T-cell Cultures Research Articles

Abstract B55: Suppression of CD8 T-cell activation by tryptophan catabolism in triple-negative breast cancer

Abstract Background: The ability of cancer cells to evade and suppress an antitumor immune response is increasingly recognized as a critical feature of aggressive, metastatic disease. Conversion of the amino acid tryptophan (trp) into the catabolite kynurenine (kyn) and the subsequent secretion of kyn is a current area of interest in the field of antitumor immunity, given that trp depletion is a trigger for T-cell death, and that kyn binds to and activates the aryl-hydrocarbon receptor (AhR) in immune cells, often with immunosuppressive consequences. In a screen to identify mechanisms promoting survival of triple-negative breast cancer (TNBC) cells in transit during the metastatic cascade, our lab discovered that trp-to-kyn catabolism is significantly increased by TNBC cells in forced-suspension culture due to upregulation of the gene encoding the rate-limiting trp-catabolizing enzyme tryptophan 2,3-dioxygenase (TDO2). Hypothesis: In addition to promoting survival of TNBC cells during metastasis, TDO2-dependent trp-to-kyn catabolism may facilitate tumor-mediated immune suppression, further contributing to the aggressive nature of this breast cancer subtype. Methods: TNBC cell lines were plated in adherent or forced-suspension culture for 24 hours, using poly (2-hydroxyethyl methacrylate)-coated plates to prevent adhesion. TDO2 mRNA (using quantitative PCR) and protein (immunoblotting) were measured in adherent versus suspended cells. Secreted kyn was measured using liquid chromatography-mass spectrometry and inhibited with a selective TDO2 inhibitor, 680C91. To test the effect of kyn on CD8 T-cell function, primary human CD8 T-cells were isolated from the blood of healthy human donors using a positive selection kit. T-cell proliferation was measured by dilution of the cell-permeable dye carboxyfluorescein succinimidyl ester and T-cell death was measured by incorporation of a fixable viability dye. Flow cytometry was used to measure both surface and intracellular T-cell proteins. Results: The rate limiting enzyme TDO2 was upregulated in BT549 cells in suspension by 20.1-fold at the mRNA level and 5.4-fold at the protein level. Secreted kyn increased by 2.4-fold in suspension culture (0.46 μM secreted kyn by adherent cells, 1.09 μM kyn secreted by suspended cells, n=3 replicates), and this increase was reversed by addition of the TDO2 inhibitor 680C91 (p<0.0001). Over the course of a five day CD3/CD28 activation, purified kyn reduced CD8 T-cell proliferation and increased CD8 T-cell death in a dose-dependent manner (n=6-9 donors, p<0.05 at 25, 50, and 100 μM kyn), and these effects were partially reversed by the addition of an AhR antagonist (n=6 donors, p<0.05 at 25 μM kyn). Kyn also reduced expression of granzyme B by live CD8 T-cells at high doses (n=4 donors, p=0.05 at 100 μM kyn), but did not alter expression of perforin or tumor necrosis factor alpha. Consistent with these results, culture of human CD8 T-cells in conditioned media from suspended BT549 cells over the course of a five day CD3/CD28 activation resulted in reduced proliferation and increased cell death in CD8 T-cells (n=6 donors, p<0.05) compared to culture in conditioned media from adherent BT549 cells. Expression of the pro-inflammatory cytokine interferon gamma by CD8 T-cells cultured in conditioned media from suspended BT549 cells may also be reduced (n=4 donors, p=0.08). Conclusions: Together, these results suggest a role for TNBC-mediated trp-to-kyn catabolism in suppressing cytotoxic T-cells. Further in vivo studies will indicate whether TDO2 activity might be a rational therapeutic target for improving antitumor immunity in patients with this aggressive subtype of breast cancer. Citation Format: Lisa I. Greene, Tullia C. Bruno, Thomas J. Rogers, Jill E. Slansky, Virginia F. Borges, Jennifer K. Richer. Suppression of CD8 T-cell activation by tryptophan catabolism in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B55.

Cytokine-mediated regulation of expression of Gfi1 and U2afll4 genes activated by T-cells with different differentiation status in vitro

The dose-dependent effects of cytokines (IL-2, IL-7, and IL-15), which have a common g-chain, on mRNA expression of U2afll4 and GFi1 genes involved in regulation of alternative splicing of the Ptprc gene, have been investigated in vitro using T-lymphocyte cultures with different degrees of differentiation. IL-2, IL-7, and IL-15 caused a similar unidirectional inhibitory effect of various severity on restimulated CD45RO+ T-cells exposed to an antigen-independent activation; they caused a dose-dependent decrease of the U2af1l4 gene expression, and an increase of Gfi1 gene expression. This may suggest formation of active forms of the CD45 receptor, and also limitation of the formation of low-molecular short splice variants of the CD45RO receptor. Under conditions of antigen-independent stimulation of naive CD45RA+-cells rIL-7 and IL-15 exhibited opposite effects on U2af1l4 and Gfi1 gene expression. The increase of IL-7 concentrations in the incubation medium of naive cells was accompanied by a decrease in expression of both genes. IL-15 IL-7 exhibited opposite effects. Cytokines possessing a common g-chain (IL-2, IL-7, and IL-15) prevented antigen-independent differentiation of naive T-cells, by preventing the formation of polyclonal "surrogate" cells. In general, the study of the molecular mechanisms of genetic control determining homeostatic processes of T-cells in response to exposure to antigenic or non-antigenic treatments may be important for construction of a general model of self-maintenance and differentiation of immune cells.

