Antigen-specific T-cell Lines Research Articles

Rare Factor VIII-reactive CD4 T cells in non-hemophilic blood characterized by ex vivo expansion and ELISPOTs with added costimulation

Abstract Background: Autoimmune responses to self-FVIII can cause a rare but potentially life-threatening autoimmune bleeding disorder referred to as “acquired hemophilia A”. Objectives: (1) To confirm the frequency and specificity of CD4 T cells recognizing factor (F)VIII in the periphery of non-hemophilic individuals; (2) to improve methodology for detecting rare self-reactive CD4 T cells that apparently escaped thymic editing. Methods: CD4 T cells were isolated from healthy non-hemophilic blood donors, incubated with autologous irradiated PBMCs, and stimulated with FVIII 15-mer peptides, anti-CD28 and anti-CD49D. Positive interferon-gamma ELISPOT responses were detected, but they were poorly reproducible due to the low frequency of these autoreactive cells. Therefore, pre-expansion of FVIII-specific T-cell lines, followed by ELISPOT assays with added co-stimulation, was carried out to increase sensitivity and accuracy. Results: Several immunodominant epitopes in FVIII restricted to H LA-DRB1* 01:01, 07:01and 15:01were identified. FVIII-specific T-cell lines generated from individual blood donors showed robust interferon-gamma secretion in response to FVIII, compared to ELISPOT assays with co-stimulation but no FVIII pre-expansion. Conclusions: Our optimized protocol, involving generation of antigen-specific T-cell lines followed by ELISPOTs with added co-stimulation, shows promise for detecting, quantifying and characterizing rare autoreactive T cells in the periphery. Intramural funds, Uniformed Services University

Increased frequency of interleukin-4 and reduced frequency of interferon-γ and IL-17-producing CD4+ and CD8+ cells in scleromyxedema.

Little is known about the pathogenesis of scleromyxedema, a life-threatening fibromucinosis disease with immunological dysregulation. To investigate on T-cell phenotype, function and cytokine biology in search of new insights supporting the immunopathogenesis of the disease. We analysed the frequency of circulating lymphocyte subsets, the T-cell maturation stage, the generation of antigen-specific T-cell lines and T-cell cytokine secretion. The analysis of T-cell maturation stage and the TCR spectratyping findings revealed that scleromyxedema patients showed clear immunological signs of long-lasting immune system activation and stimulation leading to a skewed T-cell repertoire. Moreover, these analyses showed that both CD4+ and CD8+ T cells from scleromyxedema patients have a profound deficiency (even after stimulation) relatively to the production of IFN-γ and IL17 with respect to healthy donor control cells, while they are massively skewed towards IL4 secretion after stimulation. Our data indicate that a chronic Th2-skewed T-cell response against an unknown target antigen leading to abnormally high IL4 secretion, a pro-fibrotic cytokine, is a main immunological hallmark of scleromyxedema patients. These results, never reported before, may have a translational therapeutic value due to the availability of anti-IL4 agents such as dupilumab.

Induction of an Immune-Protective T-Cell Repertoire With Diverse Genetic Coverage by a Novel Viral-Vectored Tuberculosis Vaccine in Humans

Whether a candidate tuberculosis vaccine induces clinically relevant protective T-cell repertoires in humans will not be known until the completion of costly efficacy clinical trials. We have developed an integrated immunologic approach to investigate the clinical relevance of T cells induced by a novel tuberculosis vaccine in a phase 1 trial. This approach consists of screening for likely dominant T-cell epitopes, establishing antigen-specific memory T-cell lines for identifying CD8+ and CD4+ T-cell epitopes, determining the ability of vaccine-induced T cells to inhibit mycobacterial growth in infected cells, and examining the genetic diversity of HLA recognition and the clinical relevance of identified T-cell epitopes. A single-dose immunization in BCG-primed adults with an adenovirus-based tuberculosis vaccine elicits a repertoire of memory T cells capable of recognizing multiple Ag85A epitopes. These T cells are polyfunctional and cytotoxic and can inhibit mycobacterial growth in infected target cells. Some identified T-cell epitopes are promiscuous and recognizable by the common HLA alleles. These epitopes are clinically relevant to the epitopes identified in people with latent Mycobacterium tuberculosis infection and treated patients with tuberculosis. These data support further clinical development of this candidate vaccine. Our approach helps fill the gap in clinical tuberculosis vaccine development.

