Short-term T-cell Lines Research Articles

Varicella-zoster virus proteome-wide T-cell screening demonstrates low prevalence of virus-specific CD8 T-cells in latently infected human trigeminal ganglia

BackgroundTrigeminal ganglia (TG) neurons are an important site of lifelong latent varicella-zoster virus (VZV) infection. Although VZV-specific T-cells are considered pivotal to control virus reactivation, their protective role at the site of latency remains uncharacterized.MethodsPaired blood and TG specimens were obtained from ten latent VZV-infected adults, of which nine were co-infected with herpes simplex virus type 1 (HSV-1). Short-term TG-derived T-cell lines (TG-TCL), generated by mitogenic stimulation of TG-derived T-cells, were probed for HSV-1- and VZV-specific T-cells using flow cytometry. We also performed VZV proteome-wide screening of TG-TCL to determine the fine antigenic specificity of VZV reactive T-cells. Finally, the relationship between T-cells and latent HSV-1 and VZV infections in TG was analyzed by reverse transcription quantitative PCR (RT-qPCR) and in situ analysis for T-cell proteins and latent viral transcripts.ResultsVZV proteome-wide analysis of ten TG-TCL identified two VZV antigens recognized by CD8 T-cells in two separate subjects. The first was an HSV-1/VZV cross-reactive CD8 T-cell epitope, whereas the second TG harbored CD8 T-cells reactive with VZV specifically and not the homologous peptide in HSV-1. In silico analysis showed that HSV-1/VZV cross reactivity of TG-derived CD8 T-cells reactive with ten previously identified HSV-1 epitopes was unlikely, suggesting that HSV-1/VZV cross-reactive T-cells are not a common feature in dually infected TG. Finally, no association was detected between T-cell infiltration and VZV latency transcript abundance in TG by RT-qPCR or in situ analyses.ConclusionsThe low presence of VZV- compared to HSV-1-specific CD8 T-cells in human TG suggests that VZV reactive CD8 T-cells play a limited role in maintaining VZV latency.

Phenotypic and functional characterization of T cells in white matter lesions of multiple sclerosis patients

T cells are considered pivotal in the pathology of multiple sclerosis (MS), but their function and antigen specificity are unknown. To unravel the role of T cells in MS pathology, we performed a comprehensive analysis on T cells recovered from paired blood, cerebrospinal fluid (CSF), normal-appearing white matter (NAWM) and white matter lesions (WML) from 27 MS patients with advanced disease shortly after death. The differentiation status of T cells in these compartments was determined by ex vivo flow cytometry and immunohistochemistry. T-cell reactivity in short-term T-cell lines (TCL), generated by non-specific stimulation of T cells recovered from the same compartments, was determined by intracellular cytokine flow cytometry. Central memory T cells predominated in CSF and effector memory T cells were enriched in NAWM and WML. WML-derived CD8+ T cells represent chronically activated T cells expressing a cytotoxic effector phenotype (CD95L and granzyme B) indicative for local antigenic stimulation (CD137). The same lesions also contained higher CD8+ T-cell frequencies expressing co-inhibitory (TIM3 and PD1) and co-stimulatory (ICOS) T-cell receptors, yet no evidence for T-cell senescence (CD57) was observed. The oligoclonal T-cell receptor (TCR) repertoire, particularly among CD8+ T cells, correlated between TCL generated from anatomically separated WML of the same MS patient, but not between paired NAWM and WML. Whereas no substantial T-cell reactivity was detected towards seven candidate human MS-associated autoantigens (cMSAg), brisk CD8+ T-cell reactivity was detected in multiple WML-derived TCL towards autologous Epstein–Barr virus (EBV) infected B cells (autoBLCL). In one MS patient, the T-cell response towards autoBLCL in paired intra-lesional TCL was dominated by TCRVβ2+CD8+ T cells, which were localized in the parenchyma of the respective tissues expressing a polarized TCR and CD8 expression suggesting immunological synapse formation in situ. Collectively, the data suggest the involvement of effector memory cytotoxic T cells recognizing antigens expressed by autoBLCL, but not the assayed human cMSAg, in WML of MS patients.

