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Experimental autoimmune encephalomyelitis, originally experimental allergic encephalomyelitis, is the well-known animal model of multiple sclerosis, an immune- mediated, demyelinating, inflammatory chronic disease of the central nervous system. The experimental disease is widely utilized to test new therapies in preclinical studies, to investigate new hypothesis on the possible pathogenic mechanisms of autoimmune reaction directed against the central nervous system or more generally to investigate the interactions between the immune system and the central nervous system that lead to neuroinflammation. The experimental autoimmune encephalomyelitis may be induced following different protocols in mammals, including nonhuman primates, and autoreactive CD4+ T-lymphocytes directed against myelin antigens are the main factors. Here, after introducing the model, we describe the protocol to induce active EAE in inbred mice, we report on a table the different clinical courses of EAE depending on the combination of antigen /mouse strain and we provide indications on how to evaluate the clinics and pathology of this induced disease.

Objective: Cordycepin has been shown to exhibit multiple pharmacological activities, such as antitumor, antifungi, antivirus, and immune-regulation activities, and is involved in the regulation of T cells. However, cordycepin that affects T cell activity is still not clear, and the molecular mechanism of cordycepin in regulation of TCR signaling has not yet been elucidated. In this study, the potential effect of cordycepin on T cells was observed in CFA-induced inflammation mice model, and the function of cordycepin in regulating TCR signaling cascade was investigated. Methods: A CFA-induced inflammation mice model was established for observing the effect of cordycepin on the thymus and spleen swellings, and T cell infiltration in paw tissue was detected by immunohistochemistry. The protein expression or phosphorilation was detected by western blotting, and the NFAT1 nuclear translocation was determined by fluorescence imaging. The cell proliferation, apoptosis, and IL-2 production were analyzed by CCK-8 method, flow cytometry, and ELISA. Results: In the mice model, the thymus and spleen swellings were suppressed and the T cell infiltration in paw tissue was inhibited by cordycepin at a concentration of 10 mg/kg. Although the expressions of ZAP70 and PLCγ1 were not significantly changed in the human T cell line Jurkat with cordycepin pretreatment, the CD3-antibody-induced phosphorylations of ZAP70 and PLCγ1 were markedly blocked. The protein level of p85 decreased when Jurkat cells were pretreated with cordycepin, and cordycepin blocked TCR downstream molecule Erk phosphorylation and NFAT1 nuclear translocation. Further investigation revealed that cordycepin inhibited T cell proliferation, reduced IL-2 production, and induced T cell apoptosis. Conclusions: These findings suggest that cordycepin regulates TCR signaling to inhibit excessive T cell activation in inflammation. Thus, cordycepin may be a potential therapeutic application in inflammation-associated diseases.

The Fcγ receptor IIIA (FcγRIIIA) has traditionally been known as a positive regulator of immune responses. Consistent with this, mice deficient in FcγRIIIA are protected from various inflammation-associated pathologies including several autoimmune diseases. In contrast to this accepted dogma, we show here that mice lacking FcγRIIIA developed increased rather than reduced both humoral and cellular immune responses to mucosal (sublingual) immunization with ovalbumin (OVA) given together with the strong mucosal adjuvant cholera toxin as well as to parenteral (subcutaneous) immunization with OVA in complete Freund's adjuvant. After either route of immunization, in comparison with concomitantly immunized wild-type mice, FcγRIIIA -/- mice had increased serum anti-OVA IgG (IgG1 but not IgG2) antibody responses as well as augmented cellular responses that included memory B cells and effector T cells. The increments in immune responses in FcγRIIIA -/- mice were similar to those seen in FcγRIIB -/- mice. Furthermore, OVA-pulsed FcγRIIIA -/- DCs, similar to OVA-specific FcγRIIB -/- DCs, had enhanced capacity to activate OVA-specific OT-II T cells, which was even further pronounced when DCs were pulsed with IgG1-complexed OVA. Our data support an inhibitory-regulatory role of FcγRIIIA on vaccine/adjuvant-induced immune responses and demonstrate that lack of FcγRIIIA can promote rather than suppress both humoral and cellular immune responses., (© 2017 The Foundation for the Scandinavian Journal of Immunology.)