The IDO1 selective inhibitor epacadostat enhances dendritic cell immunogenicity and lytic ability of tumor antigen-specific T cells.

Epacadostat is a novel inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1) that suppresses systemic tryptophan catabolism and is currently being evaluated in ongoing clinical trials. We investigated the effects of epacadostat on (a) human dendritic cells (DCs) with respect to maturation and ability to activate human tumor antigen-specific cytotoxic T-cell (CTL) lines, and subsequent T-cell lysis of tumor cells, (b) human regulatory T cells (Tregs), and (c) human peripheral blood mononuclear cells (PBMCs) in vitro. Simultaneous treatment with epacadostat and IFN-γ plus lipopolysaccharide (LPS) did not change the phenotype of matured human DCs, and as expected decreased the tryptophan breakdown and kynurenine production. Peptide-specific T-cell lines stimulated with DCs pulsed with peptide produced significantly more IFN-γ, TNFα, GM-CSF and IL-8 if the DCs were treated with epacadostat. These T cells also displayed higher levels of tumor cell lysis on a per cell basis. Epacadostat also significantly decreased Treg proliferation induced by IDO production from IFN-γ plus LPS matured human DCs, although the Treg phenotype did not change. Multicolor flow cytometry was performed on human PBMCs treated with epacadostat; analysis of 123 discrete immune cell subsets revealed no changes in major immune cell types, an increase in activated CD83+ conventional DCs, and a decrease in immature activated Tim3+ NK cells. These studies show for the first time several effects of epacadostat on human DCs, and subsequent effects on CTL and Tregs, and provide a rationale as to how epacadostat could potentially increase the efficacy of immunotherapeutics, including cancer vaccines.

A new small molecule for treating inflammation and chorioretinal neovascularization in relapsing-remitting and chronic experimental autoimmune uveitis.

We investigated the effect of PP-001, a new small molecule inhibitor of dihydro-orotate dehydrogenase in two experimental rat experimental autoimmune uveitis (EAU) models: a spontaneously relapsing-remitting model and a monophasic/chronic disease model that results in late chorioretinal neovascularization. Both of the diseases are induced by immunization with autoantigen peptides. Prevention was tested using daily oral applications of PP-001 after immunization with the retinal S-antigen peptide PDSAg (for induction of monophasic uveitis and neovascularization) or the interphotoreceptor retinoid-binding protein peptide R14 (for induction of spontaneously relapsing-remitting EAU). Treatment to inhibit relapses and neovascularization was tested using PP-001 daily after the first attack of R14-induced or after onset of PDSAg-induced EAU. Uveitis was graded clinically and histologically. The effect of PP-001 on cytokine secretion and proliferation was evaluated using rat T-cell lines. Preventive feeding of PP-001 abrogated both types of EAU. Starting treatment after the resolution of the first attack led to a significant reduction of the number and intensity of relapses in R14-induced EAU. PP-001-treatment initiated after onset or after peak of PDSAg-induced EAU significantly reduced neovascularization (as determined by histology). Proliferation of antigen-specific T-cell lines and secretion of IFN-γ, IL-17, IL-10, IP-10, and VEGF were efficiently suppressed by PP-001. We investigated a new dihydroorotate dehydrogenase inhibitor as treatment for primary and recurrent disease in relapsing-remitting and chronic rat models of experimental autoimmune uveitis. The small molecule compound PP-001 suppressed proliferation and cytokine secretion of autoreactive T cells (i.e., IFN-g, IL-17, and VEGF) and chorioretinal neovascularization in chronic EAU.

