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This article belongs to the Section Molecular Immunology., Tick-transmitted pathogens cause infectious diseases in both humans and animals. Different types of adaptive immune mechanisms could be induced in hosts by these microorganisms, triggered either directly by pathogen antigens or indirectly through soluble factors, such as cytokines and/or chemokines, secreted by host cells as response. Adaptive immunity effectors, such as antibody secretion and cytotoxic and/or T helper cell responses, are mainly involved in the late and long-lasting protective immune response. Proteins and/or epitopes derived from pathogens and tick vectors have been isolated and characterized for the immune response induced in different hosts. This review was focused on the interactions between tick-borne pathogenic hemoparasites and different host effector mechanisms of T- and/or B cell-mediated adaptive immunity, describing the efforts to define immunodominant proteins or epitopes for vaccine development and/or immunotherapeutic purposes. A better understanding of these mechanisms of host immunity could lead to the assessment of possible new immunotherapies for these pathogens as well as to the prediction of possible new candidate vaccine antigens., This research was funded by the Italian Ministry of Health with grants RC IZSSI 07/18 and RC IZSSI 08/19.

Natural killer T cells (NKT) are a subset of T lymphocytes bridging innate and adaptive immunity. These cells recognize self and microbial glycolipids bound to non-polymorphic and highly conserved CD1d molecules. Three NKT cell subsets, type I, II and NKT-like expressing different antigen receptors (TCR) were described and TCR activation promotes intracellular events leading to specific functional activities. NKT can exhibit different functions depending on the secretion of soluble molecules and the interaction with other cell types. NKT cells act as regulatory cells in the defence against infections but, on the other hand, their effector functions can be involved in the pathogenesis of several inflammatory disorders due to their exposure to different microbial or self antigens, respectively. A deep understanding of the biology and functions of type I, II and NKT-like cells as well as their interplay with cell types acting in innate (Neuthrophils, Innate Lymphoid cells, Machrophages and Dendritic cells) and adaptive immunity (CD4+,CD8+ and Double Negative T cells) should be important to design potential immunotherapies for infectious and autoimmune diseases.

Background Primary Sjogren Syndrome (pSS) is a chronic inflammatory disorder affecting exocrine glands. Both IL-23 and the downstream cytokines IL-17 and IL-22 are recognised as key players in the disease. Therefore, the identification of the cellular sources and inducers of IL-17 is crucial in the understanding of the drivers of inflammation in pSS. Mucosal-associated invariant T (MAIT) cells recognize riboflavin derivatives presented by the MHC class I-like molecule MR1. Objectives Recently, MAIT cells have been implicated in the pathogenesis of autoimmune disorders and found expanded in salivary glands of pSS patients. Their expression of IL7R and IL23R, makes them potential contributors to the pathogenesis of pSS. Methods Mononuclear cells from 16 patients with pSS and 14 subjects with non Sjogren Syndrome secca syndrome (nSS) were isolated from blood and salivary glands. We assessed the phenotype and cytokine expression of MAIT cells by surface and intracellular flow cytometry analysis. The function of MAIT cells was assessed upon in vitro stimulation with recombinant IL-7, IL-23 or their specific ligand lumazine (a Vitamin B2 precursor previously reported to be recognized by and specifically activate MAIT cells). Results MAIT cells were reduced in frequency in peripheral blood but not in minor salivary glands of patients with pSS, compared with nSS. In vitro stimulation of MAIT cells from pSS patients induced cytokine production which was dependent on priming with IL-7, IL-23 or specific antigen (lumazine) stimulation. Particularly, IL-7 and IL-23 guarantee IL-17 polarization of MAIT cells by two different pathways via the activation of STAT3 or RORc respectively. Conclusions This study confirms a potential role for MAIT cells in pSS and, for the first time, links IL-7 and IL-23 to the IL-17 polarization of MAIT cells in these patients. Disclosure of Interest None declared

Endothelial Progenitor Cells (EPCs) are bone marrow derived cells able to differentiate in mature endothelial cells (EC) contributing to the generation of new vessels, connecting to fibronectin, and forming colonies and/or colony forming units. Since circulating EPCs can be actively considered part of endothelial damage in several cardiovascular diseases and autoimmune disorders the possibility to have a measure for endothelium damage should be considered of interest to predict the patient out-come. At the same time the EPCs proliferative and regenerative role could be considered for therapeutic applications. Studies have been performed to elucidate the role of EPCs in Systemic Sclerosis and many review and articles published on this topic. In the present paper we aimed to review the role of EPCs in other autoimmune disorders.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the emergence of autoreactive T cells. Humans and mice with SLE have reduced numbers of CD1d-restricted invariant natural killer T (iNKT) cells, suggesting a key role for these cells in its immunopathogenesis. This subset uses an invariant TCR constituted by Valpha14 Jalpha281 chains paired with some Vbeta domains. The regulatory role for iNKT cells in non-autoimmune mice was suggested by our previous results showing that aged Jalpha281 knockout (KO) mice produce anti-dsDNA. Here we show that old Jalpha281 KO mice have proteinuria and antibodies against dsDNA and cardiolipin. Histological analysis of Jalpha281 KO mice revealed glomeruli damage and deposition of C3c and IgG, mainly of the IgG3 subclass. In spleens of aged Jalpha281 KO mice there is an increase of activated marginal zone B cells. The evolution of lesions may depend on the age-associated increase of autoantibodies production, preferentially IgG3, mainly secreted by marginal zone B cells. Our results provide the first evidence of a lupus-like syndrome in non-autoimmune mice, supporting an age-related immunoregulatory role of Jalpha281+ cells, probably associated with the activation of marginal zone B cells.