Objectives: Indoleamine 2,3-dioxygenase-1 (IDO1) is an immune-modulatory enzyme that catalyzes the degradation of tryptophan (Trp) to kynurenine (Kyn) and is strongly induced by interferon (IFN)-γ. We previously reported highly increased levels of IFN-γ and corresponding IDO activity in patients with hemophagocytic lymphohistiocytosis (HLH), a hyper-inflammatory syndrome. On the other hand, IFN-γ and IDO were low in patients with systemic juvenile idiopathic arthritis (sJIA), an autoinflammatory syndrome. As HLH can occur as a complication of sJIA, the opposing levels of both IFN-γ and IDO are remarkable. In animal models for sJIA and HLH, the role of IFN-γ differs from being protective to pathogenic. In this study, we aimed to unravel the role of IDO1 in the pathogenesis of sJIA and HLH., Methods: Wild-type and IDO1-knockout (IDO1-KO) mice were used in 3 models of sJIA or HLH: complete Freund's adjuvant (CFA)-injected mice developed an sJIA-like syndrome and secondary HLH (sHLH) was evoked by either repeated injection of unmethylated CpG oligonucleotide or by primary infection with mouse cytomegalovirus (MCMV). An anti-CD3-induced cytokine release syndrome was used as a non-sJIA/HLH control model., Results: No differences were found in clinical, laboratory and hematological features of sJIA/HLH between wild-type and IDO1-KO mice. As IDO modulates the immune response via induction of regulatory T cells and inhibition of T cell proliferation, we investigated both features in a T cell-triggered cytokine release syndrome. Again, no differences were observed in serum cytokine levels, percentages of regulatory T cells, nor of proliferating or apoptotic thymocytes and lymph node cells., Conclusions: Our data demonstrate that IDO1 deficiency does not affect inflammation in sJIA, sHLH and a T cell-triggered cytokine release model. We hypothesize that other tryptophan-catabolizing enzymes like IDO2 and tryptophan 2,3-dioxygenase (TDO) might compensate for the lack of IDO1.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis, a chronic inflammatory and demyelinating disease of the CNS. Albino Oxford (AO) rats are resistant to the induction of EAE, while the disease can be readily induced in Dark Agouti (DA) rats. Here we investigated a potential contribution of the CNS milieu in the limitation of the encephalitogenic autoimmune response. EAE was induced by immunization of the respective rat strains with spinal cord homogenate emulsified in complete Freund's adjuvant. AO rats did not exhibit clinical signs after immunization while DA rats developed severe neurologic deficits. Infiltration of immune cells into spinal cords (SC) was evident in both strains 12-14 days after the immunization. EAE lesions of AO rats contained substantially lower numbers of CD4+ T cells and CD11b+ cells compared to those in DA rats. This went together with lower levels of interferon (IFN)-γ and interleukin (IL)-17 in the cells isolated from SC. We found a dramatic increase of CXCL12 expression in SC tissue and microvessels of AO rats, whereas DA rats markedly decreased the expression of this chemokine within their CNS. Administration of the CXCL12 antagonist AMD3100 to a substrain of AO rats that developed a weak EAE led to earlier onset and exacerbation of the disease. These results suggest a role of CXCL12 in down-regulating autoimmune processes in AO rats during EAE. Therapeutic modulation of CXCL12 could be a promising strategy for the treatment of CNS autoimmunity., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Originally identified as antiviral substances produced by infected cells, type I interferons (IFN-I) are now known to have a wide range of additional activities within both the innate and adaptive immune response. Here we review properties of IFN-I contributing to their 'natural immune adjuvant' character, and their important role for the function of complete Freund's adjuvant (CFA) and the TLR9-dependent immune adjuvant IC31. We show data to demonstrate that treatment with IFN-I boosts the ability of vaccine/adjuvant combinations to induce peptide-specific CTL in both young and old mice. We view these findings in the perspective of previous clinical applications of IFN-I for vaccination.

