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Low-dose IL-2 therapy is a direct approach to boost regulatory T cells (Tregs) and promote immune tolerance in autoimmune patients. However, the mechanisms responsible for selective response of Tregs to low-dose IL-2 is not fully understood. In this study we directly assessed the contribution of CD25 and protein phosphatase 2A (PP2A) in promoting IL-2R signaling in Tregs. IL-2-induced tyrosine phosphorylation of STAT5 (pSTAT5) was proportional to CD25 levels on human CD4+ T cells and YT human NK cell line, directly demonstrating that CD25 promotes IL-2R signaling. Overexpression of the PP2A catalytic subunit (PP2Ac) by lentiviral transduction in human Tregs increased the level of IL-2R subunits and promoted tyrosine phosphorylation of Jak3 and STAT5. Interestingly, increased expression of CD25 only partially accounted for this enhanced activation of pSTAT5, indicating that PP2A promotes IL-2R signaling through multiple mechanisms. Consistent with these findings, knockdown of PP2Ac in human Tregs and impaired PP2Ac activity in mouse Tregs significantly reduced IL-2-dependent STAT5 activation. In contrast, overexpression or knockdown of PP2Ac in human T effector cells did not affect IL-2-dependent pSTAT5 activation. Overexpression of PP2Ac in human Tregs also increased the expressions of proteins related to survival, activation, and immunosuppressive function, and upregulated several IL-2-regulated genes. Collectively, these findings suggest that CD25 and PP2A cooperatively enhance the responsiveness of Tregs to IL-2, which provide potential therapeutic targets for low-dose IL-2 therapy. [ABSTRACT FROM AUTHOR]

Human herpesvirus 6 (HHV-6) infects >90% of the population and establishes a latent infection with asymptomatic episodes of reactivation. However, HHV-6 reactivation is associated with morbidity and sometimes mortality in immunocompromised patients. To date, control of the virus in healthy virus carriers and the failure to control it in patients with disease remain poorly understood. In particular, knowledge of HHV-6-specific T-cell responses is limited. Here, we characterized HHV-6A- and HHV-6Bspecific CD4+ and CD8+ T-cell responses from peripheral blood mononuclear cells (PBMCs) of healthy donors. We studied the phenotype of effector HHV-6-specific T cells ex vivo, as well as of induced specific suppressive regulatory CD4+ T cells in vitro post-stimulation, in comparison to human cytomegalovirus (HCMV) responses. Compared to that for HCMV, we show that ex vivo T-cell reactivity in peripheral blood is detectable but at very low frequency, both for HHV-6A and -6B viruses. Interestingly, the phenotype of the specific T cells also differs between the viruses. HHV-6A- and HHV-6B-specific CD4+ T lymphocytes are less differentiated than HCMV-specific T cells. Furthermore, we show a higher frequency of HHV-6-specific suppressive regulatory T cells (eTregs) than HCMV-specific eTregs in coinfected individuals. Despite the strong similarity of HHV-6 and HCMV from a virologic point of view, we observed immunological differences, particularly in relation to the frequency and phenotype of effector/memory and regulatory virus-specific T cells. This suggests that different immune factors are solicited in the control of HHV-6 infection than in that of HCMV infection. [ABSTRACT FROM AUTHOR]

The first‐in‐human clinical trial of the CD28‐specific monoclonal antibody (mAb) TGN1412 resulted in a life‐threatening cytokine release syndrome. Although TGN1412 was designed as IgG4, known for weak Fc:Fcγ receptor (FcγR) interactions, these interactions contributed to TGN1412‐induced T‐cell activation. Using cell lines (TFs) expressing human FcγRI, ‐IIa, ‐IIb, or ‐III, we show that TGN1412 and TGN1412 as IgG1 and IgG2 are bound by FcγRs as it can be deduced from literature. However, upon coculture of TGN1412‐decorated T cells with TFs or human primary blood cells, we observed that binding capacities by FcγRs do not correlate with the strength of the mediated effector function. FcγRIIa and FcγRIIb, showing no or very minor binding to TGN1412, mediated strongest T cell proliferation, while high‐affinity FcγRI, exhibiting strong TGN1412 binding, mediated hardly any T‐cell proliferation. These findings are of biological relevance because we show that FcγRI binds TGN1412, thus prevents binding to FcγRIIa or FcγRIIb, and consequently disables T‐cell proliferation. In line with this, FcγRI−FcγRII+ but not FcγRI+FcγRII+ monocytes mediate TGN1412‐induced T‐cell proliferation. Collectively, by using TGN1412 as example, our results indicate that binding of monomeric IgG subclasses does not predict the FcγR‐mediated effector function, which has major implications for the design of therapeutic mAbs. [ABSTRACT FROM AUTHOR]