Mesenchymal Stromal Cells from Osteoarthritic Synovium Are a Distinct Population Compared to Their Bone-Marrow Counterparts regarding Surface Marker Distribution and Immunomodulation of Allogeneic CD4+ T-Cell Cultures.

Introduction. The participation of an inflammatory joint milieu has been described in osteoarthritis (OA) pathogenesis. Mesenchymal stromal cells (MSCs) play an important role in modulating inflammatory processes. Based on previous studies in an allogeneic T-cell coculture model, we aimed at further determining the role of synovial MSCs in OA pathogenesis. Methods. Bone-marrow (BM) and synovial membrane (SM) MSCs from hip joints of late stage OA patients and CD4+ T-cells from healthy donors were analysed regarding surface marker expression before and after coculture. Proliferation upon CD3/CD28 stimulation and cytokine analyses were compared between MSCs. Results. SM-MSCs differed from BM-MSCs in several surface markers and their osteogenic differentiation potential. Cocultures of both MSCs with CD4+ T-cells resulted in recruitment of CD45RA+ FoxP3+ regulatory T-cells. Upon stimulation, only SM-MSCs suppressed CD4+ T-cell proliferation, while both SM-MSCs and BM-MSCs modified cytokine profiles through suppressing IL-2 and TNF-α as well as increasing IL-6 secretion. Conclusions. Synovial MSCs from OA joints are a unique fraction that can be distinguished from their bone-marrow derived counterparts. Their unique ability to suppress CD3/CD28 induced CD4+ T-cell proliferation makes them a potential target for future therapeutic approaches.

Up-regulated Circulating T-cell Angiotensin Converting Enzyme Gene Expression and Activity in Acute Coronary Syndromes

Background: T-cells are endowed with a functional cell-based renin angiotensin system (RAS), related with and independent by circulating RAS that can autonomously synthesize angiotensin (Ang) II. Angiotensing converting enzyme (ACE) is the key step for the regulation of the synthesis of Ang II by RAS and T-cell ACE gene expression was reported to be up-regulated in hypertensives with low grade inflammation. Ang II and T-cells play a major role in the systemic inflammation that occurs in unstable angina patients, but whether T-cell based RAS is directly involved is unknown. This study was aimed at measuring ACE gene expression and enzymatic activity in the cell pellet and in the supernatant of human cultured circulating T-cells obtained from control subjects, hypertensive or unstable angina patients. C reactive protein (CRP) levels as a marker of systemic inflammation were also investigated. Methods: mRNA for ACE gene expression was obtained in T cells isolated from peripheral blood and quantified by real time transcriptase–polymerase chain reaction (PCR); mRNAs for INF-gamma was semi-quantified versus the housekeeping gene glyceraldeyde-3-phosphate dehydrogenase (GAPDH) by reverse transcriptase (RT) PCR. ACE activity in cell pellet and in the culture medium (supernatant) was measured by the high performance liquid chromatography (HPLC) assay of a synthetic substrate. Plasma renin activity (PRA) and Ang II levels were measured by radioimmunoassay and high sensitive C Reactive Protein (hsCRP) by a commercial kit. Results: In hypertensive and more markedly in anginal patients increased mRNA levels for ACE, augmented cell-based ACE enzymatic activity and Ang II levels were measured in cultured circulating T-cells when compared with data obtained in T cells from controls (p<0.05 for alls). The addition of Ang II to the T-cell pellet further increase ACE activity and Ang II synthesis. In anginal patients who showed the highest ACE gene expression and enzymatic activity and hsCRP values, Ang II stimulation of T-cells induced an almost complete release of ACE in the supernatant. Conclusions: In anginal patients with hsCRP levels >3 mg/dl a marked up-regulation of circulating T-cell-based ACE gene expression and activity did occur in T-cell culture, further amplified by Ang II. According to our in vitro findings, in vivo activated T-cells could autonomously increase local de novo Ang II synthesis in tissues where they migrate and play a role in coronary plaque rupture and microvessel damage of unstable angina.