The effect of soluble antigenic (bovine serum albumin, BSA) stimulation to induce steroidogenesis in murine lymphoid organs with concomitant changes in proinflammatory or inflammatory cytokine levels and its implication in the alteration of T-cell response was studied in the mice. Male Swiss albino mice (6-8 weeks old) with average body weight (20 +/- 4 g) were randomly assigned to 3 groups and injected with BSA in presence and absence of Freund's complete or incomplete adjuvant, whereas the control group received only saline. After 3 weeks, animals were sacrificed, and serums as well as lymphoid organs were collected. From the lymphoid tissue homogenate, the activities of steroidogenic enzymes and corticosterone and cytokine levels of the serum were estimated. Steroidogenic enzyme activities in murine lymphoid organs, as well as the pro-inflammatory and inflammatory cytokines levels in serum increased after Freund's complete adjuvant-emulsified BSA administration, as compared to control. The serum corticosterone and serum cytokine profile were also elevated. Results suggested that soluble protein antigen (BSA) administration stimulated steroidogenesis in murine lymphoid tissues and rise in the pro-inflammatory or inflammatory cytokine levels might indicate monocyte recruitment as well as TH1 activation.

NY-ESO-1 is a "cancer-testis" antigen expressed in epithelial ovarian cancer (EOC) and is among the most immunogenic tumor antigens defined to date. The NY-ESO-1 peptide epitope, ESO(157-170), is recognized by HLA-DP4-restricted CD4+ T cells and HLA-A2- and A24-restricted CD8+ T cells. To test whether providing cognate helper CD4+ T cells would enhance the antitumor immune response, we conducted a phase I clinical trial of immunization with ESO(157-170) mixed with incomplete Freund's adjuvant (Montanide ISA51) in 18 HLA-DP4+ EOC patients with minimal disease burden. NY-ESO-1-specific Ab responses and/or specific HLA-A2-restricted CD8+ and HLA-DP4-restricted CD4+ T cell responses were induced by a course of at least five vaccinations at three weekly intervals in a high proportion of patients. There were no serious vaccine-related adverse events. Vaccine-induced CD8+ and CD4+ T cell clones were shown to recognize NY-ESO-1-expressing tumor targets. T cell receptor analysis indicated that tumor-recognizing CD4+ T cell clones were structurally distinct from non-tumor-recognizing clones. Long-lived and functional vaccine-elicited CD8+ and CD4+ T cells were detectable in some patients up to 12 months after immunization. These results confirm the paradigm that the provision of cognate CD4+ T cell help is important for cancer vaccine design and provides the rationale for a phase II study design using ESO(157-170) epitope or the full-length NY-ESO-1 protein for immunotherapy in patients with EOC.

Nontypeable Haemophilus influenzae (NTHI) is a common cause of otitis media in children and lower respiratory tract infection in adults with chronic lung disease. The highly conserved P6 protein of NTHI infection is under evaluation as a vaccine antigen in several animal models. To elucidate the role of cellular immune response to P6 in protective immunity, the goal of this study was to identify and characterize T cell epitope(s) on P6 and to investigate the role of these epitope(s) in eliciting antigen specific antibody responses and in mediating pulmonary clearance of NTHI. We report that T cells from BALB/c immunized with P6 recognize a single, immunodominant region, represented by 15 amino acids (residues 41-55) of the P6 protein. To verify the ability of this epitope to elicit T cell responses to the P6 protein, mice were immunized with a synthetic peptide corresponding to the sequence of dominant peptide. T cells isolated from mice primed in vivo with the peptide responded following in vitro stimulation with either the peptide or with the whole P6 molecule. Substitution of single amino acids and N or C terminal truncations of the dominant peptide resulted in complete abrogation of the response, implicating their importance to the T cell response. Furthermore, mucosal immunization of mice with a chimeric peptide that encompassed the dominant T cell epitope and a putative B cell epitope resulted in enhanced bacterial clearance following pulmonary challenge with NTHI. Collectively, these results establish that, in a mouse model, P6 contains a single immunodominant T cell epitope and this epitope plays an important role in protective immune responses induced by immunization with P6.

