Absence Of Antigen-presenting Cells Research Articles

Association of tertiary lymphoid structures with outcomes from PD-1 inhibition in advanced malignant pleural mesothelioma.

e20545 Background: Predictive biomarkers of benefit form immunotherapy in malignant mesothelioma patients remain poorly defined. The 9-15 PROMISE-MESO study (NCT02991482) of the European Thoracic Oncology Platform (ETOP) evaluated second-line pembrolizumab against chemotherapy. Pembrolizumab led to an increased response rate compared to chemotherapy, which did not translate to survival benefits. We evaluated if the presence of tertiary lymphoid structures (TLSs) with different compositions or LRRC15+ immune suppressive fibroblasts could predict the clinical benefit from pembrolizumab. Methods: We performed multiplex immune histochemistry analysis on tumor tissues. 144 patients were included in the clinical trial, from whom, 112 had available tumor tissue for analysis: 55 patients in the chemotherapy arm, and 57 patients in the immune therapy arm. We performed the following panel: calretinin or WT1 (mesothelioma cells), CD20, CD23, CD11c, LRRC15, aSMA, and DAPI. We used the Akoya Vectra Polaris platform for analysis. The IFQuant analytical pipeline developed at the Swiss Institute for Bioinformatics (SIB) was used for analyses. Results: We defined three categories of TLSs: general CD20+ TLSs independent of other markers. CD20/CD23 double-positive as mature TLSs (mTLS), and TLSs with dendritic cells (CD11c+, cTLS). Immune suppressive CAFs were defined as LRRC15+ cells/mm2. CD20+ TLSs were identified in 84% of patients' stroma, with a median of 21 TLSs/tumor tissue and 2 TLSs/mm2. mTLSs were rare, only detected in 10 of the 112 patients (9%). cTLSs with dendritic cells were more frequent and present in 45% of all patients (50/112), and roughly half of the TLS+ patients (53%) had CD11c+ cells detected in TLSs. LRRC15+ cancer-associated fibroblasts (CAFs) were detected in 85% of patients, with a median density of 61 cells/mm2. LRRC15- tumors also contained TLSs but had significantly fewer than LRRC15+ tumors (p = 0.0079). We did not find any significant association between the responses or PFS with TLS, mTLS, and LRRC15+ CAFs. We found inverse association between cTLS levels and response to immune therapy (p = 0.037) but not for PFS. Conclusions: Our data shows that advanced malignant mesotheliomas frequently harbor TLSs, but these TLSs are mostly not activated/mature. The absence of antigen-presenting cells in TLSs, like CD11c dendritic cells, is unexpectedly associated with increased response to pembrolizumab. The high prevalence of immune suppressive LRRC15+ CAFs might explain the low response rate to pembrolizumab and could be linked to the absence of maturation of TLSs. Clinical trial information: EudraCT number: 2016-002062-31 .

Reprogramming and redifferentiation of mucosal-associated invariant T cells reveal tumor inhibitory activity.

Mucosal-associated invariant T (MAIT) cells belong to a family of innate-like T cells that bridge innate and adaptive immunities. Although MAIT cells have been implicated in tumor immunity, it currently remains unclear whether they function as tumor-promoting or inhibitory cells. Therefore, we herein used induced pluripotent stem cell (iPSC) technology to investigate this issue. Murine MAIT cells were reprogrammed into iPSCs and redifferentiated towards MAIT-like cells (m-reMAIT cells). m-reMAIT cells were activated by an agonist in the presence and absence of antigen-presenting cells and MR1-tetramer, a reagent to detect MAIT cells. This activation accompanied protein tyrosine phosphorylation and the production of T helper (Th)1, Th2, and Th17 cytokines and inflammatory chemokines. Upon adoptive transfer, m-reMAIT cells migrated to different organs with maturation in mice. Furthermore, m-reMAIT cells inhibited tumor growth in the lung metastasis model and prolonged mouse survival upon tumor inoculation through the NK cell-mediated reinforcement of cytolytic activity. Collectively, the present results demonstrated the utility and role of m-reMAIT cells in tumor immunity and provide insights into the function of MAIT cells in immunity.