Clinical Activity and Persistence of Anti-CD22 Chimeric Antigen Receptor in Children and Young Adults with Relapsed/Refractory Acute Lymphoblastic Leukemia (ALL)

Background: Adoptive cellular therapy with genetically modified T-cells using viral-based vectors to express chimeric antigen receptors targeting the CD19 molecule have demonstrated dramatic clinical responses in patients ALL. However, not all patients respond and CD19-negative escape has been observed. Thus, additional targets are needed. CD22 is a B-lineage-restricted, transmembrane phosphoglycoprotein of the Ig superfamily that is widely expressed on B-precursor ALL. Therefore, CD22 represents a promising target. This phase I dose-escalation study represents the first in human testing of anti-CD22 CAR adoptive cell therapy. The primary objectives were to determine the feasibility of producing anti-CD22 CAR cells meeting established release criteria and to assess the safety of administering escalating doses of anti-CD22-CAR engineered T cells in children and young adults with relapsed or refractory CD22-expressing B cell malignancies following a cyclophosphamide/fludarabine preparative regimen. Secondary objectives include determination of anti-leukemia effects, measurement of persistence of adoptively transferred anti-CD22 CAR T cells, and evaluation of cytokine profiles.Design: Children and young adults with relapsed/refractory CD22-expressing hematologic malignancies were eligible. Study endpoints included toxicity, feasibility, and antigen-specific immune and clinical responses. All enrolled subjects underwent autologous leukopheresis for peripheral blood mononuclear cells. Cells were then CD3+ enriched and cultured in the presence of anti-CD3/-CD28 beads followed by lentiviral vector supernatant containing the anti-CD22 (M971BBz) CAR, with culture duration of 7-10 days. On Day-4 (cell infusion=Day 0), subjects began induction chemotherapy with fludarabine 25 mg/m2 on Days-4, -3 and -2 and cyclophosphamide 900 mg/m2 on day-2. The first dose level started at 3 x 105 transduced T-cells/recipient weight (kg).Results: 6 subjects, aged 7-22 years, with ALL have been treated to date. All enrolled subjects had previously undergone at least one prior allogeneic hematopoietic stem cell transplant and all had received treatment with CD19 directed CAR-T cell therapy. Five subjects had a CD19 negative antigen escape, and one subject was a non-responder to prior CD19 CAR therapy. All subjects had demonstration of CD22 expression on > 99% of their ALL, although the antigen binding capacity had variability from < 900 to > 13,000 sites/cells. All subjects underwent successful culture, expansion and infusion of anti-CD22 CAR T-cells at the first dose level. The second subject enrolled met criteria for dose-limiting toxicity by virtue of grade 3 diarrhea which led to dose expansion at the first dose-level to treat a total of 6 subjects. Two subjects had grade 1 cytokine release syndrome (CRS), one subject had grade 2 CRS and in two subjects CRS was not seen. Evidence for CAR-T cell expansion was seen in peripheral blood, bone marrow and cerebrospinal fluid (Table). Clinical responses were evaluated at day 28 (+/- 4 days) post-infusion and included two subjects who had disease progression, two with disease stabilization and one subject who attained a minimal residual disease (MRD) negative complete remission. Flow cytometric CAR persistence was detected out to 47 days post-infusion in the responding patient with remission maintained for 3 months post-infusion. One patient is actively undergoing treatment and is too early to evaluate.Conclusions: This first-in-human anti-CD22 CAR T-cell therapy is safe, feasible and clinically active in patients who have undergone previous CAR therapy. Understanding mechanisms which may determine clinical efficacy are being explored. Accrual to the next dose level at 1 x 106 transduced T cells/kg is planned. Table 1#Age/SexPrior HCTPrior anti-CD19 CARCD19 neg relapse?CD22 site densityPre-HCT disease burden (% leukemia in aspirate)Maximum CD22 CAR expansion (flow)CRSBest ResponsePBMarrowCSF122/MYYY208495-100%00n/aNonePD220/FY (2)YY134525%52.3%19.5%0%1MRD neg CR322/MYYY846>90%73%36%32%1SD422/MYYN258995%6%1%0%2SD57/FYYY283932%0%1.3%0%NonePD617/FYYY21851%n/an/an/an/an/aSD: stable disease; PD: progressive disease DisclosuresMackall:Juno: Patents & Royalties: CD22-CAR.

Stimulation with lysates of Aspergillus terreus, Candida krusei and Rhizopus oryzae maximizes cross-reactivity of anti-fungal T cells

Background aims. Invasive fungal diseases caused by filamentous fungi and yeasts are significant causes of morbidity and mortality in immunosuppressed hematology patients. We previously published a method to expand Aspergillus fumigatus–specific T cells for clinical cell therapy. In the present study, we investigated expansion of T cells specific for other fungal pathogens and creation of a broadly reactive panfungal T-cell product. Methods. Fungal strains selected were those frequently observed in the clinical hematology setting and included Aspergillus, Candida, Fusarium, Rhizopus and Lomentospora/Scedosporium. Four T-cell cultures specific to each fungus were established. We selected lysates of Aspergillus terreus, Candida krusei and Rhizopus oryzae to expand panfungal T cells. Allelic restriction of anti-fungal activity was determined through the use of specific major histocompatibility complex class II–blocking antibodies. Results. Individual T-cell cultures specific to each fungus could be expanded in vitro, generating predominantly CD4+ T cells of which 8% to 20% were fungus-specific. We successfully expanded panfungal T cells from the peripheral blood (n = 8) and granulocyte–colony-stimulating factor–primed stem cell products (n = 3) of normal donors by using a combination of lysates from Aspergillus terreus, Candida krusei and Rhizopus oryzae. Anti-fungal activity was mediated through human leukocyte antigen (HLA)-DR alleles and was maintained when antigen-presenting cells from partially HLA-DRB1–matched donors were used to stimulate T cells. Conclusions. We demonstrate a method to manufacture panfungal T-cell products with specificity against a range of clinical fungal pathogens by use of the blood and stem cells of healthy donors as the starting material. The safety and efficacy of these products will need to be tested clinically.