Purpose: An existing immunological paradigm is that high concentrations of soluble protein contribute to the maintenance of peripheral tolerance/ignorance to self protein. We tested this hypothesis in a clinical immunotherapy trial using class I-restricted peptide epitopes derived from alpha-fetoprotein (AFP). AFP is a self protein expressed by fetal liver at high levels, but transcriptionally repressed at birth. AFP is de-repressed in a majority of hepatocellular carcinomas (HCCs) and patients with active disease can have plasma levels in the mg/ml range. We previously identified four immunodominant HLA-A*0201-restricted peptides derived from human AFP that could stimulate specific T-cell responses in normal volunteer peripheral blood lymphocytes cultures. We wished to test the hypothesis that AFP peptide-reactive T cells could be expanded in vivo in HCC patients immunized with these four AFP peptides., Experimental Design: We undertook a pilot Phase I clinical trial in which HLA-A*0201 patients with AFP-positive HCC were immunized with three biweekly intradermal vaccinations of the four AFP peptides (100 microg or 500 microg each) emulsified in incomplete Freund's adjuvant., Results: All of the patients (n=6) generated T-cell responses to most or all of the peptides as measured by direct IFNgamma enzyme-linked immunospot (ELISPOT) and MHC class I tetramer assays., Conclusions: We conclude that the human T-cell repertoire is capable of recognizing AFP in the context of MHC class I even in an environment of high circulating levels of this oncofetal protein.

Background: T helper type 1 (Th1) response plays a permissive role in atherosclerosis. We hypothesized that adoptive transfer of a novel subtype of T lymphocytes called regulatory T cells type 1 (Tr1) would inhibit Th1 responses by inducing a bystander immune suppression and therefore limit the development of atherosclerosis., Methods and Results: Clones of ovalbumin (OVA)-specific Tr1 cells expanded in vitro were administered intraperitoneally (106 cells per mouse) with their cognate antigen (50 microg of OVA subcutaneously in complete Freund's adjuvant [CFA]) to female apolipoprotein E-knockout mice. A group of mice received only (OVA/CFA) immunization without Tr1 cells. Two other control groups received no immunization and were injected with either Tr1 cells or saline. After 9 weeks of treatment, mice injected with (OVA/CFA)+OVA-specific Tr1 cells showed a significant decrease in Th1 responses, as revealed by a decrease in OVA-specific IgG2a serum levels (P<0.0001), a decrease in the production of interferon-gamma (P<0.001), and an increase in interleukin-10 production (P<0.001) by cultured spleen and lymph T cells compared with controls. In addition, cytokine production by concanavalin A-stimulated spleen cells showed a clear switch to a regulatory immune response in mice treated with (OVA/CFA)+Tr1. This was associated with a significant reduction in atherosclerotic lesion size in both the thoracic aorta and aortic sinus of mice treated with (OVA/CFA)+Tr1 compared with controls (P=0.002 to P<0.0001). Plaques of mice injected with (OVA/CFA)+Tr1 showed significantly lower accumulation of macrophages and T cells than plaques of control mice., Conclusions: Tr1-type regulatory immune response reduces the development of experimental atherosclerosis.

Memory and effector T cells modulate subsequently primed T cell responses to the same antigen. However, little is known about the impact of pre-existing memory and effector T cell immunity on subsequently primed immune responses to unrelated antigens. Here, we show that an antigen-primed first wave of Th1 and Th2 immunity enhanced or inhibited the subsequently primed T cell immunity to an unrelated antigen, depending on whether the second antigen was administered in the same or opposite type of adjuvant. The regulatory effects of the first wave of T cell immunity on the subsequent T cell responses to an unrelated antigen attenuated over time. Notably, following challenge with the second antigen, there was a mutual cross-regulation between the first and second wave of humoral responses to unrelated antigens. Thus, immunization with one antigen not only primes immune responses to that antigen, but also influences subsequently primed immune responses to unrelated antigens.