Background The aim of the study was to better characterise the immunological origin and the behaviour of interleukin (IL)-23-responsive innate lymphoid cells (ILCs) in the gut, synovial fluid (SF) and bone marrow (BM) of patients with ankylosing spondylitis (AS).Methods ILC1, ILC2 and ILC3 cells were determined and characterised by confocal microscopy and flow cytometry in ileal and BM biopsies, in peripheral blood (PB) and SF mononuclear cells obtained from patients with AS and controls. Mucosal vascular addressin cell adhesion molecule 1 (MADCAM-1), IL-7, IL-15 and aggregates of lymphoid tissue inducer cells (LTi) were evaluated by immunohistochemistry. The in vitro ability of epithelial cells in driving the differentiation of ILC3 and the effect of tumour necrosis factor inhibitors (TNFi) on the frequency of ILC3 and the expression of MADCAM1 were also assessed.Results ILC3 characterised as Lyn(-)RORc(-)Tbet(+) NKp44(+) cells were significantly expanded in the gut, SF and BM of patients with AS compared with controls, produced high levels of IL-17 and IL-22 and expressed alpha 4 beta 7. MADcAM1 was overexpressed in BM and ileal high endothelial venules. IL-7 was significantly increased in AS gut, especially in the context of Paneth cells, and accompanied by the presence of aggregates of c-kit/IL-7R(+) cells (LTi). In in vitro experiments, epithelial cells from patients with AS actively induced differentiation of ILC3 from LTi. TNFi efficacy was accompanied by a significant decrease in the percentage of intestinal and circulating ILC3 and in the expression of MADCAM1.Conclusions Gut-derived IL-17(+) and IL-22(+) ILC3 are expanded in the peripheral blood, SF and inflamed BM of patients with AS, suggesting the presence of an active homing axis between the gut and the inflamed sacroiliac joints.

Objective IL-9 has been shown to be upregulated before the clinical onset of articular disease in RA. The exact role of IL-9 and Th9 cells in RA, however, has not yet been adequately studied. The aim of this study was to evaluate the expression of IL-9 and IL-9-expressing cells in RA patients. Methods IL-9, IL-9R, PU.1, IL-9, thymic stromal lymphopoietin (TSLP), IL-4 and TGF-β expression was assessed by real-time-PCR in the synovial tissues of RA and OA patients. IL-9, IL-9R, IL-4, TSLP and TGF-β were also investigated by immunohistochemistry. Peripheral CD4(+) T cell subsets were studied by flow cytometry analysis before and after incubation with citrullinated peptides. Results IL-9 was overexpressed in RA synovial tissues and correlated with the degree of histological organization of B and T cells in ectopic lymphoid structures. The majority of IL-9-producing cells were identified as CD3(+) cells. Increased mRNA and protein expression of IL-9R, IL-4, TSLP and TGF-β was also observed in RA synovial tissue. Blood peripheral Th9 cells were expanded by citrullinated peptides. Conclusion These results indicate that Th9 cells and IL-9 were frequently detected in peripheral blood mononuclear cells and synovia of RA patients. A possible pathogenic role for Th9 in RA is discussed.

Previously we have reported on the development of antibodies against MICA alleles in kidney transplant recipients. These alloantibodies have now been determined using a new assay using Luminex beads bound to soluble recombinant MICA antigens produced in insect cells. In the present study we have analyzed sera from 85 kidney transplant recipients on the waiting list and 66 patients transplanted within the last 4 years and 59 acid eluates obtained from allograft nephrectomy specimens. Many of the patients in those groups were sensitized and some had previous transplants (waiting list: 15%; post-tx: 7.6%; eluates 22%) and their sera were found to contain anti-human leukocyte antigen (HLA) and anti-MICA antibodies. Anti-MICA antibodies were detected in 21/85 (24.7%) of the waiting list patients and in 15/66 (22.7%) of the transplanted recipients; 11 of the eluates (18.6%) were found to have MICA-specific antibodies (6 of them also had anti-HLA antibodies and 5 did not). These data suggested that immunization against mismatched MICA alleles induces development of anti-MICA antibodies. The finding of MICA allele-specific antibodies in eluates of kidney transplants suggests that anti-MICA antibodies can be involved in the pathogenesis of kidney allograft rejection. Further studies will be required to determine whether patients who produce alloantibodies against MICA alleles are at risk for transplant rejection even when no HLA antibodies are detected.