Mycobacterium avium Subspecies paratuberculosis Drives an Innate Th17-Like T Cell Response Regardless of the Presence of Antigen-Presenting Cells.

The gastrointestinal disease of ruminants is clinically known as Johne's disease (JD) and is caused by Mycobacterium avium subspecies paratuberculosis (MAP). An accumulative effect by insensitive diagnostic tools, a long subclinical stage of infection, and lack of effective vaccines have made the control of JD difficult. Currently lacking in the model systems of JD are undefined correlates of protection and the sources of inflammation due to JD. As an alternative to commonly studied immune responses, such as the Th1/Th2 paradigm, a non-classical Th17 immune response to MAP has been suggested. Indeed MAP antigens induce mRNAs encoding the Th17-associated cytokines IL-17A, IL-17F, IL-22, IL-23, IL-27, and IFNγ in CD3+ T cell cultures as determined by RT-qPCR. Although not as robust as when cultured with monocyte-derived macrophages (MDMs), MAP is able to stimulate the upregulation of these cytokines from sorted CD3+ T cells in the absence of antigen-presenting cells (APCs). CD4+ and CD8+ T cells are the main contributors of IL-17A and IL-22 in the absence of APCs. However, MAP-stimulated MDMs are the main contributor of IL-23. In vivo, JD+ cows have more circulating IL-23 than JD– cows, suggesting that this proinflammatory cytokine may be important in the etiology of JD. Our data in this study continue to suggest that Th17-like cells and associated cytokines may indeed play an important role in the immune responses to MAP infection and the development or control of JD.

087 Molecular and cellular mechanisms of tolerogenic signature induction in CD4+ T cells by apoptotic cells

Apoptotic cells are typically recognized by a highly efficient phagocytic system, thus maintaining immune homeostasis. However, defective clearance of apoptotic cells has been suggested as a major driver of cutaneous autoimmune disease. We and others have demonstrated that receptors for apoptotic cells, specifically, the T cell Ig and mucin domain (Tim) family, have been linked to susceptibility to autoimmune and allergic diseases. However, the specific, direct effects of apoptotic epithelial cells on CD4+ T cells and its role in the pathogenesis of autoimmune disorders remain largely undefined. Using RNASeq, NanoString codesets, and an in vitro co-culture system, we show that apoptotic cells directly induce a tolerogenic transcriptional signature in naïve CD4+ T cells, including the production of the immune suppressive cytokine, IL-10, in the absence of antigen-presenting cells. This induction is IL-4 dependent as inhibiting IL-4 signaling, either genetically or by neutralizing antibodies, abrogates this effect. Moreover, cell contact with apoptotic cells via the co-inhibitory molecule Tim-3 on CD4+ T cells is partially required. Apoptotic cell-derived nucleic acids, signaling through TLR9 on naïve CD4+ T cells, induces IL-10 production. The adoptive transfer of apoptotic cell-treated CD4+ T cells results in a less severe form of experimental autoimmune encephalitis disease when compared to non-treated CD4+ T cells. Therefore, our data supports a model whereby phosphatidylserine on apoptotic cells binds to Tim-3 on CD4+ T cells. This allows apoptotic cell-derived DNA to stimulate TLR9 on CD4+ T cells and induces the production of IL-10, limiting an inflammatory response to apoptotic cells. Further insight into the CD4+ T cell response to apoptotic cells may provide an avenue for therapeutic intervention in cutaneous autoimmune disorders.