Objective: CD4+,CD25+ T regulatory cells may offer opportunities to intervene in the course of autoimmune disease. We wished to evaluate their potential for influencing systemic and chronic joint inflammation by investigating their involvement in collagen-induced arthritis (CIA)., Methods: We depleted DBA/1 mice of CD25+ regulatory cells by injection of a depleting monoclonal antibody specific for CD25 14 days before a single immunization with type II collagen (CII) in Freund's complete adjuvant. CD4+,CD25+ T cells were adoptively transferred to some groups of mice during immunization. Mice were then scored for signs of arthritis, and blood was taken periodically to measure the amounts of CII-specific antibodies. Splenocytes of treated mice were examined in vitro to determine the effects of depletion on proliferation to CII and control antigens., Results: CD25+ cell-depleted DBA/1 mice had significantly more severe disease than control mice following collagen immunization. The magnified severity was also accompanied by higher antibody titers against collagen, and in vitro tests showed increased proliferation of collagen-specific T cells. Adoptively transferring CD4+,CD25+ T cells into depleted mice was shown to reverse the heightened severity. Control mice, which were depleted and immunized with the neoantigen keyhole limpet hemocyanin (KLH), had neither an increased antibody response toward KLH nor an augmented proliferative response, indicating that CD25+ cell depletion preferentially affects immunity against self antigen., Conclusion: These results establish a link between CD4+,CD25+ regulatory cells and CIA and provide a rationale for investigating CD4+,CD25+ T regulatory cells in the treatment and prevention of arthritis.

Helicobacter pylori infection of the gastric mucosa is a significant cause of morbidity and mortality because of its etiologic role in symptomatic gastritis, peptic ulcer disease, and gastric adenocarcinoma. Infection occurs in young children; therefore, a prophylactic vaccine would have to be administered within the first year of life, a period thought to be immunologically privileged. We investigated vaccine formulations administered by different routes to confer protective anti-H. pylori immunity in neonatal mice. Neonatal mice immunized with a single dose of vaccine in complete Freund's adjuvant (CFA) generated antigen-specific gamma interferon-, interleukin-2 (IL-2)-, IL-4-, and IL-5-secreting T cells in numbers similar to those in immunized adult mice, while vaccine administered to neonates in incomplete Freund's adjuvant (IFA) induced such cells in reduced numbers compared to those in adult mice. Both IFA and CFA, however, provided partial protection from a challenge with infectious H. pylori when the vaccine was administered subcutaneously. Neonatal immunized mice also had reduced bacterial loads when immunized intraperitoneally with CFA. In all cases, protection was equivalent to that achieved when adult counterparts were immunized. These studies suggest that an efficacious vaccine might be successfully administered to very young children to prevent perinatal infection of H. pylori.

We conducted a randomized trial in HLA*A0201+ patientswith American Joint Committee on Cancer stage III or IV melanoma immunized with tyrosinase 368-376(370D) peptide and gp100 209-217(210M) peptide to compare the potency of three different adjuvants. Patients received 3 monthly immunizations with 500 microg of each peptide either with incomplete Freund's adjuvant (IFA), QS-21, or granulocyte macrophage colony-stimulating factor (GM-CSF). The primary end point was induction of IFN-gamma release by CD8+ T cells against tyrosinase and gp100 peptides measured by enzyme-linked immunospot assays without in vitro prestimulation measured pretreatment, 2 and 8 weeks after the third vaccination. Four of 9 and 4 of 8 patients immunized using QS-21 and GM-CSF, respectively, developed increased frequencies of CD8+ T cells against tyrosinase 370D peptide compared with 0 of 9 patients immunized using IFA (P = 0.045). T-cell responses against a gp100-related peptide showed similar results, but their relevance to T-cell reactivity against native gp100 209-217 is uncertain. These results show that: (a) QS-21 and GM-CSF are superior to IFA as immunological adjuvants for vaccination against tyrosinase 370D peptide; and (b) with appropriate adjuvants, increased frequencies of peptide-specific T cells after vaccination can be detected by enzyme-linked immunospot without prolonged prestimulation in vitro.

Lewis rats immunized with guinea pig myelin basic protein (GPBP) emulsified with incomplete Freund's adjuvant (IFA) do not develop experimental autoimmune encephalomyelitis (EAE). However, we found that GPBP/IFA with pertussis toxin (PT) administration induced full-blown EAE. By comparing the immunological status of rats immunized with GPBP/IFA plus PT [PT (+) rats] with that of rats immunized with GPBP/IFA alone [PT (-) rats], we tried to elucidate the pathomechanisms of EAE. Analysis of the TCR clonality by CDR3 spectratyping revealed that Vbeta8.2 and Vbeta10 expansion of T cells occurred in both PT (-) and PT (+) rats, indicating that activation of T cells at this level is not sufficient for the development of clinical EAE. Quantitation of cytokine mRNA and protein revealed that PT (-) rats showed a Th2-dominant, while PT (+) rats showed a Th1-dominant, cytokine profile. Furthermore, administration of IL-12, but not of IFN-gamma and TNF-alpha, induced clinical EAE in GPBP/IFA-immunized animals. Taken together, two-step activation, activation of T cells bearing a particular type of TCR by antigen immunization and subsequent overproduction of Th1 cytokines, mainly IL-12 production, induced by appropriate adjuvants is essential for the development of clinical EAE.