Characterization of a cross-reactive, immunodominant and HLA-promiscuous epitope of Mycobacterium tuberculosis-specific major antigenic protein PPE68.

PPE68 (Rv3873), a major antignic protein encoded by Mycobacteriun tuberculosis-specific genomic region of difference (RD)1, is a strong stimulator of peripheral blood mononuclear cells (PBMCs) obtained from tuberculosis patients and Mycobacterium bovis bacillus Calmette Guerin (BCG)-vaccianted healthy subjects in T helper (Th)1 cell assays, i.e. antigen-induced proliferation and interferon-gamma (IFN-γ) secretion. To confirm the antigen-specific recognition of PPE68 by T cells in IFN-γ assays, antigen-induced human T-cell lines were established from PBMCs of M. Bovis BCG-vaccinated and HLA-heterogeneous healthy subjects and tested with peptide pools of RD1 proteins. The results showed that PPE68 was recognized by antigen-specific T-cell lines from HLA-heteregeneous subjects. To further identify the immunodominant and HLA-promiscuous Th1-1 cell epitopes present in PPE68, 24 synthetic peptides covering the sequence of PPE68 were indivdually analyzed for HLA-DR binding prediction analysis and tested with PBMCs from M. bovis BCG-vaccinated and HLA-heterogeuous healthy subjects in IFN-γ assays. The results identified the peptide P9, i.e. aa 121-VLTATNFFGINTIPIALTEMDYFIR-145, as an immunodominant and HLA-DR promiscuous peptide of PPE68. Furthermore, by using deletion peptides, the immunodominant and HLA-DR promiscuous core sequence was mapped to aa 127-FFGINTIPIA-136. Interestingly, the core sequence is present in several PPE proteins of M. tuberculosis, and conserved in all sequenced strains/species of M. tuberculosis and M. tuberculosis complex, and several other pathogenic mycobacterial species, including M. leprae and M. avium-intracellulalae complex. These results suggest that the peptide aa 121–145 may be exploited as a peptide-based vaccine candidate against tuberculosis and other mycobacterial diseases.

Human CD103(+) dendritic cells promote the differentiation of Porphyromonas gingivalis heat shock protein peptide-specific regulatory T cells.

Regulatory T cells (Tregs), expressing CD4 and CD25 as well as Foxp3, are known to play a pivotal role in immunoregulatory function in autoimmune diseases, cancers, and graft rejection. Dendritic cells (DCs) are considered the major antigen-presenting cells (APCs) for initiating these T-cell immune responses, of which CD103(+) DCs are derived from precursor human peripheral blood mononuclear cells (PBMCs). The aim of the present study was to evaluate the capacity of these PBMC-derived CD103(+) DCs to promote the differentiation of antigen-specific Tregs. Monocyte-derived DCs were induced from CD14(+) monocytes from the PBMCs of 10 healthy subjects. Once the CD103(+) DCs were purified, the cell population was enriched by adding retinoic acid (RA). Peptide numbers 14 and 19 of Porphyromonas gingivalis heat shock protein 60 (HSP60) were synthesized to pulse CD103(+) DCs as a tool for presenting the peptide antigens to stimulate CD3(+) T cells that were isolated from human PBMC. Exogenous interleukin 2 was added as a coculture supplement. The antigen-specific T-cell lines established were phenotypically identified for their expression of CD4, CD25, or Foxp3. When PBMCs were used as APCs, they demonstrated only a marginal capacity to stimulate peptide-specific Tregs, whereas CD103(+) DCs showed a potent antigen presenting capability to promote the peptide-specific Tregs, especially for peptide 14. RA enhanced the conversion of CD103(+) DCs, which paralleled the antigen-specific Treg-stimulating effect, though the differences failed to reach statistical significance. We demonstrated that CD103(+) DCs can promote antigen-specific Tregs from naive T cells, when used as APCs for an epitope peptide from P. gingivalis HSP60. RA was an effective reagent that induces mature DCs with the typical phenotypic expression of CD103 that demonstrated the functional capability to promote antigen-specific Tregs.