Epithelial IL-1R2 acts as a homeostatic regulator during remission of ulcerative colitis.

Ulcerative colitis (UC) is a chronic intestinal inflammatory disease that may undergo periods of activity followed by remission. We aimed to identify the endogenous regulatory mechanisms that may promote disease remission. Transcriptional and protein analysis of the intestinal mucosa revealed that the IL-1 decoy receptor, interleukin-1 receptor type 2 (IL1R2), was upregulated in remission compared with active UC and controls. We identified epithelial cells as being responsible for increased IL-1R2 production during remission. Expression of IL1R2 was negatively regulated by Wnt/beta-catenin signals in colonic crypts or epithelial stem cell cultures; accordingly, epithelial stem cells upregulated IL-1R2 upon differentiation. Blocking IL-1R2 in isolated colonic crypt cultures of UC patients in remission and T-cell cultures stimulated with biopsy supernatant from UC patients in remission boosted IL-1β-dependent production of inflammation-related cytokines. Finally, IL1R2 transcription was significantly lower in patients that relapsed during a 1-year follow-up period compared with those in endoscopic remission. Collectively, our results reveal that the IL-1/IL-1R2 axis is differentially regulated in the remitting intestinal mucosa of UC patients. We hypothesize that IL-1R2 in the presence of low concentrations of IL-1β may act locally as a regulator of intestinal homeostasis.

Cytokine Release Patterns in Mixed Lymphocyte Culture (MLC) of T-Cells with Dendritic Cells (DC) Generated from AML Blasts Contribute to Predict anti-Leukaemic T-Cell Reactions and Patients' Response to Immunotherapy.

To enlighten interactions between autologous, allogeneic or T-cells from patients after stem cell transplantation with leukaemia-derived-dendritic-cells containing dendritic cells or blast containing mononuclear cells (n = 21, respectively), we determined cytokine-concentrations (interleukin 2, 4, 6, 10, tumor-necrosis-factor-α, interferon-γ) in supernatants of mixed-lymphocyte-culture and in serum (n = 16) of 20 patients with acute myeloid leukaemia and three patients with myelodysplastic syndromes by cytometric-bead-assay. We correlated our data with lytic capabilities of stimulated T-cells in a fluorolysis-assay and clinical data: Dendritic-cell-/mononuclear-cell-stimulation of T-cells resulted in increased cytokine-levels in culture-medium compared to serum. There were no significant differences between cytokine-patterns of cases with/without lytic T-cell-activity, response to immunotherapy (stem cell transplantation/donor-lymphocyte-infusion) or graft-versus-host-disease. However, some predictive cytokine-cut-off-values for antileukaemic T-cell-activity, patients' response to immunotherapy and graft-versus-host-disease could be defined. Cytokine-profiles alone, without functional assays, are no useful tool to predict antileukaemic T-cell-function, although they can indicate lytic T-cell-activity, patients' response to immunotherapy and graft-versus-host-disease.

Lipophilic prodrugs of nucleoside triphosphates as biochemical probes and potential antivirals.

The antiviral activity of nucleoside reverse transcriptase inhibitors is often limited by ineffective phosphorylation. We report on a nucleoside triphosphate (NTP) prodrug approach in which the γ-phosphate of NTPs is bioreversibly modified. A series of TriPPPro-compounds bearing two lipophilic masking units at the γ-phosphate and d4T as a nucleoside analogue are synthesized. Successful delivery of d4TTP is demonstrated in human CD4+ T-lymphocyte cell extracts by an enzyme-triggered mechanism with high selectivity. In antiviral assays, the compounds are potent inhibitors of HIV-1 and HIV-2 in CD4+ T-cell (CEM) cultures. Highly lipophilic acyl residues lead to higher membrane permeability that results in intracellular delivery of phosphorylated metabolites in thymidine kinase-deficient CEM/TK− cells with higher antiviral activity than the parent nucleoside.

Oxypurinol-Specific T Cells Possess Preferential TCR Clonotypes and Express Granulysin in Allopurinol-Induced Severe Cutaneous Adverse Reactions

Allopurinol, a first-line drug for treating gout and hyperuricemia, is one of the leading causes of severe cutaneous adverse reactions (SCARs). To investigate the molecular mechanism of allopurinol-induced SCAR, we enrolled 21 patients (13 Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) and 8 drug reaction with eosinophilia and systemic symptoms (DRESS)), 11 tolerant controls, and 23 healthy donors. We performed in vitro T-cell activation assays by culturing peripheral blood mononuclear cells (PBMCs) with allopurinol, oxypurinol, or febuxostat and measuring the expression of granulysin and IFN-γ in the supernatants of cultures. TCR repertoire was investigated by next-generation sequencing. Oxypurinol stimulation resulted in a significant increase in granulysin in the cultures of blood samples from SCAR patients (n=14) but not tolerant controls (n=11) or healthy donors (n=23). Oxypurinol induced T-cell response in a concentration- and time-dependent manner, whereas allopurinol or febuxostat did not. T cells from patients with allopurinol-SCAR showed no crossreactivity with febuxostat. Preferential TCR-V-β usage and clonal expansion of specific CDR3 (third complementarity-determining region) were found in the blister cells from skin lesions (n=8) and oxypurinol-activated T-cell cultures (n=4) from patients with allopurinol-SCAR. These data suggest that, in addition to HLA-B*58:01, clonotype-specific T cells expressing granulysin upon oxypurinol induction participate in the pathogenesis of allopurinol-induced SCAR.