Myelin basic protein (MBP)-specific T cells play a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a prototype for T cell-mediated autoimmunity. In PL/J and B10.PL mice (H-2(u) haplotype), the immunodominant epitope of MBP is represented by an N-terminal nonameric peptide, MBP1-9. To date, the MBP1-9-specific T cell repertoire has not been analyzed in quantitative terms. In the present study we demonstrate, using MHC class II tetramers, that 15,000-70,000 self-antigen-specific T(h) cells accumulate in the draining lymph nodes following immunization with spinal cord homogenate or MBP1-9. In contrast, MBP1-9-specific T cells are undetectable in unimmunized H-2(u) mice and represent >60% of the CD4 cells in naive mice transgenic for a TCR specific for this epitope. The results suggest that the extremely low affinity of the N-terminal peptide for I-A(u) does not limit the MBP1-9-specific T cells from expanding into a sizeable pool of autoreactive T cells. Therefore, the primary immune response to MBP1-9 does not differ quantitatively from previously reported CD4(+) T cell responses to foreign antigens.

Adoptive transfer of quiescent encephalitogenic T cells to normal syngeneic recipients was without clinical effect. RT-PCR was used to assess localization of an adoptively transferred quiescent encephalitogenic T cell clone in normal and antigen-unresponsive mice prior to or after challenge with neuroantigen/CFA. The T cell clone was not detectable in lymphoid tissues prior to challenge with neuroantigen; however, following challenge, the clone was found in the spleen, lymph nodes and spinal cord of both normal and antigen-tolerized mice. The latter animals remained clinically normal. Non-activated encephalitogenic T cells transferred to wild-type recipients pretreated i.p. with neuroantigen/IFA were rendered unresponsive. Transfer of the same T cells to alpha/beta T cell-deficient mice pretreated with neuroantigen/IFA resulted in spontaneous disease indicating that an intact alpha/beta T cell system was required for development of the unresponsive state.

We have previously reported that CD4+ T cells recognizing a peptide comprising residues 234-252 of the heat shock protein (HSP)70 of Mycobacterium tuberculosis (M.tb) in the context of RT1.B MHC class II molecule emerged in the peritoneal cavity during the course of Listeria monocytogenes infection in rats and suppressed the inflammatory responses against listerial infection via IL-10 production. We report in this work that pretreatment with peptide 234-252 of HSP70 derived from M.tb suppressed the development of adjuvant arthritis (AA) in Lewis rats induced using heat-killed M.tb. T cells from rats pretreated with peptide 234-252 produced a significant amount of IL-10 in response to the epitope. T cells from rats pretreated with the peptide and immunized with M.tb produced the larger amount of IL-10 in response to the peptide, but only a marginal level of IFN-gamma in response to purified protein derivative of M.tb. Administration of anti-IL-10 Ab partly inhibited the suppressive effect of pretreatment with peptide 234-252 on the development of AA. Furthermore, transfer of a T cell line specific for the epitope at the time of AA induction markedly suppressed AA. These findings suggested that T cells recognizing peptide 234-252 may play a regulatory role in inflammation during AA via the production of suppressive cytokines including IL-10.