Vδ2 T lymphocytes recognize nonpeptidic antigens without presentation by MHC molecules and mount both immediate effector functions and memory responses after microbial infection. However, how Vδ2 T cells mediate different facets of a memory response remains unknown. Here, we show that the expression of CD45RA and CD27 antigens defines four subsets of human Vδ2 T cells with distinctive compartmentalization routes. Naive CD45RA+CD27+ and memory CD45RA−CD27+ cells express lymph node homing receptors, abound in lymph nodes, and lack immediate effector functions. Conversely, memory CD45RA−CD27− and terminally differentiated CD45RA+CD27− cells, which express receptors for homing to inflamed tissues, are poorly represented in the lymph nodes while abounding at sites of inflammation, and display immediate effector functions. These observations and additional in vitro experiments indicate a lineage differentiation pattern for human Vδ2 T cells that generates naive cells circulating in lymph nodes, effector/memory cells patrolling the blood, and terminally differentiated effector cells residing in inflamed tissues.

Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tuberculosis is still a matter of debate. It was reported earlier that Vgamma9/Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis through a granule-dependent mechanism that results in killing of intracellular bacilli. This study found that Vgamma9/Vdelta2 T lymphocytes reduce the viability of both extracellular and intracellular M. tuberculosis. Granulysin and perforin, both detected in Vgamma9/Vdelta2 T lymphocytes, play a major role, which indicates that Vgamma9/Vdelta2 T lymphocytes directly contribute to a protective host response against M. tuberculosis infection.

Human T cells expressing Vγ9/Vδ2-encoded TCR recognize several nonpeptide phosphoantigens in the absence of major histocompatibility complex restriction. As these cells respond differentially to increasing concentrations of structurally related phosphoantigens, such ligands constitute agonists of different strengths. By analyzing early cellular events and late effector responses of γ δ T cells, we compared their patterns of stimulation by weak, medium and strong phosphoantigen agonists. We found that, although the early metabolic activation as assessed by cytosensormicrophysiometry directly reflects the intensity of subsequent effector response by γ δ cells, TCR down-modulation is dissociated from the latter. Weak and mid-range phosphoantigen agonistsinduce a time- and dose-dependent down-modulation of the γ δ TCR, whereas strong phosphoantigen agonists induce little or no TCR down-regulation. This indicates that γ δ TCR down-modulation does not match the extent of TCR signaling as assessed by microphysiometry or conventional effector responses (TNF-α production and cytotoxicity). This differential pattern of γ δ cell activation by phosphoantigens could explain the stronger potencies of some of these agonists.

An effective immune response against the intracellular pathogen Mycobacterium tuberculosis is strictly dependent on T cell activation. Although this protective response mainly depends on local release of pro-inflammatory cytokines by Th1 CD4(+) T cells, contribution of Vgamma9 / Vdelta2 T lymphocytes to immune protection against this pathogen is suggested by the antimycobacterial reactivity of this subset and its ability to produce large amounts of Th1 cytokines. Here we show that Vgamma9 / Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis. The cytotoxic activity of Vgamma9 / Vdelta2 T lymphocytes was not MHC class I or class II restricted but was blocked by anti-TCR monoclonal antibodies, thus indicating that it involved specific interaction between the TCR and the target cell. The cytotoxicity of Vgamma9 / Vdelta2 T lymphocytes was not mediated by TNF-alpha or Fas-Fas ligand, but was shown to occur through a granule-dependent mechanism that resulted in reduction of the viability of intracellular bacilli. Perforin was shown to play an important role in killing of both infected macrophages and intracellular mycobacteria. These data strongly suggest that Vgamma9 / Vdelta2 T lymphocytes contribute to the host defense against M. tuberculosis infection.

The repertoire of CD4+ T-lymphocytes was investigated in six patients affected by tuberculosis, who had a negative PPD skin test at diagnosis. Polyclonal CD4+ T-cell lines from the peripheral blood failed to proliferate to PPD and to the 16- or 38-kDa proteins of Mycobacterium tuberculosis, while CD4+ T-cell lines from the site of disease responded to PPD, and to the 16- and 38-kDa proteins, and derived epitopes in vitro. The repertoire of CD4+ T-cells accumulating at the site of disease was found to be widely heterogeneous as demonstrated by the finding that at least seven different peptides from the 16- and 38-kDa proteins were recognized by every patient. These results indicate that CD4+ T-cells localized at the site of disease in tuberculosis recognize a vast array of M. tuberculosis epitopes.