Induction of APOBEC3B cytidine deaminase gene in HTLV-1 infected T-cells of ATL model mouse

A variety of mutations are accumulated in the genome of HTLV-1 infected T-cells during ATL development. To elucidate the mechanism of ATL development a mouse model of ATL was established by infecting HTLV-1 to humanized NOG mice and the infected mice recapitulate the ATL-like symptoms and die of leukemia within several months of infection. Analysis of gene expressions in the humanized mouse model of ATL demonstrated the induction of APOBEC3B (A3B) gene in the HTLV-1 infected human T-cells. A3B is a member of the APOBEC family of cellular cytidine deaminase and was recently identified as the mutational source in multiple human cancers. We have previously shown that HTLV-1 infected CD25 (-) CD4 T-cells but not CD25 (+) CD4 T-cells in ATL model mouse express a small amount of Tax mRNA even though both cell populations consist of identical infected-cell clones. As the A3B expression in HTLV-1 infected CD25 (+) T-cells was similar to, or rather higher than that in CD25 (-)T-cells, Tax appears not to be involved in the induction A3B. In contrast, ex vivo culture of infected T-cells, in which Tax expression was highly enhanced in both CD25 (+) and CD25 (-)T-cells, resulted in the suppression of A3B expression. However, ectopic expression of HTLV-1 in Jurkat cell by the transfection of HTLV-1 molecular clone plasmid enhanced the expression of A3B gene. It is thus suggested that HTLV-1 infection but not Tax induces the A3B expression. Interplay between HTLV-1 infection and A3B and the possible involvement of A3B in the mutagenesis of host cell genome are currently investigated.

Low-cost generation of Good Manufacturing Practice–grade CD19-specific chimeric antigen receptor–expressing T cells using piggyBac gene transfer and patient-derived materials

Background aimsProtocols for the production of CD19-specific chimeric antigen receptor (CAR19) T cells are often complex and expensive because of the use of retroviral and lentiviral vectors or the need for CAR19 T-cell enrichment. We aimed to simplify the generation of CAR19 T cells from the peripheral blood of normal donors and patients using the piggyBac transposon system of gene modification. MethodsWe varied electroporation voltage, cytokines and stimulation conditions for the generation and expansion of CAR19 T cells over a 3-week culture period. ResultsUsing optimized electroporation voltage, interleukin-15 alone and co-culturing CAR T cells with peripheral blood mononuclear cells, we were able to expand CAR19 T-cell cultures by up to 765-fold over 3 weeks in normal donors and 180-fold in patients with B-cell malignancies. Final median CAR19 expression of 72% was seen in normal donors, and 81% was seen in patients with acute lymphoblastic leukaemia, chronic lymphocytic leukemia or non-Hodgkin lymphoma. CAR19 T cells produced interferon gamma on stimulation with CD19+ cell lines and efficiently lysed both CD19+ cell lines and primary leukemia cells. In addition, combining CAR expression with an inducible caspase safety switch allowed elimination of CAR19 T cells by the application of a small molecule dimerizer. DiscussionWe have produced a simple, inexpensive and easily adoptable protocol for the generation of CAR19 T cells suitable for use in clinical trials using the piggyBac transposon system. This provides a robust platform for further enhancing the T-cell product and testing new CAR technologies.

Expression and functions of long noncoding RNAs during human T helper cell differentiation.

Long noncoding RNAs (lncRNAs) regulate an array of biological processes in cells and organ systems. Less is known about their expression and function in lymphocyte lineages. Here we have identified >2000 lncRNAs expressed in human T cell cultures and those which display a TH lineage specific pattern of expression and are intragenic or adjacent to TH lineage specific genes encoding proteins with immunologic functions. One lncRNA cluster selectively expressed by the effector TH2 lineage consists of four alternatively spliced transcripts that regulate expression of TH2 cytokines, IL-4, IL-5 and IL-13. Genes encoding this lncRNA cluster in humans overlap the RAD50 gene and thus are contiguous with the previously described TH2 locus control region (LCR) in the mouse. Given its genomic synteny with the TH2 LCR, we refer to this lncRNA cluster as TH2-LCR lncRNA.