Drug fever after a single dose of amoxicillin-clavulanic acid

Although amoxicillin-clavulanic acid (AMX/CLV) can be associated with hypersensitivity reactions, ranging from minor and self-limiting to anaphylaxis, this is the first reported case of a reproducible maculopapular exathema (MPE) with drug fever occurring after a single therapeutic dose, not associated with drug reaction with eosinophilia and systemic symptoms (DRESS). This case presented with delayed onset skin hypersensitivity reactions and fever that were finally associated with increased neutrophil counts plus high C-reactive protein (CRP) levels. The patient was a 14-year-old Caucasian girl with a history of AMX/CLV-associated reactions. In 2012, during an Epstein Barr virus infection, she had an MPE during AMX/CLV therapy. In February 2014, she was given AMX/CLV for a febrile bacterial infection and noted another itchy MPE 12 hours after the first dose. She stopped the drug, and in May 2014, she was referred to the Allergy Unit of Anna Meyer Children’s Hospital for evaluation. Skin tests were performed according to the European Network for Drug Allergy recommendations in the following sequence: (a) skin prick testing (SPT) with 20 mg/mL AMX and AMX/ CLV; and (b) an intradermal (ID) testing with 1/100 and 1/10 dilutions of the full-strength (200 mg/mL) AMX and AMX/CLV concentration at 20to 30-minute intervals. Positive and negative controls for SPT and ID were obtained using histamine (ALK-Abello, Milan, Italy: 10 and 1 mg/mL) and normal saline. A positive skin test result was when the SPT wheal was greater than 3 mm and was accompanied by erythema with a negative saline control result or when the ID was greater than 3 mm from the initial wheal or an increase in the diameter of the initial wheal was associated with a flare and a negative saline control result within 15 minutes. ID tests were also read after 24-72 hours for delayed T-cell-mediated reactions. Specific IgE to amoxicillin, ampicillin, and penicillin G and V were measured (Immunocap RAST, Uppsala, Sweden) and were all negative. Because all of the skin and in vitro tests were negative, an open challenge drug provocation test (DPT) to AMX/CLV using 1/ 10, 2/10, and then 7/10 of the therapeutic dose every 30 minutes (50/mg/kg/day in 2 doses) was administered following the current guidelines. She had no acute reactions, and after 2 hours of observation, she was discharged. After 3 hours from the last dose, she was vomiting and had a fever (39.4 C), headache, and generalized erythema. After 8 hours, she had diarrhea and difficulty sleeping. She was transported to the emergency department of Anna Meyer Children’s Hospital where her vital signs, complete blood count, and CRP levels were found to be within the normal range. She was discharged with the suspicion of a concurrent viral infection. In October 2014, the ID tests with AMX/CLAV were again negative. The DPT with AMX/CLV was repeated with the same graded challenge protocol, and after 3 hours, she again noted vomiting and had a fever, headache, and an itchy MPE. Later she had difficulty sleeping and diarrhea. The next day, she was admitted to the Anna Meyer Children’s Hospital Allergy Unit, where her blood was drawn and the following results were observed: 12.3 mg/mL CRP (normal value < 0.50 mg/dL); white blood cells count (WBC) of 14,950 mmc; neutrophils 90%; and liver function and a erythrocyte sedimentation rate within the normal ranges. After 2 days, the MPE was reduced and the girl no longer had a fever (Figure 1). After a month, a lymphocyte transformation test (LTT), induction of hapten-specific short-term T-cell lines, and cytokine production measurements were performed (see this article’s Online Repository at www.jaci-inpractice.org). The LTT was strongly positive to both AMX and AMX/CLV (Figure 2, A), and the T-cell lines that were specific for AMX and AMX/CLV could be induced. The LTT positivity and induction of the hapten-specific T-cell lines were also confirmed 6 months apart from the reaction (data not shown). A flow cytometric analysis showed high percentages of TNF-a producing T-cell blasts (Figure 2, B). No significant production of CXCL8, IL-6, or IL-17 could be found (data not shown). Drug fever is a rare event occurring in only approximately 3%-5% of adverse drug reactions. In 18%-29% of patients who