We have analyzed the immune response induced by a 9mer synthetic peptide derived from the male histocompatibility antigen H-Y and containing D b -binding motifs in C57BL/6 mice. In this study we report that a single, subcutaneous injection of the peptide emulsified in IFA gave rise to the development of male-specific CD8 + T cells which displayed H-Y-specific proliferative response in vitro and showed a Tc1-type pattern of cytokine production (i.e. they secreted IFN-γ and IL-2, but not IL-4 and IL-10). Development of a strong cytotoxic activity required in vitro stimulation with specific peptide and IL-2: under these culture conditions, we were able to generate potent CD8 + CTLs that lysed both male cells and peptide-pulsed female cells. Continuous administration of soluble peptide, delivered over a 7-day period by a mini-osmotic pump implanted subcutaneously, inhibited proliferative and cytotoxic responses and IFN-γ production in lymph node cells from C57BL/6 mice subsequently primed with peptide in adjuvant. This decreased responses were associated with a strong increase in the secretion of IL-4 by antigen-specific CD8 + T lymphocytes. Subcutaneous administration of the H-Y-peptide in adjuvant significantly accelerates rejection of male skin graft, while continuous administration of peptide in soluble form did not modify the time course of rejection.

The recognition of phosphorylated nonpeptidic microbial metabolites by Vγ9Vδ2 T cells does not appear to require the presence of MHC molecules or antigen processing, permitting rapid responses against microbial pathogens. These may constitute an important area of natural anti-infectious immunity. To provide evidence of their involvement in immune reactivities against mycobacteria, we measured the responsiveness of peripheral blood Vγ9Vδ2 T cells in children with primary Mycobacterium tuberculosis (MTB) infections. Peripheral blood mononuclear cells from 22 children with MTB infections and 16 positivity of tuberculin (PPD)-negative healthy children were exposed to nonpeptidic antigens in vitro and the reactivity of the Vγ9Vδ2 T cell subset with these antigens was determined using proliferation and cytokine assays. Also, responses of γδ T cells from rhesus monkeys stimulated with phosphoantigens in vivo were measured. The Vγ9Vδ2 T cell responses were highly increased in infected children in comparison with age-matched controls. This augmented Vγ9Vδ2 T cell reactivity subsided after successful antibiotic chemotherapy, suggesting that persistent exposure to mycobacterial antigens is required for the maintenance of γδ T cell activation in vivo. The in vivo reactivity of Vγ9Vδ2 T cells to phosphoantigens was also analyzed in a rhesus monkey model system. Intravenous injections of phosphoantigens induced an activated state of simian Vγ9Vδ2 T cells which decreased after 2 months, i.e., with a time course similar to that seen in MTB-infected children. The increased reactivity of Vγ9Vδ2 T cells to phosphoantigens appears to be dependent on constant antigenic exposure. Consequently, the assessment of Vγ9Vδ2 responses may be useful for monitoring the efficacy of antimycobacterial therapies.

Ptak and Askenase showed that both alphabeta and gammadelta cells are required for transfer of contact sensitivity (CS). This study confirms that day 4 immune cells depleted of gammadelta cells fail to transfer CS to trinitrochlorobenzene (TNP-Cl) systemically and demonstrates that administration of anti-gammadelta monoclonal antibodies (mAb) in vivo abolishes the CS reaction. Moreover, gammadelta cells accumulate at the antigen challenge site: these cells have the unusual phenotype CD8alpha+, CD8beta-, IL-4 R+ which we suggest is due to their state of activation. Following immunization with contact sensitizer on the skin, the absolute number of gammadelta cells increases in the regional lymph nodes with a peak at 4 days. Of the gammadelta cells, 80 %, both in the lymph nodes of TNP-Cl-immune mice and accumulating at the antigen challenge site are Vgamma3+. The gammadelta cells expressing Vgamma3, which is characteristic of dendritic epithelial T cells (DETC), obtained 4 days after sensitization, proliferate in response to interleukin (IL)-7, but only poorly to IL-2 and IL-4. They also respond to concanavalin A and immobilized anti-gammadelta mAb, but not to haptens or heat-shocked syngeneic spleen cells. Furthermore, injection of mice with mAb to IL-7 inhibits accumulation of Vgamma3+ cells both in the lymph nodes after skin sensitization and at the antigen-challenge site. Altogether, these results strongly support the view that DETC are related to, or the original source of, the gammadelta cells found in the lymph node after skin sensitization and at the site of challenge, and that IL-7 is implicated in these phenomena.

Phenotypic and functional heterogeneity are the hallmarks of effector and memory T cells. Upon antigen stimulation, γδ T cells differentiate into two major types of memory T cells: central memory cells, which patrol the blood and secondary lymphoid organs, and effector memory cells, which migrate to peripheral tissues. γδ T cells display in vitro a certain degree of plasticity in their function that is reminiscent of that which is observed in conventional CD4 T cells. Similar to CD4 T cells, in which a plethora of specialized subsets affect the host response, γδ T cells may readily and rapidly assume distinct Th1-, Th2-, Th17-, T(FH) and T regulatory-like effector functions, suggesting that they profoundly influence cell-mediated and humoral immune responses. In addition to differences in cytokine repertoire, γδ T cells exhibit diversity in homing, such as migration to lymph node follicles, to help B cells versus migration to inflamed tissues. Here, we review our current understanding of γδ T-cell lineage heterogeneity and flexibility, with an emphasis on the human system, and propose a classification of effector γδ T cells based on distinct functional phenotypes.