Mixed functional characteristics correlating with TCR‐ligand koff‐rate of MHC‐tetramer reactive T cells within the naive T‐cell repertoire

The low frequency of antigen-specific naïve T cells has challenged numerous laboratories to develop various techniques to study the naïve T-cell repertoire. Here, we combine the generation of naïve repertoire-derived antigen-specific T-cell lines based on MHC-tetramer staining and magnetic-bead enrichment with in-depth functional assessment of the isolated T cells. Cytomegalovirus (CMV) specific T-cell lines were generated from seronegative individuals. Generated T-cell lines consisted of a variety of immunodominant CMV-epitope-specific oligoclonal T-cell populations restricted to various HLA-molecules (HLA-A1, A2, B7, B8, and B40), and the functional and structural avidity of the CMV-specific T cells was studied. Although all CMV-specific T cells were isolated based on their reactivity toward a specific peptide-MHC complex, we observed a large variation in the functional avidity of the MHC-tetramer positive T-cell populations, which correlated with the structural avidity measured by the recently developed Streptamer koff -rate assay. Our data demonstrate that MHC-tetramer staining is not always predictive for specific T-cell reactivity, and challenge the sole use of MHC-tetramers as an indication of the peripheral T-cell repertoire, independent of the analysis of functional activity or structural avidity parameters.

Adiponectin is a negative regulator of antigen‐activated T cells

Adiponectin (APN), a cytokine constitutively produced in fat tissue, has been shown to exert anti-inflammatory effects in various disease models. While the influence of APN on monocytic cells has been extensively studied in vitro, little is known about its role in T cells. In this study, we show that while <10% of human peripheral blood T cells express adiponectin receptors (AdipoRs) on their surface, most T cells store AdipoRs in intracellular compartments. AdipoRs colocalized with immune regulatory molecules CTLA-4 and TIRC7 within clathrin-coated vesicles. After stimulation, the expression of adiponectin receptor 1 (AdipoR1) and AdipoR2 was upregulated on the surface of antigen-specific T cells, as determined by tetramer or CD137 staining, and AdipoR1 and AdipoR2 coexpressed with CTLA-4. Addition of APN resulted in a significant diminution of antigen-specific T-cell expansion. Mechanistically, APN enhanced apoptosis and inhibited proliferation of antigen-specific T-cell lines. Further, APN directly inhibited cytokine production in response to antigen stimulation. In line with the in vitro data, APN-deficient (knockout, KO) mice had higher frequencies of CD137(+) T cells upon Coxsackie B virus infection. Altogether, our data suggest that APN is a novel negative T-cell regulator. In contrast to the CTLA-4 ligand B7 only expressed on APCs, APN is abundant in human plasma.

Telomerase and primary T cells: biology and immortalization for adoptive immunotherapy.

Telomeres are specialized repeats, present at the end of chromosomes, whose loss during cell division is followed by growth arrest, a central mechanism of replicative senescence in human cells. Telomere length in stem cells is maintained by telomerase, a specialized reverse transcriptase, whose function is to restore shortening telomeres. Unlike most somatic cell types, human T lymphocytes are capable of briefly reactivating telomerase expression at the time of stimulation. Telomerase expression in T lymphocytes is modulated by a variety of external stimuli and by viral infections. However, telomerase reactivation in stimulated, proliferating human T lymphocytes is limited and cannot prevent the ultimate onset of senescence. Ectopic telomerase expression can rescue human and macaque antigen-specific T cells from senescence. Primary T cells have been engineered with telomerase to have substantially extended replicative lifespans without the loss of primary cell functions or malignant transformation. 'Immortal' antigen-specific T-cell lines and clones overexpressing telomerase are an invaluable source of well-characterized quasi-primary T cells for research of T-cell biology and are potentially useful for immunotherapy of cancer and AIDS.