Direct Engagement of TLR9 Ligand with T Helper Cells Leads to Cell Proliferation & Up-regulation of Cytokines

ABSTRACTPurpose: Toll like receptor (TLR) engagement is primarily a function of the innate immune cells. The purpose of the study was to assess direct uptake of ODN 2216 in T helper cells and effects on cell proliferation and cytokine expression.Methods: We isolated CD4+ CD25- T helper cells by magnetic sorting and studied the uptake of ODN 2216 using flow cytometry and confocal microscopy. We then studied the effect of ODN 2216 engagement on cell proliferation and cytokine expression using flow cytometry and gene expression of TLR9 signaling genes using real time RT-PCR.Results: We made a chance observation that purified T helper cells from healthy individuals consistently bind to the TLR9 ligand ODN 2216. In PBMCs, on the other hand, 98% of monocytes preferentially bound to ODN 2216 FITC, indicating that they competed with the lymphocytes. We confirmed intracellular localization of ODN 2216 FITC as well as intracellular expression of TLR9 in Thelper cells. Furthermore, ODN 2216 FITC was also co-localized with the lysosomal membrane associated protein 1. The uptake of TLR9 ligand culminated in cellular proliferation, up-regulation of cytokines and increased mRNA expression of TLR9 and IRF7 in T helper cells, in the absence of antigen presenting cells. ODN 2216 uptake was inhibited by promethazine as well as by TLR9 antagonist.Conclusions: Our results show a direct engagement of TLR9 ligand in T helper cells and suggest involvement of TLR9 signalling in CD4+T cells, which may envisage novel targets for TLR inhibitors.

066 Glucocorticoids promote intrinsic human Th17 differentiation

Although Th17 cells are critical to host protection at epithelial barriers, they can play a pathogenic role in inflammatory and autoimmune diseases. Glucocorticoids (GC) are therapeutically used to suppress undesired inflammation, but the common persistence or even increase of Th17 responses in GC-treated patients led us to investigate the effect of GC on Th17 development. We found that GC trigger a Th17 differentiation program in naïve human CD4 T cells in the absence of antigen-presenting cells or exogenous cytokines. GC-induced Th17 cells expressed IL-10 and their development was associated with an inhibition of Th2, and particularly Th1 differentiation. GC induction of Th17 differentiation was not linked to increased IL-1β, IL-6 and TGF-β, yet GC downregulated IL-2. Consistently, the addition of exogenous IL-2 prevented the GC-induced increase in Th17/Th1 ratios. Also, IL-2/IL-2R blocking enhanced Th17/Th1 ratios, altogether suggesting a mechanistic role for IL-2 regulation in GC-induced Th17 polarization. Functionally, supernatants of GC-differentiated T cells were superior to those of non-GC-differentiated T cells at inducing antimicrobial peptides and pro-IL-1β in keratinocytes, despite decreased IFN-γ and increased IL-10. Finally, GC also favored Th17 over Th1 among memory T cells from blood and skin. Altogether, our data define GC as human Th17 polarizing factors.

S1P1 Agonist SEW2871 Inhibits human Th17 cell generation and functions

Abstract S1P1 is a G-protein coupled receptor expressed by lymphocytes and is required for their egress out of secondary lymphoid organs. Fingolimod, an agonist of four S1P receptors including S1P1, is used in multiple sclerosis (MS) treatment in humans; however, less is known regarding the impact of S1P1 agonists on the egress-independent biology and functions of human Th17 and Treg cells. In this study, we explored the effects of S1P1 agonists SEW2871 and FTY720 on the ex vivo generation and functions of human Th17 and Treg cells. We report that a selective S1P1 agonist SEW2871 inhibited human Th17 development ex vivo, from naïve T cells, associated with reduced IL-17 and IL-22 production, demonstrated by ELISA and intracellular cytokine staining. On the other hand, Treg generation was augmented in SEW2871+ cultures. Additionally, SEW2871-exposed monocytes and dendritic cells produced less IL-23, a cytokine required for Th17 cell-expansion and pathogenicity. Th17 differentiation in the presence or absence of antigen presenting cells revealed that SEW2871 could inhibit Th17 generation either by blocking APC-derived IL-23 or directly affecting T cells. Our data provide novel information as to egress-independent effects of S1P1 agonists on Th17 cells.