In this paper we focus our attention on the role of two families of receptors, Toll-like receptors (TLR) and decoy receptors (DcR) involved in the generation of systemic lupus erythematosus (SLE) and lupus-like syndromes in human and mouse models. To date, these molecules were described in several autoimmune disorders such as rheumatoid arthritis, antiphospholipids syndrome, bowel inflammation, and SLE. Here, we summarize the findings of recent investigations on TLR and DcR and their role in the immunopathogenesis of the SLE.

Vγ9Vδ2 T lymphocytes recognize nonpeptidic antigens without presentation by MHC molecules and display pleiotropic features. Here we report that coculture of Vγ9Vδ2 cells with phosphoantigen and IL-21 leads to selective expression of the transcription repressor Bcl-6 and polarization toward a lymphocyte subset displaying features of follicular B-helper T (T(FH)) cells. T(FH) like Vγ9Vδ2 cells have a predominant central memory (CD27(+)CD45RA(-)) phenotype and express ICOS, CD40L and CXCR5. Upon antigen activation, they secrete IL-4, IL-10 and CXCL13, and provide B-cell help for antibody production in vitro. Our findings delineate a subset of human Vγ9Vδ2 lymphocytes, which, upon interaction with IL-21-producing CD4 T(FH) cells and B cells in secondary lymphoid organs, is implicated in the production of high affinity antibodies against microbial pathogens.

Decoy receptors are "silent scavengers" of CC chemokines and cytokines, which play a key role in damping inflammation and tissue damage. In this review we discuss on recent findings demonstrating that these receptors set the balance between antimicrobial resistance, immune activation and inflammatory response in Mycobacterium tuberculosis infection.

D6 is a decoy and scavenger receptor for inflammatory CC chemokines. D6-deficient mice were rapidly killed by intranasal administration of low doses of Mycobacterium tuberculosis. The death of D6(-/-) mice was associated with a dramatic local and systemic inflammatory response with levels of M. tuberculosis colony-forming units similar to control D6-proficient mice. D6-deficient mice showed an increased numbers of mononuclear cells (macrophages, dendritic cells, and CD4 and CD8 T lymphocytes) infiltrating inflamed tissues and lymph nodes, as well as abnormal increased concentrations of CC chemokines (CCL2, CCL3, CCL4, and CCL5) and proinflammatory cytokines (tumor necrosis factor alpha, interleukin 1beta, and interferon gamma) in bronchoalveolar lavage and serum. High levels of inflammatory cytokines in D6(-/-) infected mice were associated with liver and kidney damage, resulting in both liver and renal failure. Blocking inflammatory CC chemokines with a cocktail of antibodies reversed the inflammatory phenotype of D6(-/-) mice but led to less controlled growth of M. tuberculosis. Thus, the D6 decoy receptor plays a key role in setting the balance between antimicrobial resistance, immune activation, and inflammation in M. tuberculosis infection.

Serum responses against Mycobacterium tuberculosis HSP16 were determined for children with tuberculosis (TB) and for healthy purified protein derivative (PPD)-positive and PPD-negative children. Immunoglobulin G (IgG) and IgM responses were higher for TB patients than for other groups. After chemotherapy, IgM and IgG responses decreased for TB patients and PPD-positive subjects. Monitoring of anti- M. tuberculosis HSP16 responses could assist in the management of pediatric TB.

α−galactosylceramide, a natural killer T cell ligand, and its synthetic homolog, KRN7000, consistently influence IFN-γ and TNF-α release, both mediators of LPS-induced shock. To modify the course of endotoxin shock, we injected KRN7000 at different time points of experimental systemic Shwartzman reaction. Mice treated with KRN7000 survived when it was injected within 2 h before and after LPS challenge. Mice survival was associated with low levels of T helper 1 (Th1) cytokines, such as IFN-γ and TNF-α. By contrast, protection from endotoxin shock was associated with an increase of T helper 2 (Th2) cytokines, like IL-4 and IL-10. A role of Th2 cytokines in counteracting LPS-induced shock was supported by experiments in which the protection against Shwartzman reaction by KRN7000 was abrogated by in vivo coadministration of anti-Th2 cytokines antibodies. In addition, cytofluorimetric analysis showed that surviving animals have higher percentages of NKT-IL-10-positive cells and lower percentages of NKT-IFN-γ and macrophages/TNF-α-stained cells than nonprotected mice. Taken together, our data demonstrate that KRN7000 treatment given at times near LPS challenge is protective for endotoxin shock inhibiting IFN-γ and TNF-α release. Moreover, KRN7000-mediated protection occurs through an increased production of IL-4 and IL-10, which are mainly secreted by NKT cells. Since IFN-γ release by NKT requires a longer TCR stimulation than that required for Th2 cytokines production, we demonstrate that timing of KRN7000 in vivo exposure affect the pattern of cytokines expression protecting animals by endotoxin shock.