Cytokine-mediated regulation of expression of Gfi1 and U2afll4 genes by activated T-cells with various differentiation status in vitro

The dose-dependent effects of cytokines (IL-2, IL-7, and IL-15), which have a common γ-chain, on mRNA expression of U2afll4 and GFi1 genes involved in regulation of alternative splicing of the Ptprc gene, have been investigated in vivo using T-lymphocyte cultures with different degrees of differentiation. IL-2, IL-7, and IL-15 caused a similar unidirectional inhibitory effect of various severity on restimulated CD45RO+ T-cells exposed to an antigen-independent activation; they caused a dose-dependent decrease of the U2af1l4 gene expression, and an increase of Gfi1 gene expression. This may suggest formation of active forms of the CD45 receptor, and also limitation of the formation of low-molecular short splice variants of the CD45RO receptor. Under conditions of antigen-independent stimulation of naive CD45RA+ - cells rIL-7 and IL-15 exhibited opposite effects on U2af1l4 and Gfi1 gene expression. The increase of IL-7 concentrations in the incubation medium of naive cells was accompanied by a decrease in expression of both genes. IL-15 IL-7 exhibited opposite effects. Cytokines possessing a common Γ-chain (IL-2, IL-7, and IL-15) prevented antigen-independent differentiation of naive T-cells, by preventing the formation of polyclonal “surrogate” cells. In general, the study of the molecular mechanisms of genetic control determining homeostatic processes of T-cells in response to exposure to antigenic or non-antigenic treatments may be important in the construction of a general model of self-maintenance and differentiation of immune cells.

Generation of Human B-Cell Lines Dependent on CD40-Ligation and Interleukin-4.

WHERE WE STARTED The description of factor-dependent cytotoxic T-cell lines in the late 1970s transformed T-cell biology (1). Among other events, it led to the cloning of a cDNA encoding IL-2 (2). It also led to the identification of T-cell subsets and formulation of the Th1/Th2 concept in the 80s (3). However, comparable advances in B-cell biology were lacking, partly because of the lack of availability of factor-dependent Bcell lines. This was the case despite the fact that B-cell-specific trophic factors, including BSF (B-cell stimulation Factor), BCGF (B-cell growth factor), and BCDF (B-cell differentiation factor) had been described in the supernatants of activated T cells. The cloning at DNAX, our sister institute acquired by Schering–Plough, of a cDNA encoding BSF-1, later renamed IL4, in mouse (4) and in human (5) was a first step forward to the definition of the molecules controlling B-cell growth and differentiation. In our laboratory, based in Dardilly near Lyon (France), we found that cultured purified human B-cells triggered with anti-B-cell receptor (BCR) and IL-4 resulted in significant B-cell proliferation as measured by tritiated thymidine counts, a common way of measuring Bcell proliferation in the 1980–1990s (6). These cultures yielded more B-cells than did naive cultures or those exposed to antiBCR alone or IL-4 alone. Yet, these cultures established with anti-BCR plus IL-4 yielded less viable B-cells than were input. Thus, we, B-cell biologists had not yet been able to reproduce with B-cells the factor-dependent growth of T cells that our colleagues T-cell biologists have been able to achieve. FEEDER CELLS AND NEW MONOCLONAL ANTIBODIES YIELD MORE ROBUST B-CELL CULTURES A possible explanation for our lack of success was the absence of feeder cells, which had become part of the T-cell culture system and proved necessary to allow for the expansion of human T-cell lines and clones. Meanwhile, Kevin Moore and his colleagues at DNAX, cloned a human cDNA coding for FcγRII/CD32 and found that FcγRII/CD32-transfected fibroblast cell lines could present monoclonal antibodies in a manner that allowed for cross-linking of the target molecule of the relevant cell (7, 8). More specifically, antibodies to the T-cell CD3 complex presented by these transfected cells together with IL-2 could induce prolonged T-cell proliferation (9). Thus, we wondered whether the presentation of monoclonal antibodies specifically directed at B-cell surface molecules in the presence of B-cell tropic cytokines would lead to the proliferation and expansion of Bcells. By the end of the 80s, we, investigators from Schering–Plough/DNAX had cloned cDNAs encoding human GM-CSF (10), IL-4, IL-5 (5, 6), and FcgR/CD32 (8). We had also generated a number of monoclonal antibodies that would recognize Bcells including a CD40 antibody (11) and an anti-B7 antibody now known as CD86 (12). When Paolo de Paoli came to our lab to perfect his flow cytometry skills, he took a side project to refine methods for culturing sorted B-cells using both classical and new approaches, including the addition of a feeder-layer of CD32/FcγR-transfected cells as discussed above (9). To this end, 96-well-plate microwells were first seeded with the irradiated fibroblast line. A few thousand B-cells were then added along with a few selected monoclonal antibodies with or without IL-2 or IL-4. Cultures were harvested 3–5 days later after a brief pulse with tritiated thymidine. It very quickly became apparent that the combination of the CD40 antibody Mab 89 (11) and IL-4 could induce unusually strong B-cell proliferation. The wellknown CD40 antibody G28-5 made by Ed Clark and Jeff Ledbetter also proved highly effective in this system (13, 14). Curiously, IL-2 was unable to enhance CD40induced B-cell proliferation, although it did enhance the proliferation of B-cells activated through their BCR. Furthermore, the fibroblast layer provided some feeder effect, as cross-linking the CD40 antibody on plastic was never as effective in inducing prolonged B-cell proliferation as presenting it with the CD32-transfected fibroblast.