Interleukin 6 Is Not Made By Chimeric Antigen Receptor T Cells and Does Not Impact Their Function

Introduction:Anti-CD19 chimeric antigen receptor T cells (CART19) generate unprecedented complete response rates of up to 90% in relapsing/refractory acute lymphoblastic leukemia. Associated with this therapy is a multi-symptom toxicity known as cytokine release syndrome (CRS). Clinically CRS resembles macrophage activation syndrome (MAS), with a pattern of cytokines in serum coupled with other biomarkers (such as ferritin) and physical findings (fever, sudden organomegaly, confusion). Clinical interest has focused on interleukin-6 (IL-6), as blocking this pathway with tocilizumab (an IL-6R antagonist) has relieved the most life-threatening aspects of CRS in patients. Nothing is known, however, about the mechanism behind the triggering of CAR associated CRS/MAS nor the cellular sources of the toxic cytokines. This is a critical lack of knowledge, as each CAR product may result in a different CRS resulting in different clinical outcomes and management strategies. We sought to identify the cellular source of IL-6 and other MAS cytokines specifically during a 41BB CAR mediated CRS, and cross validate with investigations into patient peripheral blood PBMCs during CRS.Results: Using a xenograft model of a primary patient leukemia and CART19 from a patient that experienced Grade 4 CRS, we measured cytokine production in the serum of animals 3 and 7 days post CART19. While we could detect GMCSF, IL-2 and IFNg easily, IL-6 was not detected and the animals did not appear ill during this phase despite disease response. Given the clinical similarities of CRS to MAS, we performed co-culture of CART19 T cells, Nalm-6 leukemia and cells derived in vitro from peripheral blood monocytes (which were autologous to the CAR T cells) including immature dendritic cells (iDC), mature dendritic cells (mDC) and macrophages. Similar to the in vivo results, coculture of CART19 and Nalm-6 produced high levels of GMCSF, IFNg, IL-2 and IL-10 but no detectable IL-6 or IL-8. Only in the presence of the monocyte lineage antigen presenting cells (APCs) did we observe IL-6 and IL-8 release (more than 100 fold increase over controls). Transwell in vitro experiments separating CART19/Nalm-6 from the APCs showed the same pattern, indicating the CART19 mediated killing of target cells induces the IL-6 release from APCs in a contact independent manner. Nanostring RNA analysis of separated cell populations indicated that IL-6 and IL-8 are exclusively produced by APCs, not CART19 or Nalm-6 (Figure 1). Both CD107a degranulation and the total Nanostring RNA profile of CART19 was not different in the presence or absence of APCs, indicating that an MAS-like CRS is likely not part of CART19 efficacy. Finally, we analyzed the peripheral blood mononuclear cells of 18 patients receiving CART19 for pediatric ALL by Nanostring. Patients with Grade 4 CRS and only circulating T cells showed no IL-6 or IL-8 RNA, confirming in vivo that CART19 cells are not the cellular source of IL-6 during CRS. Unsupervised clustering of the Nanostring profiles also revealed four distinct gene signatures: one for patients with only circulating leukemic blasts, two for Grade 2-3 CRS that clustered separately and one for Grade 4 CRS (Figure 2).Conclusions: Here we demonstrate that IL-6 as part of CRS is produced by APCs and not T cells in response to CART19 mediated killing of leukemia, and that CART19 cells do not seem affected by the presence of CRS cytokines either in transcriptional profile or killing potential. This data provides the rationale for blocking this toxic cytokine before symptoms appear without changing CART19 efficacy, in addition to supporting a rapid Nanostring based profile to identify prospectively the patients at risk for Grade 4 CRS. [Display omitted] [Display omitted] DisclosuresBarrett:Novartis: Research Funding. Grupp:Novartis: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy.

Apurinic/Apyrimidinic Endonuclease 1/Redox Factor-1 (Ape1/Ref-1) Modulates Antigen Presenting Cell-mediated T Helper Cell Type 1 Responses