The isoquinoline quaternary alkaloid Berberine possesses a variety of pharmacological properties that suggests its promising application for an anticancer delivery system design utilizing its ability to intercalate DNA. In the current work, we have investigated the effects of Berberine on the human T cell leukemia cell line in vitro. Fluorescent microscopy of leukemic cells revealed Berberine nuclear localization. The results showed that Berberine inhibited leukemic cell growth in a time- and dose-dependent manner, that was associated with reactive oxygen species production intensification and caspase 3/7 activity increase with followed apoptosis induction. Berberine was used as a toxic and phototoxic agent for triple system synthesis along with DNA as a carrier and nanosilver as a plasmonic accelerator of Berberine electronic transitions and high energy emission absorbent centers. The proposed method allows to obtain the complex of DNA with Berberine molecules and silver nanoparticles. The optical properties of free components as well as their various combinations, including the final triple system DNA-Nanosilver-Berberine, were investigated. Obtained results support the possibility to use the triple system DNA-Nanosilver-Berberine as an alternative therapeutic agent for cancer treatment. [ABSTRACT FROM AUTHOR]

The live attenuated mycobacterial strain BCG, in use as vaccine against tuberculosis, is considered the gold standard for primary therapy of carcinoma in situ of the bladder. Despite its limitations, to date it has not been surpassed by any other treatment. Our group has developed a recombinant BCG strain expressing the detoxified S1 pertussis toxin (rBCG-S1PT) that proved more effective than wild type BCG (WT-BCG) in increasing survival time in an experimental mouse model of bladder cancer, due to the well-known adjuvant properties of pertussis toxin. Here, we investigated the capacity of rBCG-S1PT to stimulate human immune responses, in comparison to WT-BCG, using an in vitro stimulation assay based on human whole blood cells that allows for a comprehensive evaluation of leukocyte activation. Blood leukocytes stimulated with rBCG-S1PT produced increased levels of IL-6, IL-8, and IL-10 as compared to WT-BCG, but comparable levels of IL-1β, IL-2, IFN-γ, and TNF-α. Stimulation of blood cells with the recombinant BCG strain also enhanced the expression of CD25 and CD69 on human CD4+ T cells. PBMC stimulated with rBCG-S1PT induced higher cytotoxicity to MB49 bladder cancer cells than WT-BCG-stimulated PBMC. These results suggest that the rBCG-S1PT strain is able to activate an immune response in human leukocytes that is higher than that induced by WT-BCG for parameters linked to better prognosis in bladder cancer (regulation of immune and early inflammatory responses), while fully comparable to WT-BCG for classical inflammatory parameters. This establishes rBCG-S1PT as a new highly effective candidate as immunotherapeutic agent against bladder cancer. [ABSTRACT FROM AUTHOR]

Mutations in ZBTB24 and CDCA7 cause the Immunodeficiency, Centromeric Instability and Facial Anomalies syndrome type 2 and 3 (ICF2/3), respectively. Most ICF2 patients carry ZBTB24 nonsense mutations and are thus ZBTB24-deficient. Although the immune deficiency in ICF2 patients is primarily regarded as a B-cell defect due to the greatly reduced serum antibodies and circulating memory B cells, the reduced expansions of PBMCs stimulated by mitogens or recall antigens suggest a T-cell defect in these patients as well. However, the molecular mechanisms behind this T-cell dysfunction remain unknown. In the present study, we demonstrated that ZBTB24-deficiency significantly represses the proliferation of human T cells by promoting TRAIL-induced cell death. Downregulation of ZBTB24 in both Jurkat and human primary T cells upregulates the expression of TRAIL and/or its death receptors (TRAIL-R1/2), and induces significant amount of cells to undergo apoptosis. The profound survival defects of ZBTB24-deficient cells are largely reversed by blocking TRAIL/TRAIL-R interactions with exogenous recombinant TRAIL-R2. Moreover, ZBTB24-downregulation reduces the expression of CDCA7, and knockdown of the latter in human T cells results in a phenotype resembling that caused by ZBTB24-depletion. Functionally, overexpression of CDCA7 abrogates the increased apoptosis in ZBTB24-depleted Jurkat T cells. Together, these data indicated that ZBTB24 regulates human T-cell apoptosis via CDCA7/TRAIL-R axis. Our study thus not only provides a molecular explanation for the T-cell defects in ZBTB24-deficient ICF2 patients, but also highlights a convergence between ZBTB24 and CDCA7, the two ICF genes, in modulating the functions of T cells. • Knockdown of ZBTB24 or CDCA7 upregulates TRAIL & TRAIL-Rs in human T cells. • Blocking TRAIL-signaling abrogates apoptosis induced by ZBTB24/CDCA7-depletions. • ZBTB24 activates the expression of CDCA7 in T cells. • Overexpression of CDCA7 reverses the survival defects caused by ZBTB24-depletions. [ABSTRACT FROM AUTHOR]