Background iNKT cells represent a T cell subset at the bridge between innate and adaptive immunity, playing a role in regulating auto-antibody-producing B cells before their entry into germinal centers. Therefore the absence and/or reduction of iNKT cells seem to increase auto-reactive B cell activation. Primary Sjogren9s syndrome (pSS) is a systemic autoimmune disease in which lymphocyte infiltration and organization in lymphoid structures of inflamed salivary glands occur. Objectives The aim of this study was to investigate the frequency of iNKT in the salivary glands and peripheral blood of patients with pSS and their function by using CD1d/aGalactosylceramide (aGalaCer) tetramers. Methods Flow cytometry analysis of ex vivo frequency of tetramer+ cells, producing IFN-g and IL-17, and quantitative gene expression analysis by real-time PCR for Va24 chain expression was performed on salivary glands and peripheral blood obtained from patients and controls. Flow cytometric analysis and immune fluorescence for autoreactive B lymphocytes and ELISA for anti-SSA detection were also performed. Results A significant expansion of IL-17+- and IFN-γ+-producing iNKT cells, expressing low levels of the chemokine receptors CXCR3, CCR6 and CCR5, was observed in the peripheral blood of pSS patients. In the inflamed salivary glands, iNKT were absent whereas a significant increase of SSA specific B cells occurs. Moreover, co-culture experiments in vitro demonstrate that activated iNKT inhibit autoantibody production by autoreactive B cells from pSS patients. Conclusions An impaired tissue migration of iNKT may lead to the expansion of autoreactive B cells specific for SSA -antigen in salivary glands of patients, suggesting an important role of iNKT in regulating B cells activation in pSS. References Bendelac, A., P. B. Savage, and L. Teyton. The biology of NKT cells. Annu Rev Immunol 2007;25: 297-336. Yang JQ, Wen X, Kim PJ, Singh RR. Invariant NKT cells inhibit autoreactive B cells in a contact- and CD1d-dependentmanner. Invariant NKT cells inhibit autoreactive B cells in a contact- and CD1d-dependent manner. J Immunol. 2011 Feb 1;186(3):1512-20. Wu L, Gabriel CL, Parekh VV, Van Kaer L. Invariant natural killer T cells: innate-like T cells with potent immunomodulatory activities. Tissue Antigens 2009 Jun;73(6):535-45. Disclosure of Interest None declared

Transgenic mice expressing human major histocompatibility complex (MHC) class II molecules would provide a valuable model system for studying murine anti-human MHC immune response. We have previously shown that skin from HLA-DR1 transgenic mice was rejected by control littermates and spleen cells from rejecting mice were able to proliferate to donor cells. The aim of this paper is to analyze the mechanism of recognition of this xenoantigen and the possible involvement of antibody response in anti-HLA-DR1 immune response. Control littermates were immunized with spleen cells from HLA-DR1 transgenic (TG) mice; at indicated times, xenoantigen-specific proliferation and IFNgamma production was assessed using APC obtained from HLA-DR1 TG mice. Mixed direct-indirect pathway of xenoantigen recognition was suggested by the following findings: i)T cell response to HLA-DR1 was inhibited adding in culture monoclonal antibodies directed either to donor (HLA-DR) or to recipient MHC (I-A); ii) APC from control mice pulsed with purified DR1 molecules were able to induce proliferation by FVB/N mice immunized with transgenic spleen cells. HLA-DR1 recognition permits DR peptide-specific T cell response by lymphocytes of control littermates immunized with the xenoantigen. In addition, we detected xenoreactive IgM and IgG2 antibodies. Our data suggest that HLA-DR1 xenoantigen may be recognized through direct or indirect pathway and provide additional information on mouse anti-human HLA immune response.

Much evidence now indicates that human leucocyte antigen (HLA) class I and class II transgenic (Tg) mice can be of value in analysing HLA-restricted presentation of T-cell epitopes relevant to experimental models of autoimmune diseases. One area where this has been applied is the characterization of myelin epitopes presented by HLA class II molecules in experimental model of multiple sclerosis (experimental allergic encephalomyelitis (EAE)). As a first step towards humanized disease models in HLA Tg mice, we have analysed immune response of lymph node cells of HLA-DR1 Tg mice immunized with the human myelin basic protein (MBP) peptides 13–33, 87–106 and 139–154 bound by HLA-DR1. We report here that HLA-DR1 Tg mice display a hierarchy of response in vivo and in vitro to MBP epitopes depending on the binding affinity to DRB*0101 molecule. In fact, the 13–33 epitope induced a strong T helper 1 (Th1) response accompanied by high T-cell precursor frequency and caused mild EAE, while the two other epitopes gave poor (139–154) or no disease (87–106), and these data correlate with in vitro Th1 response. These data could prove a useful tool in understanding the role played by different MBP epitopes in EAE.