T-Cell Responses to Tyrosinase-Derived Self-Peptides in Patients with Leukoderma Induced by Rhododendrol: Implications for Immunotherapy Targeting Melanoma

Background: Rhododendrol, a phenolic compound contained in lightening/whitening cosmetics, can bind and inhibit tyrosinase and was reported to induce leukoderma in Japan. Only 2% of the cosmetics users are affected, and tacrolimus is effective in treatment of the condition. Objective: To test the hypothesis that the disease is an autoimmune disorder. Methods: Short-term T-cell lines were established using peripheral blood mononuclear cells from 8 patients with human melanoma-associated and tyrosinase-derived synthetic peptides. The effects of rhododendrol on melanoma immunization were also examined. Results: Seven out of 8 patients were positive for HLA-DR4. Both class I- and class II-restricted and tyrosinase peptide-specific T-cell responses were observed. Immunization of mice with rhododendrol-treated and irradiated B16 melanoma cells successfully delayed the growth of melanoma cells in vivo. Conclusion: Rhododendrol-induced leukoderma is an autoimmune disorder, with rhododendrol as an environmental factor and HLA-DR4 as a genetic factor. Rhododendrol might be effective in treating melanomas.

Tolerogenic effect of mesenchymal stromal cells on gliadin-specific T lymphocytes in celiac disease

Background aimsCeliac disease is caused by a dysregulated immune response toward dietary gluten, whose only treatment is a lifelong gluten-free diet. We investigated the effects of mesenchymal stromal cells (MSCs) on gliadin-specific T cells, which are known to induce intestinal lesions, in view of a possible use as new therapy. MethodsBone marrow–derived MSCs and gliadin-specific T-cell lines were obtained from allogeneic donors and mucosal specimens of celiac patients, respectively. The immunosuppressant effect of MSCs was evaluated in terms of proliferative response and interferon (IFN)-γ production upon gliadin stimulation of long-term T-cell lines; the immunomodulant effect was assessed in terms of apoptotic rate, immunophenotype and cytokine profile of short-term T-cell lines generated in the presence of MSCs. Different MSC:T-cell ratios were applied, and statistics were performed as appropriate. ResultsMSCs inhibited both proliferative response and IFN-γ production of long-term T-cell lines in a dose-dependent manner while limiting the expansion of short-term T-cell lines by increasing the apoptotic rate. Moreover, a reduction of the CD4+ population and expansion of the regulatory FoxP3+ subset were found in T-cell lines cultured with MSCs, in which a significant decrease of interleukin (IL)-21, IFN-γ and IL-10 paralleled by an upregulation of transforming growth factor-β1, IL-6 and IL-8 were observed. Finally, an increase of the indoleamine 2,3-dioxygenase activity was found, possibly playing a key role in mediating these effects. ConclusionsMSCs exert potent immunomodulant effects on gliadin-specific T cells, which may be exploited for future therapeutic application in celiac disease.

Cathepsin W expressed exclusively in CD8 + T cells and NK cells, is secreted during target cell killing but is not essential for cytotoxicity in human CTLs

Cathepsin W (CatW, lymphopain) is a putative cysteine protease with restricted expression to natural killer (NK) cells and CD8(+) T cells and so far unknown function and properties. Here, we characterize in detail, the regulation of human CatW during T-cell development in response to different stimuli and its functional involvement in cytotoxic lymphocyte effector function. Western blots and real time polymerase chain reaction of sorted, unstimulated, and stimulated cell subsets (thymocytes, T cells, NK cells) and their culture supernatants were used to study regulation and expression of CatW. Primary CD8(+) T cells and short-term T-cell lines were transfected with small interfering RNA to study the involvement of CatW in effector function such as target cell killing and interferon-gamma production. Levels of CatW expression correlate closely with cytotoxic capacity both during development and in response to factors influencing cytotoxicity. Furthermore, CatW is secreted during specific target cell killing. However, knockdown of CatW expression by small interfering RNA neither influences target cell killing nor interferon-gamma production. Despite being expressed in the effector subset of CD8(+) and NK cells and of being released during target cell killing, our functional inhibition studies exclude an essential role of CatW in the process of cytotoxicity.