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ape1/Ref-1) is a multifunctional protein possessing DNA repair, redox control, and transcriptional regulatory activities. Although Ape1/Ref-1 plays multiple roles in the immune system, its functions in helper T (Th) cell activation and differentiation are largely unknown. In this study, the function of Ape1/Ref-1 in Th cell activation was analyzed using an Ape1/Ref-1 redox-specific inhibitor, E3330. When splenocytes from OT-II mice, which are ovalbumin (OVA)-specific T-cell receptor transgenic mice, were activated with OVA in the presence of E3330, the induction of IFN-γ-producing OT-II T cells was significantly increased. In contrast, E3330 did not enhance IFN-γ production from plate-bound anti-CD3 antibody-stimulated CD4+ T cells in the absence of antigen presenting cells (APCs). Furthermore, E3330-pretreated and OVA-pulsed APCs also enhanced the IFN-γ production from OT-II T cells. These results suggested that E3330 enhances Th1 responses by modifying APC function. E3330 did not alter the surface expression of MHC-II or the co-stimulatory molecules CD80 and CD86 on APCs. On the other hand, E3330 up-regulated the IL-12 p35 and p40 gene expression, and IL-12 surface retention, but decreased the IL-12 secretion from Toll-like receptor (TLR) ligand-stimulated APCs. These results were confirmed with Ape1/Ref-1 knockdown experiments. Taken together, our findings indicated that the suppression of Ape1/Ref-1 redox function leads to an increased cell surface retention of IL-12 and enhances Th1 responses. This is the first study to demonstrate that Ape1/Ref-1 modulates the IL-12 production and secretion from APCs and controls Th1 immune responses.

Murine Splenic Natural Killer Cells Do Not Develop Immunological Memory after Re-Encounter with Mycobacterium bovis BCG.

Several lines of evidence have recently suggested that natural killer (NK) cells develop immunological memory against viral infections. However, there is no apparent evidence that NK cells acquire specific memory against Mycobacterium bovis bacillus Calmette—Guérin (BCG), the only currently licensed vaccine for preventing tuberculosis. In the present study, we investigated whether murine splenic NK cells can be activated by BCG in a dendritic cell (DC)-independent or -dependent manner, and furthermore examined whether these NK cells acquire specific memory following BCG vaccination. NK cells isolated from spleens of BCG-immunized mice produced interferon (IFN)γ through direct BCG stimulation in the absence of antigen-presenting cells; however, NK cells from control animals similarly directly responded to BCG, and the response level was not statistically significant between the immunized and the naïve NK cells. When purified NK cells that had been exposed to BCG were cocultured with RAW murine macrophages infected with BCG, the antibacterial activity of the macrophages was strongly enhanced; however, its level was similar to that by naïve NK cells, which had not been exposed to BCG. When splenocytes harvested from BCG-immunized mice were stimulated with purified protein derivative (PPD) derived from Mycobacterium tuberculosis, a specific IFNγ response was clearly observed, mainly attributed to NK cells and memory CD4+ T cells. To investigate whether these NK cells as well as the T cells are activated by cell−cell interaction with DCs presenting mycobacterial antigens, NK cells isolated from BCG-immunized mice were cocultured with splenocytes harvested from naïve mice in the presence of PPD stimulation. However, no IFNγ response was found in the NK cells. These results suggest that murine splenic NK cells do not develop BCG-specific immunological memory in either a DC-independent or -dependent manner.

Direct tumor recognition by a human CD4(+) T-cell subset potently mediates tumor growth inhibition and orchestrates anti-tumor immune responses.

Tumor antigen-specific CD4+ T cells generally orchestrate and regulate immune cells to provide immune surveillance against malignancy. However, activation of antigen-specific CD4+ T cells is restricted at local tumor sites where antigen-presenting cells (APCs) are frequently dysfunctional, which can cause rapid exhaustion of anti-tumor immune responses. Herein, we characterize anti-tumor effects of a unique human CD4+ helper T-cell subset that directly recognizes the cytoplasmic tumor antigen, NY-ESO-1, presented by MHC class II on cancer cells. Upon direct recognition of cancer cells, tumor-recognizing CD4+ T cells (TR-CD4) potently induced IFN-γ-dependent growth arrest in cancer cells. In addition, direct recognition of cancer cells triggers TR-CD4 to provide help to NY-ESO-1-specific CD8+ T cells by enhancing cytotoxic activity, and improving viability and proliferation in the absence of APCs. Notably, the TR-CD4 either alone or in collaboration with CD8+ T cells significantly inhibited tumor growth in vivo in a xenograft model. Finally, retroviral gene-engineering with T cell receptor (TCR) derived from TR-CD4 produced large numbers of functional TR-CD4. These observations provide mechanistic insights into the role of TR-CD4 in tumor immunity, and suggest that approaches to utilize TR-CD4 will augment anti-tumor immune responses for durable therapeutic efficacy in cancer patients.