CD8 T cells are infrequently considered part of germinal center reactions. Yet, a distinct CXCR5+ CD8 T cell subset identified within the B cell follicle and germinal center in situations of chronic antigen has recently been defined. CXCR5+ CD8 T cells maintain transcriptional and phenotypic features consistent with the CD8 T cell nomenclature of a non-exhausted, effector memory population. CD8 T cell localization to the B cell follicle suggests a functional profile similar to CD4 T follicular helper cells that are licensed to promote B cell responses. The functional mechanisms defined under different immune settings, while largely similar, differentially control disease pathogenesis. CXCR5+ CD8 T cells control viral load during infection, and also promote antibody-mediated autoimmune disease progression. The existence of this novel CXCR5+ CD8 T cell subset in human and murine models of disease may provide a paradigm shift in our understanding of germinal center reactions. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) are required for the maintenance of immune tolerance and adoptive Treg infusion therapy has become a promising approach to suppress immune responses in diseases such as autoimmunity and transplant rejection. However, one critical challenge of Treg therapy is the requirement of in vitro expansion of functionally stable Tregs while preventing either the contamination of T effector and/or emergence of unstable pathogenic Tregs. Recent studies showing distinct metabolic requirements of T effectors and Tregs suggest that manipulation of cell metabolism may be an attractive strategy to achieve this goal. Here we show that human thymically derived Tregs (tTregs) and in vitro induced Tregs (iTregs) from naive T cells engage glycolysis equivalently upon activation. However, inhibiting glucose metabolism via 2-deoxy-D-glucose (2DG) has distinct effects on each of these subsets. While 2DG treatment at the onset of activation significantly reduced the proliferation and expression of suppressive molecules such as ICOS and CTLA-4 in tTregs, its effect on FOXP3 expression was small. In contrast, 2DG treatment during iTreg induction modestly decreased their proliferation but strongly reduced both ICOS and FOXP3 expression. Importantly, both Treg subsets became insensitive to 2DG after day 3 post activation with little effect on either proliferation or FOXP3 expression while T conventional Th0 cells showed reduced proliferation under the same conditions. Moreover, 2DG treatment at day 3 did not impair the suppressive capabilities of Treg subsets. Collectively, these findings suggest that there is a distinct temporal requirement of glycolysis in each of the activated human Treg subsets and T conventional cells. Furthermore, 2DG treatment at the onset as a strategy to impair contaminating T effector cell proliferation is unfavorable for optimal Treg generation as well. [ABSTRACT FROM AUTHOR]

Although remarkable results have been attained by adoptively transferring T cells expressing fully murine and/or humanized anti‐CD19 chimeric antigen receptors (CARs) to treat B cell malignancies, evidence of human anti‐mouse immune responses against CARs provides a rationale for the development of less immunogenic CARs. By developing a fully human CAR (huCAR), these human anti‐mouse immune responses are likely eliminated. This, perhaps, not only increases the persistence of anti‐CD19 CAR T cells—thereby reducing the risk of tumor relapse—but also facilitates administration of multiple, temporally separated doses of CAR T cells to the same recipient. To these ends, we have designed and constructed a second‐generation fully human anti‐CD19 CAR (or huCAR19) containing a fully human single‐chain variable fragment (ScFv) fused with a CD8a hinge, a 4‐1BB transmembrane domain and intracellular T cell signaling domains of 4‐1BB and CD3z. T cells expressing this CAR specifically recognized and lysed CD19+ target cells produced cytokines and proliferated in vitro. Moreover, cell volume data revealed that our huCAR construct cannot induce antigen‐independent tonic signaling in the absence of cognate antigen. Considering our results, our anti‐CD19 huCAR may overcome issues of transgene immunogenicity that plague trials utilizing CARs containing mouse‐derived ScFvs. These results suggest that this huCAR19 be safely and effectively applied for adaptive T cell immunotherapy in clinical practice. [ABSTRACT FROM AUTHOR]

We investigated human T-cell repertoire formation using high throughput TCRβ CDR3 sequencing in immunodeficient mice receiving human hematopoietic stem cells (HSCs) and human thymus grafts. Replicate humanized mice generated diverse and highly divergent repertoires. Repertoire narrowing and increased CDR3β sharing was observed during thymocyte selection. While hydrophobicity analysis implicated self-peptides in positive selection of the overall repertoire, positive selection favored shorter shared sequences that had reduced hydrophobicity at positions 6 and 7 of CDR3βs, suggesting weaker interactions with self-peptides than unshared sequences, possibly allowing escape from negative selection. Sharing was similar between autologous and allogeneic thymi and occurred between different cell subsets. Shared sequences were enriched for allo-crossreactive CDR3βs and for Type 1 diabetes-associated autoreactive CDR3βs. Single-cell TCR-sequencing showed increased sharing of CDR3αs compared to CDR3βs between mice. Our data collectively implicate preferential positive selection for shared human CDR3βs that are highly cross-reactive. While previous studies suggested a role for recombination bias in producing "public" sequences in mice, our study is the first to demonstrate a role for thymic selection. Our results implicate positive selection for promiscuous TCRβ sequences that likely evade negative selection, due to their low affinity for self-ligands, in the abundance of "public" human TCRβ sequences. [ABSTRACT FROM AUTHOR]