Tryptophan 2,3-dioxygenase (TDO)-reactive T cells differ in their functional characteristics in health and cancer

Tryptophan-2,3-dioxygenase (TDO) physiologically regulates systemic tryptophan levels in the liver. However, numerous studies have linked cancer with activation of local and systemic tryptophan metabolism. Indeed, similar to other heme dioxygenases TDO is constitutively expressed in many cancers. In the present study, we detected the presence of both CD8+ and CD4+ T-cell reactivity toward TDO in peripheral blood of patients with malignant melanoma (MM) or breast cancer (BC) as well as healthy subjects. However, TDO-reactive CD4+ T cells constituted distinct functional phenotypes in health and disease. In healthy subjects these cells predominately comprised interferon (IFN)γ and tumor necrosis factor (TNF)-α producing Th1 cells, while in cancer patients TDO-reactive CD4+ T-cells were more differentiated with release of not only IFNγ and TNFα, but also interleukin (IL)-17 and IL-10 in response to TDO-derived MHC-class II restricted peptides. Hence, in healthy donors (HD) a Th1 helper response was predominant, whereas in cancer patients CD4+ T-cell responses were skewed toward a regulatory T cell (Treg) response. Furthermore, MM patients hosting a TDO-specific IL-17 response showed a trend toward an improved overall survival (OS) compared to MM patients with IL-10 producing, TDO-reactive CD4+ T cells. For further characterization, we isolated and expanded both CD8+ and CD4+ TDO-reactive T cells in vitro. TDO-reactive CD8+ T cells were able to kill HLA-matched tumor cells of different origin. Interestingly, the processed and presented TDO-derived epitopes varied between different cancer cells. With respect to CD4+ TDO-reactive T cells, in vitro expanded T-cell cultures comprised a Th1 and/or a Treg phenotype. In summary, our data demonstrate that the immune modulating enzyme TDO is a target for CD8+ and CD4+ T cell responses both in healthy subjects as well as patients with cancer; notably, however, the functional phenotype of these T-cell responses differ depending on the respective conditions of the host.

Seasonal modulation of immunity by melatonin and gonadal steroids in a short day breeder goat Capra hircus

Role of melatonin in regulation of immunity and reproduction has never been studied in detail in goats. The aim of the present study was to explore hormonal regulation of immunity in goats with special reference to melatonin. Plasma of male and female goats (n = 18 per sex per season) was processed for hormonal (estrogen, testostrone, and melatonin) and cytokine (interleukin [IL-2], IL-6, and tumor necrosis factor α) measurements during three seasons, i.e., summer, monsoon, and winter. To assess cell-mediated immune response, percent stimulation ratio of thymocytes was recorded during three seasons. To support and establish the modulation by hormones, Western blot analysis for expressions of melatonin receptors (MT1, MT2), androgen receptor, and estrogen receptor α and estimations of marker enzymes, arylalkylamine N-acetyltransferase for melatonin and 3β-hydroxysteroid dehydrogenase activities for steroidogenesis were performed in thymus. All the hormones and cytokines were estimated by commercial kits. Biochemical, immunologic, and Western blot analyses were done by standardized protocols. We noted a significant increase in estrogen and testosterone levels (P < 0.05) in circulation during monsoon along with melatonin (P < 0.05) presenting a parallel relationship. Expressions of melatonin receptors (MT1 and MT2) in thymus of both the sexes were significantly high (P < 0.01) during winter. Estrogen receptor α expression in female thymus was significantly high during monsoon (P < 0.05). However, androgen receptor showed almost static expression pattern in male thymus during three seasons. Further, both arylalkylamineN-acetyltransferase and 3β-hydroxysteroid dehydrogenase enzyme activities were significantly high (P < 0.05; P < 0.01, respectively) during monsoon. These results suggest that there may be a functional parallelism between gonadal steroids and melatonin as melatonin is progonadotrophic in goats. Cell-mediated immune parameters (percent stimulation ratio of thymocytes) and circulatory levels of cytokines (IL-2, IL-6, and tumor necrosis factor α) were significantly high (P < 0.01) during monsoon. In vitro supplementation of gonadal steroids to T-cell culture suppressed immunity but cosupplementation with melatonin restored it. Further, we may also suggest that reproductive and immune seasonality are maintained by variations in circulatory hormones and local synthesis of melatonin and gonadal steroids. These functional interactions between melatonin and gonadal steroid might be of great importance in regulating the goat immunity by developing some hormonal microcircuit (gonadal steroid and melatonin) in lymphatic organs.