Amaranthus leucocarpus lectin recognizes a moesin-like O-glycoprotein and costimulates murine CD3-activated CD4(+) T cells.

The Galβ1,3GalNAcα1,O-Ser/Thr specific lectin from Amaranthus leucocarpus (ALL) binds a ∼70 kDa glycoprotein on murine T cell surface. We show that in the absence of antigen presenting cells, murine CD4+ T cells activated by an anti-CD3 antibody plus ALL enhanced cell proliferation similar to those cells activated via CD3/CD28 at 48 h of culture. Moreover, ALL induced the production of IL-4, IL-10, TNF-alpha, and TGF-beta in CD3-activated cells. Proteomic assay using two-dimensional electrophoresis and far-Western blotting, ALL recognized two prominent proteins associated to the lipid raft microdomains in CD3/CD28-activated CD4+ T cells. By mass spectrometry, the peptide fragments from ALL-recognized proteins showed sequences with 33% homology to matricin (gi|347839 NCBInr) and 41% identity to an unnamed protein related to moesin (gi|74186081 NCBInr). Confocal microscopy analysis of CD3/CD28-activated CD4+ T cells confirmed that staining by ALL colocalized with anti-moesin FERM domain antibody along the plasma membrane and in the intercellular contact sites. Our findings suggest that a moesin-like O-glycoprotein is the ALL-recognized molecule in lipid rats, which induces costimulatory signals on CD4+ T cells.

Promiscuous T-cell responses to drugs and drug-haptens

Promiscuous T-cell responses to drugs and drug-haptens

Interferon-β suppresses murine Th1 cell function in the absence of antigen-presenting cells.

Interferon (IFN)-β is a front-line therapy for the treatment of the relapsing-remitting form of multiple sclerosis. However, its immunosuppressive mechanism of function remains incompletely understood. While it has been proposed that IFN-β suppresses the function of inflammatory myelin antigen-reactive T cells by promoting the release of immunomodulatory cytokines such as IL-27 from antigen-presenting cells (APCs), its direct effects on inflammatory CD4+ Th1 cells are less clear. Here, we establish that IFN-β inhibits mouse IFN-γ+ Th1 cell function in the absence of APCs. CD4+ T cells express the type I interferon receptor, and IFN-β can suppress Th1 cell proliferation under APC-free stimulation conditions. IFN-β-treated myelin antigen-specific Th1 cells are impaired in their ability to induce severe experimental autoimmune encephalomyelitis (EAE) upon transfer to lymphocyte-deficient Rag1-/- mice. Polarized Th1 cells downregulate IFN-γ and IL-2, and upregulate the negative regulatory receptor Tim-3, when treated with IFN-β in the absence of APCs. Further, IFN-β treatment of Th1 cells upregulates phosphorylation of Stat1, and downregulates phosphorylation of Stat4. Our data indicate that IFN-γ-producing Th1 cells are directly responsive to IFN-β and point to a novel mechanism of IFN-β-mediated T cell suppression that is independent of APC-derived signals.

The immunomodulating effect of seminal plasma on T cells

Seminal plasma (SP) contains immunomodulatory factors that may contribute to the formation of a tolerogenic environment at the embryo implantation site. The main focus of this study was to investigate the influence of SP on female T cells in the presence and absence of antigen-presenting cells (APCs) in an in vitro model. Female PBMCs and T cells were incubated with SP from seminal fluid samples of known and variable sperm quality. The immediate effect of SP on the mRNA expression of CD25, IL-10, IFN-γ, and Foxp3 was measured. Furthermore, proliferative responses, cytokine production, and CD25 expression were determined. Exposure to SP leads to increased mRNA expression of CD25, IL-10, and Foxp3 in T cells. Induction of mRNA for IL-10 and CD25 was dependent on the presence of APCs. Both PBMCs and T cells exposed to SP showed a proliferative response and produced several cytokines. The proliferative effects of SP on T cells observed were independent of sperm quality parameters, cytokines or soluble HLA molecules in SP. Furthermore, the presence of SP induced a higher expression of CD25 on the membrane of CD4+ T cells. SP has a direct immunomodulatory effect on T cells, as reflected in a proliferative response and upregulation of Foxp3. The presence of APCs is needed to induce IL-10 and CD25 upregulation in T cells exposed to SP. In conclusion, SP has both a direct and an indirect effect mediated through APCs on T cells.