The experimental conditions influencing the use of Valphabeta TCR families were examined in lymph node (LN) cells from peptide-immunized C57BL/6 and Vbeta8.2 transgenic mice. Expanded proportions of Vbeta5, Vbeta8.2, Vbeta9, Vbeta12 and Vbeta14 positive cells and an association of Vbeta8.2 with Valpha11 was found in freshly harvested 8-day or 34-day immune LN cells. In contrast, peptide-specific T-cell lines generated in vitro from 8-day immune lymph node cells were found to be almost exclusively of the Valpha2/Vbeta12 family. However, T-cell lines originating from Vbeta8.2 transgenic mice did not show preferential Valpha usage. Anti-Vbeta8.2 antibody produced different effects: when added to cultures of LN cells from C57BL/6 or Vbeta8.2 transgenic strains, the peptide-induced proliferation was suppressed; however, following the injection of mice, subsequent in vitro proliferation and cytokine production induced by both peptide and Concanavalin A was suppressed in Vbeta8.2 transgenic, but much less in C57BL/6 mice. Hence, compensatory expansion of different Vbeta gene products occurred in vivo, but not under the employed in vitro conditions. In conclusion, these results suggest that the TCR family usage is influenced by the experimental conditions in which the T cells are selected and expanded and by the genetic potentials of the precursor pool.

Antigen (trinitrochlorobenzene)-specific T cell lines were obtained by repeated stimulation of lymph node cells from immune mice with antigen in vitro. These T cell lines, consisting of more than 90% CD4+ Vbeta8.2+ and 6 to 9% gammadelta+ T lymphocytes, transfer contact sensitivity (CS) locally when injected at the same site as the challenge antigen, but fail to mediate a systemic passive transfer when injected i.v. Injection of T cell lines together with spleen cells from mice immunized 1 day beforehand (1-day cells) allowed a successful, specific systemic transfer of CS. Phenotypic analysis showed that the 1-day immune cell was alphabeta+, gammadelta-, sIg-, CD3+, CD4-, CD8-, CD5+, B220 (CD45R)+, Thy 1.2+. The effect of 1-day immune cells occurred through a mechanism involving IL-4, as 1-day immune cells failed to allow systemic transfer of CS by T cell lines in recipient mice treated with mAb to IL-4. These observations strongly indicate that three cell subsets are required for the systemic passive transfer of CS by T cell lines: alphabeta+ CD4+, gammadelta+, and a third cell subset, which is CD45R+, alphabeta+, CD3+, but double (CD4, CD8) negative.

We have recently demonstrated a remarkable selection of in vitro cultivated, TNP-specific polyclonal T cell lines for the expression of a TCR beta chain encoded by the V beta 8.2 gene. The goal of the present study was to analyse V alpha usage in V beta 8.2 T cells responsive to TNP, using TNP-specific T cell lines derived from three common strains of mice, as well as from V beta 8.2 transgenic mice. Results indicate that in vitro TNP stimulation of T cells from TNP-immune mice results in significant skewing of V alpha usage among responding V beta 8.2+ T cells, with overexpression observed for V alpha 3.2 and V alpha 8. These results indicate that V alpha expression influences recognition of TNP by T cells, and suggest that the hapten TNP might be recognized like typical peptide antigens by combinatorial TCR alpha and beta contact sites.

This paper describes a novel primary in vivo/in vitro culture system which allows analysis of the effect of IL-4 added to culture 1 day after immunization on the production of IFN-gamma. Mice are immunized epicutaneously with picryl chloride (TNP) and draining lymph node cells were harvested 1 day later. These cells (1 day lymph node cells), when cultured in vitro for 3 days in the presence of IL-2, either continuously or as a pulse, give an IFN-gamma response on reexposure to antigen 3 days later. This production of IFN-gamma is both antigen-specific and genetically (MHC)-restricted and is due to both CD8+ and CD4+ T cells. However, if 1 day lymph node cells are cultured with both IL-2 and IL-4, no IFN-gamma is produced on subsequent reexposure to antigen, but the cells acquire the ability to produce IL-4 and IL-10. Moreover, cells pulsed with IL-4 blocked IFN-gamma production when cocultured with cells pulsed with IL-2. IL-4 only exerted its inhibitory activity on IFN-gamma production when added on the first or second day of culture and had no effect at later times. Finally, the inhibitory activity of IL-4 on IFN-gamma production may depend on the production of IL-10, induced by the IL-4, as the inhibitory effect of IL-4 is reversed by mAb against IL-10.

Lymph node cells of CBA (H-2k), but not of BALB/c (H-2d) mice immunized epicutaneously with picryl chloryde secrete interleukin (IL)-5 when stimulated with the specific antigen in vitro. The low IL-5 production in BALB/c mice persists when either picryl chloride or the unrelated antigen oxazolone are used, when the amount of antigen in vitro is varied and when a secondary response is studied. The difference in IL-5 production maps to the major histocompatibility complex (MHC) in the congenic BALB/b, BALB/c and BALB/k mice. Furthermore, lymph node cells from (k × d) F1 mice produce IL-5 when stimulated by antigen presented on H-2k but not on H-2d antigen-presenting cells. Finally, the low IL-5 production in vitro in BALB/c mice is correlated with low picryl-specific IgA levels in vivo, which otherwise are ten times greater in CBA and BALB/k mice. The influence of MHC on IL-5 production and IgA secretion in the mouse might be a possible basis for the association of MHC with IgA deficiency in humans.