The therapeutic effect of glatiramer acetate in a murine model of inflammatory bowel disease is mediated by anti-inflammatory T-cells

Inflammatory bowel diseases (IBDs) are complex multifactorial immunological disorders characterized by dysregulated immune reactivity in the gut and imbalance between pro-inflammatory and anti-inflammatory reactivity. The therapeutic effect of the immunomodulatory drug glatiramer acetate (GA, Copaxone, copolymer 1) has been established in several IBD models, including trinitrobenzene sulfonic acid (TNBS) and dextran sulfate sodium (DSS)-induced colitis, as well as in a spontaneous colitis model. In the present study we investigated the mechanism of action of GA and cells specifically induced by it. Immunization of naive mice by GA, generated a lymphocyte population of the Th2/3 subtype, that drastically reduced disease manifestations upon their adoptive transfer to mice with DSS colitis. This was demonstrated by the substantial decrease in weight loss, intestinal bleeding and diarrhea, as well as by the prevention of macroscopic and microscopic colonic damage. In contrast, adoptive transfer of control lysozyme-specific cells did not induce any beneficial effect on the disease. Moreover, GA-specific short-term T-cell lines, either exogenously labeled or genetically marked, adoptively transferred by the intraperitoneal route to colitis-induced mice, localized in the inner layers of the colon and secreted in situ the regulatory cytokine TGF-β. These results demonstrate the accumulation of GA-specific Th2/3 cells secreting regulatory cytokines in the injured colon, and thus draw a direct linkage between the therapeutic effect of GA in IBD and an immunomodulatory effect at the site in which the pathological process occurs.

HCV-Specific T-Cell Response in Relation to Viral Kinetics and Treatment Outcome (DITTO-HCV Project)

The second slope of viral decline induced by interferon treatment has been suggested to be influenced mainly by the hepatitis C virus (HCV)-specific T-cell response; however, this hypothesis needs to be validated by results derived from experimental studies. To address this issue, the HCV-specific T-cell response of 32 genotype-1-infected patients of the 270 patients enrolled in the dynamically individualized treatment of hepatitis C infection and correlates of viral/host dynamics phase III, open-label, randomized, multicenter trial was studied in relation to viral kinetics and treatment outcome. Greater proliferative responses by HCV-specific CD8 cells were found before treatment in patients with a fast viral decline and with a sustained viral response. However, no significant improvement of HCV-specific CD8 responses was observed in the first weeks of therapy in both rapid viral responder and non-rapid viral responder patients. A mild enhancement of proliferative T-cell responses and a partial restoration of the cytotoxic T-cell potential was expressed only late during treatment, likely favored by HCV clearance. Early restoration of an efficient T-cell response does not seem to be an essential requirement for a rapid viral decline in the first weeks of treatment. However, patients presenting a better HCV-specific CD8 cell proliferative potential at baseline are more likely to present a rapid and sustained viral response. Therefore, future treatment protocols should consider the development of strategies aimed at improving HCV-specific T-cell responses.

Novel self-epitopes derived from aggrecan, fibrillin, and matrix metalloproteinase-3 drive distinct autoreactive T-cell responses in juvenile idiopathic arthritis and in health

