better protection than the parental BCG strain only when this antigen is surface expressed, and (iii) the HPE-ΔMPT64-BCG-induced MPT64-specific T cell repertoire when characterized by β chain variable region-β chain joining region (BV-BJ) spectratyping indicates that protection is correlated with the ability to recruit gamma interferon (IFN-γ)-secreting T cells carrying the BV8.3-BJ1.5 (172 bp) shared rearrangement. These results demonstrate that HPE-ΔMPT64-BCG is one of the most effective new vaccines tested so far in the mouse model of tuberculosis and underscore the impact of antigen cellular localization on the induction of the specific immune response induced by rBCG.

Administration of PLP139–151 Primes T Cells Distinct from Those Spontaneously Responsive In Vitro to This Antigen

We examined the TCR repertoire used by naive SJL mice in their in vitro spontaneous response to proteolipid protein (PLP) 139–151 by Vβ-Jβ spectratyping and compared it to that used after immunization with the peptide. T cells from immunized mice use the public rearrangement Vβ10-Jβ1.1, but naive mice do not; in contrast, TCR CDR3-β rearrangements of Vβ18-Jβ1.2 and Vβ19-Jβ1.2 consistently are associated with the spontaneous response. T cells involved in spontaneous and induced responses can each recognize PLP139–151 presented in vivo, but its s.c. administration has different consequences for the two repertoires. Four days after immunization, T cells associated with spontaneous responsiveness appear in the draining lymph nodes but disappear by day 10 and never appear elsewhere. Simultaneously, Vβ10-Jβ1.1 T cells are likewise activated in the lymph nodes by day 4 and spread to the spleen by day 10. Eight- to 10-wk-old naive mice use a narrower repertoire of TCRs than do immunized age-matched mice. Induced Vβ10-Jβ1.1 T cells home to the CNS during experimental autoimmune encephalomyelitis, whereas we failed to detect Vβ18-Jβ1.2 and Vβ19-Jβ1.2 TCR rearrangements in the CNS. Thus, we observe that administration of PLP139–151 primes a T cell repertoire distinct from the one responsible for spontaneous responsiveness. This “immunized” repertoire substitutes for the naive one and becomes dominant at the time of disease onset.

Mycobacterium smegmatis Expressing 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

We infected SJL mice with a recombinant Mycobacterium smegmatis expressing a chimeric protein containing the self-epitope of proteolipid protein 139–151 (p139) fused to MPT64, a secreted protein of Mycobacterium tuberculosis (rMSp139). Infected mice developed a relapsing experimental autoimmune encephalomyelitis (EAE), showing a prevailing demyelination of the CNS, and disease severity was significantly lower in comparison with the one that follows immunization with p139. rMSp139 was not detected in lymph node or spleen in the course of clinical disease development or in the CNS during relapse. Infection with rMSp139 modified the p139-specific T cell repertoire, recruiting the spontaneous p139-specific repertoire and activating CD4+ T cells carrying the BV4 semiprivate rearrangement. T cells carrying the public BV10 rearrangement that are consistently found in the CNS during flares of disease were not activated by infection with rMSp139 because lymph node APCs infected with rMSp139 selectively fail to present the epitope for which BV10 cells are specific. Simultaneously, rMSp139 expanded p139-specific CD8+ cells more efficiently than immunization with peptide in adjuvant. SJL mice vaccinated against the CDR3 sequence of the BV10 public rearrangement reduced usage of the BV10 cells and displayed reduced symptoms during bouts of EAE. Thus, transient peripheral infection with a CNS-cross–reactive nonpathogenic Mycobacterium induces a relapsing EAE that continues long after clearance of the infectious agent. The composition of the self-reactive repertoire activated determines severity and histology of the resulting disease.

Collagen Specific T-Cell Repertoire and HLA-DR Alleles: Biomarkers of Active Refractory Rheumatoid Arthritis

Rheumatoid arthritis (RA) is characterized by chronic joint inflammation and associates with HLA-DRB1*04. The Collagen IIp261-273-specific T cell repertoire in the peripheral blood of DR4 + patients at the onset of the disease shows a restricted TCR-beta chain usage among which the most frequent is TRBV25. To define whether this group of DR4-restricted collagen-specific shared T cell could represent markers of active-severe disease and response to therapy, 90 subjects affected by early-RA were enrolled in the study; peripheral blood mononuclear cells were cultured with or without the human collagen II peptide p261-273 and were examined by immunoscope analysis for the usage of the previously identified shared TCR-beta chains. We report that the presence of T cells carrying rearrangement TRBV25 associated with HLA-DR haplotype and disease activity. HLA-DRB1* haplotypes 04–04, 04–01 and 04–11 were significantly associated with usage of TRBV25, higher disease activity at the onset of disease and poor response to DMARDs. Finally, the HLA-DRB1* haplotype appeared complementary with current serologic tools to predict good and poor responders in a treat to target strategy. The data reported here offer clues to predict the course of the disease and to foresee personalized treatments in RA patients.