Immune stimulating complexes (iscoms) are 40 nm particles combining adjuvant-active Quillaja saponins and multimeric presentation of antigens. The distribution in mice of influenza virus iscoms and the resulting T cell responses in the lymph nodes (LN) and spleen were characterized. After a single subcutaneous injection, iscoms were delivered to the draining LN where they induced a transient population of LN cells which responded with proliferation and secretion of interleukin-2 (IL-2), gamma-interferon (IFN-gamma) and interleukin-4 (IL-4) after restimulation. The response in the spleen developed more slowly, sustained for 12 weeks and was characterized by cells producing in particular IL-2 and IFN-gamma but also IL-4. A booster resulted in a dramatic enhancement of the production of IFN-gamma, indicating that iscoms efficiently recruit cells with Th1 properties. Comparisons of T cell responses to iscoms and to influenza virus antigen in Freund's complete adjuvant demonstrate that these adjuvants affect both the localization and cytokine profile of T cell responses.

This study identifies activation characteristics of PPD-responsive T-cells that emerge after treatment with anti-CD4 monoclonal antibody (Mab). PPD-stimulated T-cell proliferations, OX40 phenotype and protein tyrosine phosphorylations involving p56lck (pp56lck) were compared to Con A stimulations using T-cells isolated from spleen and draining lymph node of CFA/PPD-immunized rats either untreated or treated in vivo with anti-CD4 Mab. Splenocytes stimulated by concanavalin A (Con A) showed correlated increases in proliferation, levels of pp56lck, and OX40 expression; these parameters were not correlated in splenocytes after PPD-stimulations. T-cells isolated from lymph nodes draining the site of CFA/PPD immunization proliferated in response to stimulation by either PPD or Con A, but only PPD-responsive cells showed correlation to the OX40 activation phenotype and increased levels of pp56lck. CD4+ T-cells isolated from either tissue compartment after anti-CD4 Mab treatments showed higher background and PPD-stimulated proliferations, and expressed lower levels of OX40. In contrast, anti-CD4 Mab treatments reduced (60%) and abolished Con A-stimulated proliferations of splenocytes and lymph node T-cells, respectively. The effects of anti-CD4 Mab treatment on pp56lck levels correlated only to the changes observed for Con A stimulations of splenocytes. These results demonstrate that PPD antigen-specific T-cell populations recovered from different tissue compartments were resistant to in vivo anti-CD4 Mab treatments and did not show the activation changes characteristics of CD4+ T-cells after Con A stimulation.

We have studied the reactivity of chicken T cells to mycobacterial antigens. Neither peripheral blood nor splenic lymphocytes isolated from unprimed chickens proliferated in response to mycobacterial antigens (mycobacterial sonicate, purified protein derivative, or recombinant 65-kDa heat-shock protein HSP65). After immunization with complete Freund's adjuvant (CFA) a strong response appeared, and a transient increase of peripheral blood gamma delta T cells was observed. Analysis of spleen cells isolated from CFA-primed chickens showed that both alpha beta and gamma delta T cells were activated by the mycobacterial antigens, apparently at equal levels. Both subsets also responded to HSP65, but no preference of gamma delta T cells to respond to it or any of the other mycobacterial antigens was observed. These results indicate that although HSP65 is an important mycobacterial antigen, it is not dominant in the chicken gamma delta T cell repertoire. Moreover, the results show a clear distinction between the naive and primed repertoire of gamma delta T cells.

We have recently reported antigenic (B-cell) cross-reactivity between the mycobacterial 65 kDa heat shock protein (hsp65) and human lactoferrin (LF) and we suggested that this cross-reactivity might have a role in mycobacteria-associated autoimmune disease. Here, we have searched for anti-LF T-cell reactivity in Lewis rats submitted to a mycobacteria-triggered autoaggressive disorder (adjuvant arthritis, AA), an autoimmune disorder characterized by high anti-hsp65 reactivity. We have quantified the in vitro proliferative response to LF of lymph node and spleen cells of Lewis rats killed 9, 14 and 21 days after the immunization with the AA-triggering, mycobacteria-containing adjuvant (complete Freund's adjuvant, CFA). We found that LF induced significant proliferation of lymph node T cells of rats undergoing AA. This T-cell proliferation was not as marked as the one provoked by hsp65; it was, nevertheless, significantly higher (P < 0.05) than that produced by a non-arthritogenic antigen (i.e. albumin). T cells from naive or mineral oil (incomplete Freund's adjuvant, IFA) injected rats did not respond to LF or hsp65. These data indicate that LF may work as an accessory stimulatory factor of the T-cell autoreactivity associated with mycobacteria-induced arthritis.