Juvenile idiopathic arthritis (JIA) is a heterogeneous autoimmune disease characterized by chronic joint inflammation. Knowing which antigens drive the autoreactive T-cell response in JIA is crucial for the understanding of disease pathogenesis and additionally may provide targets for antigen-specific immune therapy. In this study, we tested 9 self-peptides derived from joint-related autoantigens for T-cell recognition (T-cell proliferative responses and cytokine production) in 36 JIA patients and 15 healthy controls. Positive T-cell proliferative responses (stimulation index ≥2) to one or more peptides were detected in peripheral blood mononuclear cells (PBMC) of 69% of JIA patients irrespective of major histocompatibility complex (MHC) genotype. The peptides derived from aggrecan, fibrillin, and matrix metalloproteinase (MMP)-3 yielded the highest frequency of T-cell proliferative responses in JIA patients. In both the oligoarticular and polyarticular subtypes of JIA, the aggrecan peptide induced T-cell proliferative responses that were inversely related with disease duration. The fibrillin peptide, to our knowledge, is the first identified autoantigen that is primarily recognized in polyarticular JIA patients. Finally, the epitope derived from MMP-3 elicited immune responses in both subtypes of JIA and in healthy controls. Cytokine production in short-term peptide-specific T-cell lines revealed production of interferon-γ (aggrecan/MMP-3) and interleukin (IL)-17 (aggrecan) and inhibition of IL-10 production (aggrecan). Here, we have identified a triplet of self-epitopes, each with distinct patterns of T-cell recognition in JIA patients. Additional experiments need to be performed to explore their qualities and role in disease pathogenesis in further detail.

Identification of a naturally processed cytotoxic CD8 T-cell epitope of coxsackievirus B4, presented by HLA-A2.1 and located in the PEVKEK region of the P2C nonstructural protein.

The adaptive immune system generates CD8 cytotoxic T lymphocytes (CTLs) as a major component of the protective response against viruses. Knowledge regarding the nature of the peptide sequences presented by HLA class I molecules and recognized by CTLs is thus important for understanding host-pathogen interactions. In this study, we focused on identification of a CTL epitope generated from coxsackievirus B4 (CVB4), a member of the enterovirus group responsible for several inflammatory diseases in humans and often implicated in the triggering and/or acceleration of the autoimmune disease type 1 diabetes. We identified a 9-mer peptide epitope that can be generated from the P2C nonstructural protein of CVB4 (P2C(1137-1145)) and from whole virus by antigen-presenting cells and presented by HLA-A2.1. This epitope is recognized by effector memory (gamma interferon [IFN-gamma]-producing) CD8 T cells in the peripheral blood at a frequency of responders that suggests that it is a major focus of the anti-CVB4 response. Short-term CD8 T-cell lines generated against P2C(1137-1145) are cytotoxic against peptide-loaded target cells. Of particular interest, the epitope lies within a region of viral homology with the diabetes-related autoantigen, glutamic acid decarboxylase-65 (GAD(65)). However, P2C(1137-1145)-specific cytotoxic T lymphocyte (CTL) lines were not activated to produce IFN-gamma by the GAD(65) peptide homologue and did not show cytotoxic activity in the presence of appropriately labeled targets. These results describe the first CD8 T-cell epitope of CVB4 that will prove useful in the study of CVB4-associated disease.

Glatiramer acetate-reactive T cells produce brain-derived neurotrophic factor

Experimental and MRI evidence suggest that glatiramer acetate's (Copaxone) therapeutic effect in multiple sclerosis (MS) could be mediated by anti-inflammatory GA-reactive Th2 cells that enter the brain, cross-react with myelin antigens, and produce bystander suppression. Furthermore, a neuroprotective effect, possibly mediated by neurotrophic factors such as BDNF, has been suggested based on experimental evidence in animal models, and the observation that inflammatory cells can elaborate BDNF. Therefore, we examined BDNF production in 73 GA, 13 MBP, and 22 TT-reactive short-term T-cell lines from 12 MS patients treated with GA. Ten of 73 GA-TCL (14%), 1 of the MBP-TCL (3%), and 2 of the TT-TCL (9%) produced BDNF levels two standard deviations above the mean levels produced by resting TCL. RT-PCR analysis confirmed BDNF expression in some GA- and MBP-reactive TCL. The mean BDNF level produced by GA-TCL was significantly higher than that for MBP-TCL, or TT-TCL when lines originating from the same patients were compared ( P=0.033). All 10 high BDNF-producing GA-reactive TCL were Th2-biased as determined by the IL-5/IFN-γ levels ratio. A positive correlation was observed between BDNF and IL-5 (Th2 indicator) ( P=0.006) but not with IFN-γ Th1 indicator) levels in GA-TCL derived from MS patients during but not pre-treatment. We conclude that while BDNF production by T cells is not antigen-specific, GA-reactive TCL are more likely to produce BDNF, and to be Th2-biased.