Characterization of the T-Cell Epitopes of the Major Peach Allergen Pru p 3

Background: Pru p 3 is the major peach allergen recognized by more than 90% of peach-allergic individuals of the Mediterranean area. Identification of the dominant Pru p 3 T-cell epitopes can improve our understanding of the immune responses against this protein and could be helpful in the development of hypoallergenic immunotherapy. For this purpose, we examined the phenotypes, specificities and cytokine secretion profiles of proliferating T cells in response to Pru p 3 in peach-allergic individuals. Methods: Peripheral blood mononuclear cells from 15 peach-allergic patients were incubated with Pru p 3. The proliferation of antigen-specific T-cell lines (TCLs) was assessed by tritiated methylthymidine incorporation. T-cell epitopes were identified by analyzing the reactivity of TCLs against 8 overlapping peptides spanning the entire length of Pru p 3. We characterized the phenotype of Pru-p-3-specific TCLs by flow cytometry and analyzed their production of interleukin (IL) 4 and γ-interferon (IFN-γ) by ELISA. Results: Ninety-two Pru-p-3-specific TCLs were isolated (stimulation index ≧5). These TCLs proliferated mainly in response to Pru p 3_12–27 and Pru p 3_57–72. Pru-p-3-specific TCLs were mainly CD4+ (81%) and expressed cell surface CD30. In addition, TCLs produced high levels of IL-4 and low levels of IFN-γ, indicating a Th2 phenotype. Conclusions: Two immunodominant T-cell-reactive regions of Pru p 3 were identified: Pru p 3_12–27 and Pru p 3_57–72. These peptides showed a differential ability to elicit a Th2 response. Taken together, our results provide a better understanding of the immunological T-cell reactivity against Pru p 3.

Ex vivo expansion of tumor specific lymphocytes with IL-15 and IL-21 for adoptive immunotherapy in melanoma

Although T Central Memory cells have been described as the most effective T-cell subtype against tumor growth, little is known about the requirements needed for their optimal ex vivo generation. Hence, our goal is to establish a protocol that will lead to consistent ex vivo generation of lymphocytes skewed toward a Central Memory phenotype. Antigen-specific T-cell lines were generated by ex vivo stimulation with Class-I and Class-II melanoma peptide pulsed Dendritic Cells in the presence of either IL-2 or IL-15 plus IL-21. Tumor specific lymphocytes of both Central Memory and effector characteristics were consistently generated from healthy donors and melanoma patients. IL15/IL21 cultures result in a cell population with a lower proportion of CD4 +CD25 highFoxP3 + regulatory cells and higher number of CD8 + and CD56 + cells, and consequently render a higher yield of cells with a greater cytolytic activity and IFN-γ production against melanoma cell lines.

Detection of interferon gamma-secreting CD8+ T lymphocytes in humans specific for three PE/PPE proteins of Mycobacterium tuberculosis

The PE and PPE family of proteins of Mycobacterium tuberculosis have been hypothesized to play important roles in the biology of the organism and some proteins have been shown to be involved in eliciting T-cell responses. Earlier, we had identified putative HLA class I binding epitopes of the PE and PPE proteins of Mycobacterium tuberculosis employing computational and molecular modeling approaches. In the present work, three of the PE/PPE family proteins, coded by Rv1818c, Rv3812 and Rv3018c genes, were selected based on the computational analysis for testing human immune responses. PBMCs from patients with active tuberculosis and healthy, BCG vaccinated, PPD-positive individuals were tested for in vitro proliferative response and gamma-interferon production using synthetic peptides derived from the chosen proteins. Significant differences were seen in the responsiveness between healthy controls and patients. Antigen-specific T-cell lines were established from the PBMCs of healthy controls and their responses to peptide-specific CD8+ T-cell effectors were shown to be present at high frequency in the PBMCs of PPD+ controls. The T-cell lines also showed cytotoxic activity against the peptide pulsed monocytes.