Insulin-dependent diabetes mellitus is an autoimmune disease that is characterized by the destruction of insulin-producing beta cells in the islet of Langerhans. We have recently reported that the induction of the disease in nonobese diabetic (NOD) mice can be prevented by a single injection of CFA. In this study, we have explored the cellular basis and the time course of the disease protection. Since CFA contains a mycobacterial cell wall that has adjuvant property, we investigated the protective role of mycobacteria in young NOD mice. Mice injected with Mycobacterium tuberculosis or Mycobacterium bovis (BCG vaccine) at 4 wk of age were also found to be protected from diabetes. We have found that complete protection from diabetes is only achieved by administration of CFA between 4 and 10 wk of age. Draining lymph node cells or spleen cells from CFA-treated NOD mice transfer the protection. Adoptive transfer of spleen cells from CFA-treated mice with spleen cells from acutely diabetic mice delayed the induction of disease into irradiated recipient mice. CFA-treated old NOD mice were also resistant to passive transfer of disease by spleen cells from acutely diabetic mice. Depletion of the Thy 1.2+ cells or CD4(+)-bearing T cells abrogated the protection. However, disease can be induced in the protected mice by cyclophosphamide treatment. We also found that thymocytes from NOD mice responded only weakly to mitogen Con A. CFA treatment, however, restored the ability of these cells to respond to Con A. Finally, our results suggest that T cells induced after CFA treatment of NOD mice prevent both the induction and effector phases of the disease.

Injection of incomplete Freund's adjuvant (IFA) into the footpads of BALB/c mice induced an acute inflammation. Draining popliteal lymph nodes showed major histocompatibility complex (MHC) class II-restricted proliferation when challenged in vitro with recombinant Mycobacterium bovis 65-kDa heat shock protein (hsp65). alpha beta T cell receptor-positive, CD4+, hsp65-specific T cell lines and clones were generated from these lymph nodes, and 87% of clones responded to a beta galactosidase fusion protein containing residues 238-573 of human hsp60. Seventy percent of these hsp60-responsive clones also responded to a synthetic peptide corresponding to residues 412-423 of the mouse hsp60. This peptide also induced significant responses in IFA-primed lymph node cells but not in lymphoid cells from unimmunized mice. These results demonstrate that T cells specific for epitopes in self hsp60 are activated during inflammatory responses induced in the absence of exogenous bacterial hsp65. The findings of this study may provide a basis for understanding the often reported isolation of mycobacterial hsp65-responsive T cells from inflammatory sites of arthritis patients, and the protective effects of preimmunization with hsp65 in experimental models of arthritis.

Initial studies determined whether intraperitoneal (i.p.) injection of BALB/c mice with 0.1, 1.0, and 10 mg of adriamycin (a cardiotoxic anthracycline antibiotic) for times ranging between 1 and 9 weeks prior to i.p. injection of 10(5) PFU of coxsackievirus B3 (CVB3) would alter the severity of virus-induced myocarditis. Prior adriamycin exposure enhanced pathogenicity of a poorly pathogenic CVB3 variant (H310A1) but had no effect on myocarditis produced by the pathogenic variant (H3). Cardiac virus concentrations were equivalent in H3- and H310A1-infected mice irrespective of adriamycin treatment. BALB/c mice treated with either 0.1 ml of complete Freund's adjuvant (CFA), 10 mg of adriamycin, or 10(5) PFU of H3 and H310A1 i.p. developed cytolytic Thy 1.2+ lymphocytes (CTL) to H3-infected myocytes 7 days later. CFA-, adriamycin-, and H3-treated mice developed CTL expressing the gamma delta+ T-cell receptors, while H310A1-infected animals did not. Only H3- and H310A1-infected mice developed alpha beta+ CTL. Treatment of BALB/c mice with 0.1 ml of CFA 5 days prior to H310A1 infection dramatically increased myocarditis. Selective depletion of gamma delta+ T cells abrogated this effect. The ability of gamma delta+ T cells to augment the pathogenicity of H310A1 infection was confirmed by adoptive transfer of CFA-stimulated T cells depleted of either gamma delta- or gamma delta+ cells into H310A1-infected recipients.