Characterization of primary human T‐cell responses to p‐phenylenediamine and Bandrowski's base

p-Phenylenediamine (PPD) is a component of certain henna tattoo and hair dye formulations. Human exposure to PPD containing products is associated with the development of T-cell mediated allergic contact dermatitis. Antigen-specific T-cells from allergic contact dermatitis patients are activated with either PPD or Bandrowskis base (BB), the metabolized trimer of PPD. In non-allergic individuals T-cells that are activated by BB, but not by PPD, are readily detectable. Antigen-specific T-cells from patients with allergic contact dermatitis show increased secretion of Th2 cytokines. The aim of the current study was to use an in vitro T-cell priming assay that utilizes peripheral blood mononuclear cells from healthy donors to study the activation of memory (CD45RO+) and naive (CD45RA+) T-cells with PPD and BB. T-cells were cloned from the priming assay and the phenotype and function of antigen-specific cells was compared with T-cells from patients with allergic contact dermatitis. The T-cell priming assay requires cell separation from whole peripheral blood mononuclear cells using magnetic bead sorting, followed by a period of memory/naive T-cell culture with CD14+ monocyte-derived dendritic cells and PPD or BB. After this, antigen-specific (PPD 2.5-10μM; BB 2.5-10μM) proliferation was measured by [3H]thymidine incorporation and cytokine release by IFNg, IL-13, and IL-22 ELISpot. Cell phenotype was characterised by flow cytometry. Concentration-dependent BB-specific T-cell proliferative responses were detected with both naive and memory T-cells from healthy donors. Activation of the T-cells was associated with the secretion of IFNg, IL-13 and granzyme B. In contrast, PPD-specific T-cells were not detected from either the memory or naive compartment. T-cell clones generated from the BB-responsive cultures were activated in the presence of BB, but not PPD. As described previously, T-cell proliferative responses and cytokine release to PPD and BB were detected with peripheral blood mononuclear cells from allergic patients and PPD and BB specific clones were generated. In conclusion these data show that BB stimulates pre-existing memory T-cells isolated from allergic patients and healthy donors. Furthermore, BB primes naive T-cells when the chemical is presented by autologous dendritic cells. In contrast, priming naive T-cells against PPD failed which suggests an important individual susceptibility factor is missing from the existing T-cell priming assay.

S100A4 regulates the Src-tyrosine kinase dependent differentiation of Th17 cells in rheumatoid arthritis

Objectives: To evaluate the role of S100A4, a calcium-binding regulator of nonmuscle myosin assembly, for T-cell responses in rheumatoid arthritis. Methods: Arthritis was induced in the methylated bovine serum albumin (mBSA)-immunized mice lacking the entire S100A4 protein (S100A4KO) and in wild-type counterparts treated with short hairpin ribonucleic acid (shRNA)-lentiviral constructs targeting S100A4 (S100A4-shRNA). The severity of arthritis was evaluated morphologically. T-cell subsets were characterized by the expression of master transcription factors, and functionally by proliferation activity and cytokine production. The activity of the Scr-kinases Fyn and Lck was assessed by the autophosphorylation of C-terminal thyrosine and by the phosphorylation of the CD5 cytodomain. The interaction between S100A4 and the CD5 cytodomain was analysed by nuclear magnetic resonance spectrophotometry. Results: S100A4-deficient mice (S100A4KO and S100A4-shRNA) had significantly alleviated morphological signs of arthritis and joint damage. Leukocyte infiltrates in the arthritic joints of S100A4-deficient mice accumulated Foxp3+ Treg cells, while the number of RORγt+ and (pTyr705)STAT3+ cells was reduced. S100A4-deficient mice had a limited formation of Th17-cells with low retinoic acid orphan receptor gamma t (RORγt) mRNA and IL17 production in T-cell cultures. S100A4-deficient mice had a low expression and activity of T-cell receptor (TCR) inhibitor CD5 and low (pTyr705)STAT3 (signal transducer and activator of transcription 3), which led to increased (pTyr352)ZAP-70 (theta-chain associated protein kinase of 70kDa), lymphocyte proliferation and production of IL2. In vitro experiments showed that S100A4 directly binds Lck and Fyn and reciprocally regulates their kinase activity towards the CD5 cytodomain. Spectrometry demonstrates an interaction between the CD5 cytodomain and EF2-binding sites of S100A4. Conclusion: The present study demonstrates that S100A4 plays an important part in the pathogenesis of arthritis. It controls CD5-dependent differentiation of Th17 cells by regulating the activity of the Src-family kinases Lck and Fyn.

IL-12 and IL-18 induce interferon-γ production and de novo CD2 expression in porcine γδ T cells

γδ T cells are highly abundant in the blood and spleen of pigs but little is known about their functional differentiation. In this study the potential of the type-1 polarizing cytokines IL-12 and IL-18 in combination with IL-2 and Concanavalin A (ConA) to stimulate porcine γδ T cells was investigated. Stimulation of purified γδ T cells with ConA and IL-2 induced a strong proliferation of CD2− γδ T cells, whereas additional stimulation with IL-12 and IL-18 caused a stronger proliferation of CD2+ γδ T cells. IFN-γ could only be detected in supernatants of γδ T-cell cultures supplemented with IL-12 and IL-18. Experiments with sorted CD2/SWC5-defined γδ T-cell subsets revealed that CD2+SWC5− γδ T cells are the main producers of IFN-γ following stimulation with IL-2/IL-12/IL-18. Additional stimulation with ConA led to an upregulation of CD2 within the CD2− γδ T cell subsets, indicating a previously unnoticed plasticity of CD2-defined γδ T cell subsets.