Human papillomavirus type 16-specific T cell responses and their association with recurrence of cervical disease following treatment

Human papillomavirus type 16 (HPV-16) L1- and E7-specific T cell responses were measured in 58 women with abnormal cervical cytology in a prospective study. On recruitment, patients responded most frequently and with the highest numbers of responding cells to the L1 region aa 311-345 and this response was significantly associated with the presence of cervical disease (P=0.041). Responses to the L1 peptide aa 281-295 were significantly higher in patients with CIN III than in those with HPV/CIN I or CIN II lesions (P=0.027). The E7 region aa 70-98 was the most immunogenic in patients with squamous intraepithelial lesions of the cervix (SIL) but the responses detected were not significantly higher than in patients without SIL. Following treatment, the T cell response profiles of patient groups did not change significantly. However, on analysis of the responses of individual patients with and without recurrent disease on follow-up, significant differences were found. Recurrence of disease was associated with T cell responses to the E7 region aa 70-98 at the patient's first clinic visit (P=0.017). Recurrence of disease was also accompanied by an increase in the total number of L1-specific short-term T cell lines (STLs) at follow-up, whereas absence of disease was accompanied by a decrease in L1-specific STLs. The data also suggested a possible link between E7 70-98-specific responses and acquisition of disease by patients who were previously disease-free. Further studies are warranted to determine whether this response could be useful as a marker of recurrent disease in some patients.

HLA class II polymorphisms and susceptibility to recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is characterised by multiple laryngeal papillomas. Left untreated, the lesions enlarge, spread, and endanger the airway. Medical treatments are unsatisfactory, and repeated surgery remains the mainstay of therapy. RRP is caused by human papillomavirus (HPV) infection. However, since oral HPV infection is common and RRP is rare, other host and/or viral factors may contribute to pathogenesis. In an attempt to identify such factors, we have investigated 60 patients. The patients were HLA class I, II, and tumor necrosis factor TNF typed by sequence-specific primer PCR, and the results compared to those for 554 healthy controls by using Fisher's exact test. Peripheral blood mononuclear cell proliferative responses of 25 controls and 10 patients to HPV-11 L1 virus-like particles (VLP) were compared. Short-term VLP-specific T-cell lines were established, and recognition of L1 was analyzed. Finally, the L1 open reading frames of HPV isolates from four patients were sequenced. Susceptibility to RRP was associated with HLA DRB1*0301 (33 of 60 patients versus 136 of 554 controls, P < 0.0001). The three most severely affected patients were homozygous for this allele. A range of T-cell proliferative responses to HPV-11 VLP were observed in DRB1*0301-positive healthy donors which were comparable to those in DRB1*0301-negative controls. Individuals with juvenile-onset RRP also mounted a range of VLP responses, and their magnitude was negatively correlated with the clinical staging score (P = 0.012 by the Spearman rank correlation). DRB1*0301-positive patients who responded to L1 recognized the same epitope as did matched controls and produced similar cytokines. Sequencing of clinical isolates excluded the possibility that nonresponsiveness was the result of mutation(s) in L1.

Autoreactivity to myelin antigens related to HLA associations with multiple sclerosis.

Multiple sclerosis (MS) is considered as an immune process influenced by genetic and environmental factors. HLA-DR2 and -DR4 have been documented to be associated with MS. The HLA-dependent differences of immune response to myelin and other antigens might point out some relevant mechanisms in MS development The responses to myelin antigens and to PPD have been compared in 21 MS patients and 25 healthy controls (HCs) by primary proliferation and by short-term T-cell lines. There was a significantly higher response to MBP in DR2+ HCs compared to MS patients (SI: 5.9 versus 1.5, p = 0.02). In short-term T-cell lines, we observed a higher response to PLP30-49 in both DR4+ HCs and MS patients This response was significantly more frequent in DR4+ MS patients (34.6%) than both DR2+ MS patients (0%, p = 0.0001) and DR4+ HCs (7.7%, p = 0.001). The comparison between DR2+ and DR4+ MS patients has revealed that the response to MBP was also increased in DR4+ (p = 0.02). Among DR4+ groups, an increased PPD response was detected in HCs compared to MS (65.2% versus 33.3%, p = 0.01). These results may indicate that HLA-related differences to specific and recall antigens are detectable in MS and these differences may have implications in the disease pathogenesis.