M tuberculosis in the Adjuvant Modulates Time of Appearance of CNS-Specific Effector T Cells in the Spleen through a Polymorphic Site of TLR2

DC deliver information regulating trafficking of effector T cells along T-cell priming. However, the role of pathogen-derived motives in the regulation of movement of T cells has not been studied. We hereinafter report that amount of M tuberculosis in the adjuvant modulates relocation of PLP139-151 specific T cells. In the presence of a low dose of M tuberculosis in the adjuvant, T cells (detected by CDR3 BV-BJ spectratyping, the so-called “immunoscope”) mostly reach the spleen by day 28 after immunization (“late relocation”) in the SJL strain, whereas T cells reach the spleen by d 14 with a high dose of M tuberculosis (“early relocation”). The C57Bl/6 background confers a dominant “early relocation” phenotype to F1 (SJL×C57Bl/6) mice, allowing early relocation of T cells in the presence of low dose M tuberculosis. A single non-synonymous polymorphism of TLR2 is responsible for “early/late” relocation phenotype. Egress of T lymphocytes is regulated by TLR2 expressed on T cells. Thus, pathogens engaging TLR2 on T cells regulate directly T-cell trafficking, and polymorphisms of TLR2 condition T-cell trafficking upon a limiting concentration of ligand.

OP0222 (Collagen Specific-)T Cell Related Parameters Provide Information for the Management of Rheumatoid Arthritis Patients

Background The developmental risk of Rheumatoid Arthritis (RA) is strongly linked to HLA-DRB1*04 (DR4) and *01 (DR1). In RA, activated immune cells, as CD4+T cells, B cells, dendritic cells and macrophages infiltrate the synovial tissue. In a previous work, we found that huCollp261-273-specific T cell repertoire analyzed in peripheral blood of DR4+ RA patients was restricted to a limited number of TCR rearrangements. This collagen-specific repertoire appears modulated by disease activity (1). The two most used TCR-beta chains were TRBV25-TRBJ2.2 and TRBV6-4-TRBJ2-3. Objectives To examine the frequency and the distribution of huCollp261-273-specific T cells and the role of DR alleles in RA patients in order to find new biomarker for the management of the disease. Methods HLA genotyping was performed using the Innogenetics® kit, following the manufacturers protocol. TCR repertoire analysis: Peripheral blood mononuclear cells (PBMCs) were purified from whole blood and TCR BV-JB analysis was performed as previously described. (1) Patients: we analyzed 100 samples from RA patients, 57 with a high disease activity (DAS >3.7) and 43 with a low disease activity (DAS≤2.4). Results The presence of TRBV25-TRBJ2.2 and TRBV6-4-TRBJ2-3 in T cells was significantly associated with disease activity in the group of DR4+ RA patients. The usage of these two receptors described two non-overlapping groups of RA patients, with subjects using TRBV6-4+ displaying a significantly higher disease activity. The combination of TCR usage in DR4+ subjects and of DR haplotypes in RA patients described four distinct responses to treatment with Disease Modifying Antirheumatic Drugs (DMARDs) and biologic agents. Fifty percent of DR4+ TRBV25- patients responded to DMARDs and the remaining responded well to biologic agents; DR4+ TRBV25+ patients failed to respond to DMARDs but displayed a good response to biologic agents and this latter group might therefore be candidate to treatment with biologic agents. In the group of DR4-, DR1- or DR7-patients, the DR11 allele appeared highly enriched in subjects displaying a high disease activity. Accordingly, within the group of DR4-, DR1- DR7- RA patients, we observed that 50% of all patients achieved a good response after DMARDs treatment; among patients non-responder to DMARDs, however, it appeared that DR11+ subjects responded to a lesser extent than DR11- patients to biologic agents. Conclusions These findings showed that the analysis of collagen specific T cells repertoire and HLA-DR haplotype can provide useful information to a personalized treatment in RA patients. References Ria F., et al. Arthritis Research & Therapy, 2008. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4311