The involvement of specific t cells in the pathogenesis of metamizole‐induced agranulocytosis

Background Non-chemotherapy related, drug-induced agranulocytosis is a rare idiosyncratic reaction, which may be fatal. Along with betalactames, the analgesic metamizole is reported to occasionally cause agranulocytosis. The disease results in a severe reduction of granulocytes rendering affected patients susceptible to bacterial and fungal infections. The pathogenesis of drug-induced agranulocytosis is complex as non-immune (mainly toxic) and immune mechanisms might be involved. Aim The identification and characterization of metamizolespecific T cells in patients with drug-induced agranulocytosis by generating metamizole-specific T cell lines. Methods PBMCs from metamizole-allergic and metamizole-tolerant subjects were induced with 100ug/ml metamizole. Cultures were supplemented with IL-2 and were restimulated every 14 days. Drug-specific cell activation was determined by flow cytometry after a 6h restimulation phase. Results After two restimulation rounds, metamizole-specific T cells were identified in a metamizole-allergic and in a metamizole-tolerant individual. In both cases, CD8+ T cells but no CD4+ cells reacted to the drug. Reactive cells secreted IFN and upregulated CD107a upon drug exposure. Interestingly, T cells were activated exclusively by metamizole in solution. Autologous antigen presenting cells incubated overnight in 100ug/ml metamizole prior to restimulation failed to activate CD8+ T cells. Furthermore, metamizole-specific T cells from both donors, allergic and tolerant, were self-reactive, i.e. reacted to the drug even in the absence of antigen presenting cells. Conclusion

T cell Suppression In Vitro During Toxoplasma gondii Infection is the Result of IL‐2 Competition Between Tregs and T cells Leading to Death of Proliferating T cells

A reduced proliferation to T cell mitogens is observed in vitro in murine cells isolated during the acute phase of Toxoplasma gondii infection. Foxp3(+) regulatory T cells (Tregs) mediate this suppression, which is interleukin (IL)-2 dependent. In this work, we analysed the mechanism of this Treg-mediated suppression. We found that removal of antigen-presenting cells (APC) from spleen cells from infected mice did not modify suppression but further elimination of Tregs led to a restored proliferation, demonstrating that Tregs mediate suppression in the absence of APC. Production of IL-2 by T cells from infected animals was abolished but partially restored when Tregs were removed. However, IL-2 levels and T cell proliferation were restored when Tregs and T cells were separated by transwells, indicating that Tregs require close proximity with T cells to induce suppression. Tregs from infected mice were able to reduce proliferation of CTLL-2 cells in the classical IL-2 bioassay, strongly suggesting that Tregs compete with T cells for IL-2. We found that T cells from infected mice died after a few rounds of division in vitro, but addition of recombinant IL-2 or removal of Tregs abolished this effect. Our results showed that suppression of T cell proliferation during acute Toxoplasma gondii infection is the result of death of proliferating T cells by Treg-mediated IL-2 competition. Thus, immunosuppression is due to death of proliferating T cells as a consequence of low IL-2 availability.