The fine specificity of human T cell lines towards myelin basic protein peptides in southern Italian multiple sclerosis patients.

We studied the relationship between the HLA specificities associated with multiple sclerosis (MS) susceptibility in southern Italy and the reactivity of the human myelin basic protein (hMBP) immunogenic peptides 84-98 and 143-168, using short-term T-cell lines established from 9 MS patients and from 8 healthy individuals. In our population, DR15 was significantly associated with MS (34.9% in MS versus 13.7% in healthy controls, P < 0.05). This result is in agreement with the association found in northern Europe, but not with data obtained in a population from the island of Sardinia (Italy). In MS patients the frequency of reactive T-cell lines (TCL), tested for fine specificity against the immunodominant hMBP peptides 84-98 and 143-168, was increased for the hMBP 143-168 peptide (P < 0.05) but not for the 84-98 peptide. Although this reactivity was higher in DR15+ MS patients than in DR 15- MS patients, it seemed not to be associated with DR15 specificity in the MS population. Furthermore, there were no significant differences in frequency of reactive TCL to hMBP peptide 84-98 in DR15-positive or DR15-negative MS patients. Consequently, it appears that peptide 84-98, considered as a relevant autoantigen, is not implicated in the pathogenesis of MS in our population from southern Italy.

Propagation of lewis rat encephalitogenic T cell lines: T-cell-growth-factor is superior to recombinant IL-2

This study was designed to test the process of selecting encephalitogenic T cell lines in the Lewis rat using recombinant human IL-2 (rhIL-2) in comparison to TCGF. The lines were tested for growth, antigen induced proliferation, cytokine production, V-beta 8.2 expression and pathogenicity. We now report that rhIL-2 and TCGF were equally effective in supporting short-term pathogenic T-cell lines with similar proportion of V-beta 8.2 usage. For the maintenance of long term lines, however, TCGF was superior to IL-2. The concentration of rhIL-2 influenced the cultures: 10 units/ml led to more T-cell proliferation than either 2 or 50 units/ml. However, 50 units/ml of IL-2 led to enhanced Th1 polarization. Thus, the type and concentration of growth factors can influence both the propagation of T cells and their phenotype.

A disease-associated cellular immune response in type 1 diabetics to an immunodominant epitope of insulin.

The 9-23 amino acid region of the insulin B chain (B9-23) is a dominant epitope recognized by pathogenic T lymphocytes in nonobese diabetic mice, the animal model for type 1 diabetes. We describe herein similar (B9-23)-specific T-cell responses in peripheral lymphocytes obtained from patients with recent-onset type 1 diabetes and from prediabetic subjects at high risk for disease. Short-term T-cell lines generated from patient peripheral lymphocytes showed significant proliferative responses to (B9-23), whereas lymphocytes isolated from HLA and/or age-matched nondiabetic normal controls were unresponsive. Antibody-mediated blockade demonstrated that the response was HLA class II restricted. Use of the highly sensitive cytokine-detection ELISPOT assay revealed that these (B9-23)-specific cells were present in freshly isolated lymphocytes from only the type 1 diabetics and prediabetics and produced the proinflammatory cytokine IFN-gamma. This study is, to our knowledge, the first demonstration of a cellular response to the (B9-23) insulin epitope in human type 1 diabetes and suggests that the mouse and human diseases have strikingly similar autoantigenic targets, a feature that should facilitate development of antigen-based therapeutics.