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.

Tumor Antigen–Specific T-Cell Expansion Is Greatly Facilitated byIn vivoPriming

Adoptive T-cell therapy is a promising strategy for the treatment of patients with established tumors but is often limited to specific cancers where tumor-infiltrating lymphocytes, the source of T cells for ex vivo culture, can be obtained. In this study, we evaluated the feasibility of expanding HER-2/neu-specific T cells derived from peripheral blood ex vivo following in vivo priming with a HER-2/neu peptide vaccine. Peripheral blood mononuclear cells from cytomegalovirus (CMV)-seronegative and CMV-seropositive donors as well as HER-2/neu-positive cancer patients who had or had not been vaccinated with a HER-2/neu peptide-based vaccine was used as a source of T lymphocytes. Antigen-specific T-cell lines were generated by in vitro stimulation with antigen followed by nonspecific expansion on CD3/CD28 beads. The ability to expand antigen-specific T cells was assessed using IFN-gamma and granzyme B enzyme-linked immunosorbent spot. The phenotype of the resultant T-cell lines was evaluated by flow cytometry, including the presence of FOXP3-expressing CD4(+) T cells. The frequencies of CMV-specific T cells generated from CMV(+) donors were >11-fold higher than the frequencies from CMV(-) donors (P = 0.001), with 22-fold increase of total number of CD3(+) T cells. The frequencies of HER-2/neu-specific T cells generated from the primed patients were >25-fold higher than the frequencies from unvaccinated patients (P = 0.006), with an average of a 19-fold increase of total number of CD3(+) T cells. Using peripheral blood as the source of T cells did not result in concurrent expansion of FOXP3(+)CD4(+) regulatory T cells despite the use of interleukin-2 in in vitro culture. Both CD4(+) and CD8(+) HER-2/neu-specific T cells could be expanded. The extent of ex vivo expansion correlated with the magnitude of immunity achieved during immunization (P = 0.008). Tumor-specific T cells can be efficiently expanded from the peripheral blood ex vivo following in vivo priming with a vaccine. This approach provides an effective method to generate tumor-specific polyclonal T cells for therapeutic use that could be applied to cancer patients with any tumor type.

Histamine H4 receptor agonists have more activities than H4 agonism in antigen‐specific human T‐cell responses

Histamine not only mediates immediate allergic reactions, it also regulates cellular immune responses. H4R is the most recently identified histamine receptor (HR). In the present study, we examined the in vitro effect of histamine and H4R agonists on the responses of human T cells to purified protein derivative from Mycobacterium tuberculosis (PPD) and to Cry j1, the major allergen of Cryptomeria japonica pollen. Dimaprit, clobenpropit and clozapine, which are H4R agonists, dose-dependently blocked both PPD-induced interferon-gamma and Cry j1-induced interleukin-5 production by both peripheral blood mononuclear cells (PBMCs) and antigen-specific T-cell lines. However, the addition of thioperamide, an H3R/H4R antagonist, as well as a mixture of d-chlropheniramine, famotidine and thioperamide, did not reverse the inhibition. Pretreatment of PBMCs with SQ22536 and 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, had varying abilities to reverse the inhibitory effects of H4R agonists, except for clobenpropit. Moreover, the addition of H4R agonists induced annexin-V expression on PBMCs, especially in CD19(+) and CD4(+) cells. cDNA microarray analysis revealed that, among 16,600 genes tested, increased expression following treatment with clozapine was seen in 0 x 8% of the genes, whereas decreased expression was seen in 3 x 0% of the genes. These results suggest that H4R agonists inhibit antigen-specific human T-cell responses, although H4R does not appear to be important for this effect. In addition, the present study indicated that there may be orphan receptors or HR subtypes which can bind dimaprit, clobenpropit and clozapine, and that can exert an inhibitory effect on antigen-specific cellular responses via a cAMP/cAMP-dependent protein kinase-dependent, apoptotic pathway.