T-Cells from HLA-B*57:01+ Human Subjects Are Activated with Abacavir through Two Independent Pathways and Induce Cell Death by Multiple Mechanisms

Susceptibility to abacavir hypersensitivity has been attributed to possession of the specific human leukocyte antigen allele HLA-B*57:01. HLA-B*57:01-restricted activation of CD8+ T-cells provides a link between the genetic association and the iatrogenic disease. The objectives of this study were to characterize the functionality of drug-responsive CD8+ T-cell clones generated from HLA-B*57:01+ drug-naive subjects and to explore the relationship between abacavir accumulation in antigen presenting cells and the T-cell response. Seventy-four CD8+ clones expressing different Vβ receptors were shown to proliferate and kill target cells via different mechanisms when exposed to abacavir. Certain clones were activated with abacavir in the absence of antigen presenting cells. Analysis of the remaining clones revealed two pathways of drug-dependent T-cell activation. Overnight incubation of antigen presenting cells with abacavir, followed by repeated washing to remove soluble drug, activated approximately 50% of the clones, and the response was blocked by glutaraldehyde fixation. In contrast, a 1 h antigen presenting cell pulse did not activate any of the clones. Accumulation of abacavir in antigen presenting cells was rapid (less than 1 h), and the intracellular concentrations were maintained for 16 h. However, intracellular abacavir was not detectable by mass spectrometry after pulsing. These data suggest that T-cells can be activated by abacavir through a direct interaction with surface and intracellular major histocompatibility complex (MHC) molecules. With the former, abacavir seemingly participates in the MHC T-cell receptor binding interaction. In contrast, the latter pathway likely involves MHC binding peptides displayed as a consequence of abacavir exposure, but not abacavir itself.

Abrogation of spontaneous liver tolerance during immune response to Candida albicans: contribution of NKT and hepatic mononuclear cells

Hepatic mononuclear cells (HMC) are a heterogeneous population with innate immune properties involved in the response to several pathogens. Herein, during the primary infection with Candida albicans, we observed dynamic changes in CD3+, NK+ and NKT+ intrahepatic lymphoid subsets and a significant increase in the absolute number of antigen-presenting cells (APC). The liver tolerogenic microenvironment sustained by higher levels of IL-10, transforming growth factor-β and IL-4 was severely modified upon the robust IFN-γ production after the fungal colonization. NKT cells purified from infected animals released significant amounts of IFN-γ and the production of this cytokine was exacerbated after a second contact with the fungus. Interestingly, C. albicans per se was unable to activate tolerogenic NKT cells from naive animals. In vitro experiments performed with HMC cells depleted of the CD11b/c+ population revealed that in the absence of APC, NKT cells are unable to produce IFN-γ in response to C. albicans. Our findings constitute the first evidence that this innate lymphocyte population is involved in the pathogenesis of C. albicans infection.

1,25-Dihydroxyvitamin D3 inhibits proliferation but not the suppressive function of regulatory T cells in the absence of antigen-presenting cells

Vitamin D3 is known to induce regulatory T (Treg) cells by rendering antigen-presenting cells tolerogenic, its direct effect on human naturally occurring Treg cells is unclear. Here, we investigated if and how 1,25-dihydroxyvitamin D(3) [1,25(OH)2D3] can directly affect the proliferation and function of human naturally occurring Treg cells in vitro. First, we demonstrated that these Treg cells express vitamin D receptors that were up-regulated following anti-CD3/CD28-bead stimulation. 1,25(OH)2D3 inhibited proliferation of Treg cells even when exogenous interleukin-2 was provided. Treg cells were more susceptible to the inhibitory effect of 1,25(OH)2D3 than conventional T cells(.) 1,25(OH)2D3 neither affected the anergic state nor the suppressive function of Treg cells but induced a subtle increase in interleukin-10-secreting cells. The cell-division-inhibiting effect of 1,25(OH)2D3 on Treg cells was also demonstrated in vivo by supplementing vitamin D-deficient HIV-1-infected patients with 2000 IU cholecalciferol (vitamin D3). Increased serum 1,25(OH)2D3 levels were associated with a drop in the number and percentage of Treg cells, which may be attributed to a decrease in the proliferating Foxp3+ Treg cell population. In conclusion, 1,25(OH)2D3 directly affects Treg cell growth and promotes interleukin-10 production without apparent effects on activation status and suppressive phenotype whereas in vivo, high serum 1,25(OH)2D3 levels are associated with reduced Treg cell proliferation and a reduced number of Treg cells.