Lymph node cells collected from CBA/J mice 4 days after painting with picryl chloride induce contact sensitivity in naive recipient mice by virtue of hapten IgM immuno complexes. The immunizing capacity of these cells (“4-day” cells) is abolished after incubation of the cells with a C4-deficient guinea pig serum reconstituted with plasma or purified C4 from mice with high C4 levels (C4h), but not with plasma or purified C4 from mice with low C4 levels (C41). The inhibition of the immunizing capacity of 4-day cells is due to the activation of the early components of the classical complement pathway which is likely to result in the solubilization of membrane-bound immunocomplexes. However, the same amounts of CBA/J and BALB/c C4 have a different effect in inhibiting the induction of contact sensitivity by 4-day cells. In fact, by doseresponse experiments, we have found that the amount of C41 able to inhibit the induction of contact sensitivity is about threefold higher than that of C4h. Analysis of the covalent binding ability of C4h and C41 reveals that C4h is able to bind to the surface of 4-day cells more efficiently than C41 and this probably accounts for the difference of the two C4 molecules in inhibiting the immunizing capacity of 4-day cells. Results are discussed in terms of different reactivities of C4h and C41 with the surface of 4-day cells.

This study investigated the origin of the different hemolytic activity of two allotypes of murine C4, C4H (C4-high) and C4L (C4-low) in the presence of IgM-coated erythrocytes. C4H displayed a threefold higher hemolytic titer (expressed in hemolytic units/microgram protein) than C4L. No difference was found between c4H and C4L either in stability at 37 degrees C at different pH values and in the rate of C4H and C4L hydrolysis by activated Cl. The major functional difference was found in the covalent binding capacity to IgM-coated erythrocytes, with the amount of C4H bound being about threefold higher than that of C4L. A marked difference in the reactivity of the C4b fragment of C4H and C4L toward amino and hydroxyl groups was detected. Using glycine and glucose to competitively inhibit the binding of C4H and C4L, it was observed that C4L selects preferentially for the amino groups of glycine, whereas C4H reacted preferentially with the hydroxyl groups of glucose. Furthermore, the chemical modification of the red cells amino groups by ethylacetimidate, had no appreciable effect on the binding of C4H, but reduced that of C4L. Southern blot hybridization experiments using Hind III-digested liver DNA and a cDNA probe derived from the 5' end of the mRNA (clone pAT-A) indicated that the C4H allele was associated with a 5-kb Hind III fragment, whereas the C4L allele was associated with a 15-kb Hind III fragment. The possibility that a structural difference between C4H and C4L is responsible for their different reactivities is discussed.

SUMMARY The present paper reports evidence indicating that the level of the third complement component (C3) is regulated by the S region of the murine H-2 complex. In fact, using congenic strains of mice we demonstrate that mice with the k haplotype at the S region show high C3 levels, whereas mice with the d haplotype at the S region show low C3 levels.

This review describes the phenomenon of the major histocompatibility complex (MHC) control of cytokine production both in experimental animals and in humans. H-2 (mouse MHC) regulates which type of cytokine is selectively produced in response to the hapten trinitrophenyl (TNP). T cells from TNP-immune H-2k mice produce interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-3, IL-5, tumor necrosis factor-alpha (TNF-alpha), IL-10, and very low levels of IL-4 on reexposure to the specific antigen in vitro. By contrast, T cells from H-2d mice produce IL-3, TNF-alpha, IL-10, and IL-4 but very low levels of IL-2, IL-5 and IFN-gamma. As MHC-congenic matched strains (BALB/k and BALB/c) are used, this makes it unlikely that non-MHC genes influence the class of response observed. A similar pattern of haplotype regulation of cytokine production is observed in humans. In fact, peripheral blood mononuclear cells from HLA-B8,DR3-positive and negative individuals differ in their ability to produce IL-2, IL-5, and IFN-gamma on stimulation with the mitogen phytohemagglutinin while producing similar amounts of IL-4, IL-6, and IL-10. The following main considerations emerge from these observations. The MHC/peptide complex generated after antigen immunization, indicates which class of cytokine production is preferentially induced and, therefore, the outcome of the immune response. Furthermore, MHC genotype may affect cytokine production (and then immune responses) by completely different mechanism(s), that is, by an antigen-nonspecific control that does not depend on the ability of MHC molecules to bind in different ways the different peptides. Accurate control of the functional repertoire of an immune response is a critical parameter in response to infections as well as in immunopathology. MHC control of the class of the immune response at the level of cytokine production is a sophisticated way in which this occurs. This control might be involved in adaptive immune responses to infections as well as in immunopathology.