Human Bone Marrow Stromal Cells Hamper Specific Interactions of CD4 and CD8 T Lymphocytes with Antigen-Presenting Cells

Bone marrow stromal cells (BMSCs) may inhibit T-cell functions in vitro and thus have been proposed as immunoregulators to control in vivo graft-versus-host disease (GVHD) in haploidentical hemopoietic stem cell transplants. To better investigate this phenomenon, we used a defined experimental system in which responding T cells are antigen-specific and devoid of alloreactivity against BMSC from a different subject. Thus, we established antigen-specific human CD4 and CD8 T-cell lines as the readout system. Antigen-dependent proliferation was reduced with both T-cell subsets cultured on confluent BMSCs, and also on confluent human skin fibroblasts (HSF) inhibited T-cell proliferation with similar efficiency. Morphological observations of the cocultures showed impairment of physical interactions between T-cell and antigen-presenting cells in the presence of BMSC, with lack of formation of antigen-dependent clusters of T cells and antigen-presenting cells (APCs). In contrast, no effects were seen with BMSC-conditioned medium. Since suppression was seen only with confluent mesenchymal cells, this phenomenon may not be relevant in vivo, where BMSCs are at low frequency. In addition, if the reported suppressive effect of BMSCs on GVHD in vivo is confirmed, a different in vitro system should be envisaged to better understand and exploit the underlying mechanism.

Capture of antigen-specific T lymphocytes from human blood by selective immortalization to establish long-term T-cell lines maintaining primary cell characteristics

To establish long-term, antigen-specific T-cell lines and clones, we selectively immortalized antigen-responsive T cells from human peripheral blood mononuclear cells (PBMCs). PBMCs were stimulated with either alloantigen or soluble antigen, then infected with a murine leukemia virus-based retroviral vector carrying an immortalizing gene, either the Tax gene from human T-cell leukemia virus type 1, or the human telomerase–reverse transcriptase gene. Since such vectors can only integrate in dividing cells, only antigen-activated T cells are efficiently transduced. This approach generated immortalized antigen-specific CD4 + and CD8 + T-cell lines that maintained strictly IL-2-dependent growth and HLA-restricted, antigen-specific responsiveness, some of which have been in continuous culture for longer than 1 year, far in excess of the survival of parallel control non-immortalized cultures. Clones derived from these lines showed antigen-specific proliferation with induced cytokine and chemokine production, and, in the case of a CD8 + T-cell clone, antigen-specific cytolytic activity. This approach provides a convenient, reproducible means for generating a stable, continuously renewable source of antigen-specific T lymphocytes for a variety of studies of T cell biology.

Multiple sclerosis: Mitoxantrone promotes differential effects on immunocompetent cells in vitro

Mitoxantrone is an anti-neoplastic anthracenedione derivative that, based on its immunosuppressive properties, is approved for the treatment of severe forms of relapsing–remitting or secondary progressive multiple sclerosis (MS). Whether the beneficial clinical effects of mitoxantrone in MS are due to a broad immunosuppression, or whether there is a specific mechanism of action remains unknown. Peripheral blood mononuclear cells (PBMCs) from untreated or interferon-β-treated patients with MS or from healthy donors were stimulated in the presence or absence of mitoxantrone. Irrespective of the source of the cells and the cellular phenotype, mitoxantrone inhibited proliferation of activated PBMCs, B lymphocytes, or antigen-specific T-cell lines (TCLs) stimulated on antigen-presenting cells (APCs) in a dose-dependent manner. For functional analysis, TCLs or APCs were incubated separately with mitoxantrone. Pre-incubation of APC more effectively impaired TCL proliferation than pre-incubation of TCLs. Production of cytokines, expression of activation markers, matrix metalloproteinases, and chemokine receptors were not influenced substantially by mitoxantrone. In contrast, in dendritic cells (DCs), mitoxantrone interfered with the antigen-presenting capabilities. For evaluation of apoptotic cell death of target cells, annexin-V-conjugates and a DNA fragmentation assay were applied. Mitoxantrone induced apoptosis of PBMCs, monocytes and DCs at low concentrations, whereas higher doses caused cell lysis. Our observations suggest that the beneficial effects of mitoxantrone in MS result (i) from its immunosuppressive action based on nonspecific cytotoxic effects on lymphocytes, (ii) by inducing programmed cell death of professional APCs, such as DCs.