CD4 + T-cell responses to bovine viral diarrhoea virus in cattle

Friesian calves were infected with one of three isolates of bovine viral diarrhoea virus (BVDV) and used to establish parameters for an in vitro model of BVDV-reactive T-cell responses in cattle. The study assessed virus clearance, seroconversion, maturation of lymphoproliferative responses (both during and following disease resolution) and the antigen-specificity of CD4 + T cells from recovered animals. Seroconversion and virus-specific lymphoproliferation were not detected until viraemia had resolved. Interestingly, lymphoproliferation was detected earlier in the animals infected with cytopathic viruses than in those infected with noncytopathic virus despite broadly similar rates of virus clearance and seroconversion for both biotypes. CD4 + and CD8 + T cells were induced to proliferate by virus-infected stimulator cells whereas only CD4 + T cells responded to non-infectious antigens. Lymphoproliferation was strain cross-reactive and MHC-restricted. Induction of T-cell proliferation by recombinant proteins identified the major envelope proteins E rns and E2 and the nonstructural (NS) 2-3 protein as T-cell determinants. In addition, the capsid (C) and/or the amino-terminal proteinase, N pro were identified as T-cell determinants from the responses of short-term T-cell lines. Thus, in this model, the CD4 + T-cell repertoire induce by acute BVDV infection includes at least the major envelope proteins, NS2-3, and capsid and/or N pro.

Cross-Reactivity of Borrelia burgdorferi and Myelin Basic Protein-Specific T Cells Is Not Observed in Borrelial Encephalomyelitis

Borrelial encephalomyelitis, a rare manifestation of Lyme borreliosis, may present as a multiple sclerosis (MS)-like disease. It is postulated that in MS, inflammation of the white matter is caused by a T-cell response directed to myelin antigens. Here, we examined whether a T-cell autoimmune response may play a pathogenetic role in Borrelia-associated white matter disease mediated by cross-reactivity between myelin basic protein (MBP) and B. burgdorferi. We generated a total of 1760 short-term T-cell lines against B. burgdorferi or MBP from two patients with Borrelial encephalomyelitis and compared these with three patients with late Lyme disease, one patient with transverse myelitis, eight patients with MS, and four healthy controls. While a few T-cell lines recognized both B. burgdorferi and MBP, T-cell clones from these lines responded only to the antigen of the original stimulation. Thus, our data do not provide evidence for cross-reactivity between MBP and B. burgdorferi.

Analysis of murine CD8(+) T-cell clones specific for the Dengue virus NS3 protein: flavivirus cross-reactivity and influence of infecting serotype.

Serotype-cross-reactive dengue virus-specific cytotoxic T lymphocytes (CTL) induced during a primary dengue virus infection are thought to play a role in the immunopathogenesis of dengue hemorrhagic fever (DHF) during a secondary dengue virus infection. Although there is no animal model of DHF, we previously reported that murine dengue virus-specific CTL responses are qualitatively similar to human dengue virus-specific CTL responses. We used BALB/c mice to study the specificity of the CTL response to an immunodominant epitope on the dengue virus NS3 protein. We mapped the minimal H-2Kd-restricted CTL epitope to residues 298 to 306 of the dengue type 2 virus NS3 protein. In short-term T-cell lines and clones, the predominant CD8(+) CTL to this epitope in mice immunized with dengue type 2 virus or vaccinia virus expressing the dengue type 4 virus NS3 protein were cross-reactive with dengue type 2 or type 4 virus, while broadly serotype-cross-reactive CTL were a minority population. In dengue type 3 virus-immunized mice, the predominant CTL response to this epitope was broadly serotype cross-reactive. All of the dengue virus-specific CTL clones studied also recognized the homologous NS3 sequences of one or more closely related flaviviruses, such as Kunjin virus. The critical contact residues for the CTL clones with different specificities were mapped with peptides having single amino acid substitutions. These data demonstrate that primary dengue virus infection induces a complex population of flavivirus-cross-reactive NS3-specific CTL clones in mice and suggest that CTL responses are influenced by the viral serotype. These findings suggest an additional mechanism by which the order of sequential flavivirus infections may influence disease manifestations.