Cd8+ T-Cells Specifically Cytotoxic to Renal Cell Carcinoma (RCC) Cells Can Be Isolated from Patients with Regressing Metastatic Kidney Cancer after Allogeneic Nonmyeloablative Hematopoietic Cell Transplantation (NMHCT).

Graft-vs-tumor (GVT) effects following NMHCT induce disease regression in a subset of patients (pts) with advanced metastatic kidney cancer. At present, little is known about the antigens serving as tumor targets in responding pts. In an effort to characterize GVT effectors and their tumor antigens, we generated RCC cell lines to use as targets in cytotoxicity assays in three pts (one non-responder and two responders) undergoing a cyclophosphamide/fludarabine-based NMHCT from HLA matched siblings. Peripheral blood lymphocytes (PBL) were collected from pts at multiple time-points after transplantation and were expanded in-vitro with either irradiated patient (pre-transplant) PBL/EBV-LCL or autologous tumor cells. RCC pt #1 developed grade II skin GVHD on day 22 but did not have an objective tumor response and died from progressive tumor on day 203. In a Cr51 release assay, minor histocompatibility antigen specific (mHa) T-cell lines generated using pre-transplant pt PBL/EBV-LCL stimulators lysed 98% of pt CD40-ligand stimulated B cells (CD40L-B) but did not kill pt RCC cells. RCC pt #2 developed grade II skin GVHD on Day 51 and was noted to have regression of lung metastasis on day 183. Using PBL obtained during tumor regression, mHa- reactive T-cell lines were generated (pre-transplant pt PBL/EBV-LCL used as stimulators) that lysed 76% and 12% of pt EBV-LCL and autologous RCC tumor cells at a 20:1 E:T ratio. Following limiting dilution cloning, 6 CD8+ T cell clones were expanded that killed pt EBV-LCL (but not donor) including one MHC class-1 restricted T cell clone that lysed both pt EBV-LCL and pt tumor cells. RCC pt #3 developed grade III skin GVHD on day 120, had regression of lung metastasis on day 160, and survives more than 4 years after transplantation. PBL collected from this pt 40 months after transplant contained CD8+ T-cell populations that secreted IFN-g (0.9% by intracellular cytokine staining) when co-cultured with pt RCC cells but not after co-culture with pt CD40L-B cells. CD3+/CD8+ CTL were expanded from these PBL using pt RCC cells as stimulators; in vitro, these CTL killed the pt's RCC cells but did not kill pt fibroblasts or pt EBV-LCL (Figure A). Following co-culture with tumor, intracellular IFN-g staining combined with TCR Vb antibodies revealed 3 tumor reactive CD8+ T-cell populations (TCR Vb7+, TCRVb5.1+, and TCRVb non-staining); 25.2% of these CTL were TCRVb7+. IFN-g secretion by TCR Vb7+ T cells was blocked when tumor cells were pre-incubated with mAbs to CD8, pan MHC class I and HLA-A11 (Figure B) consistent with recognition of an HLA-11 restricted tumor antigen. Following limiting dilution cloning, several CD8+ T-cell clones (both TCR Vb7+ and TCR Vb non-staining) with RCC-specific cytotoxicity were identified. We conclude that donor T-cells with both broad alloreactivity and tumor specificity play a role in mediating GVT effects in RCC pts having disease regression following NMHCT. [Display omitted]