Graft-versus-host disease (GVHD) is a frequent complication following allogeneic hematopoietic stem cell transplantation (HSCT) with current therapies limited to general immunosuppression. Humanized mouse models of GVHD are emerging as valuable intermediaries to allow translation of findings from allogeneic mouse models to humans to prevent and treat this disease, but such models require further characterization. In this study, humanized mice were generated by injecting immunodeficient non-obese diabetic severe combined immunodeficiency interleukin (IL)-2 receptor γ common chain null (NSG) mice with human peripheral blood mononuclear cells (hPBMCs). Clinical GVHD development was assessed using established scoring criteria (weight loss, posture, activity, fur texture and skin integrity). Differences between humanized NSG mice that developed clinical or subclinical GVHD were then compared. Both groups of mice demonstrated similar frequencies of human leukocyte engraftment. In contrast, mice that developed clinical GVHD demonstrated increased histological damage compared to mice with subclinical GVHD. Furthermore, mice with clinical GVHD exhibited increases in the splenic human CD4+:CD8+ T cell ratio, serum human interferon (IFN)-γ and intestinal human IL-17 expression compared to mice with subclinical GVHD. These cellular and molecular changes could be used as potential markers of disease progression in this preclinical model. This study also provides further insights into GVHD development which may be relevant to human HSCT recipients. • GVHD development in humanized mice is variable as most mice develop clinical disease but some develop subclinical disease. • Mice with clinical GVHD demonstrate increased splenic human CD4+:CD8+ T cell ratios compared to mice with subclinical GVHD. • Mice with clinical GVHD exhibit increased serum human interferon (IFN)-γ compared to mice with subclinical GVHD. [ABSTRACT FROM AUTHOR]

• Only 12.3% of patients with ATLL underwent alloHSCT. • No suitable donor was available for 25% of alloHSCT HLA-typed eligible patients. • Early mortality, loss to follow-up, uninsured status, and lack of available suitable donors, including haploidentical, were major barriers to alloHSCT. In the United States adult T cell lymphoma–leukemia (ATLL) carries a dismal prognosis and mainly affects immigrants from human T cell lymphotropic virus 1 endemic areas. Allogeneic hematopoietic stem cell transplant (alloHSCT) can be effective and is recommended as an upfront treatment in the National Comprehensive Cancer Network guidelines. We studied the barriers to alloHSCT in one of the largest ATLL populations in the United States. Comprehensive chart and donor registry reviews were conducted for 88 ATLL patients treated at Montefiore Medical Center from 2003 to 2018. Among 49 patients with acute and 32 with lymphomatous subtypes, 48 (59.5%) were ineligible for alloHSCT because of early mortality (52%), loss to follow-up (21%), uninsured status (15%), patient declination (10%), and frailty (2%). Among 28 HLA-typed eligible patients (34.6%), matched related donors were identified for 7 (25%). A matched unrelated donor (MUD) search yielded HLA-matched in 2 patients (9.5%), HLA mismatched in 6 (28.5%), and no options in 13 (62%). Haploidentical donors were identified for 6 patients (46%) with no unrelated options. There were no suitable donors for 7 (25%) alloHSCT-eligible patients. The main limitation for alloHSCT after donor identification was death from progressive disease (82%). AlloHSCT was performed in 10 patients (12.3%) and was associated with better relapse-free survival (26 versus 11 months, P =.04) and overall survival (47 versus 10 months, P =.03). Early mortality and progressive disease are the main barriers to alloHSCT, but poor follow-up, uninsured status, and lack of suitable donor, including haploidentical, are also substantial limitations that might disproportionally affect this vulnerable population. AlloHSCT can achieve long-term remissions, and strategies aiming to overcome these barriers are urgently needed to improve outcomes in ATLL. [ABSTRACT FROM AUTHOR]

Sexual HIV-1 transmission occurs primarily in the presence of semen. Although data from macaque studies suggests CCR5+CD4+ T cells are initial targets for HIV-1 infection, the impact of semen on T cell CCR5 expression and ligand production remains inconclusive. To determine if semen modulates the lymphocyte CCR5 receptor/ligand axis, primary human T cell CCR5 expression and natural killer (NK) cell anti-HIV-1 antibody-dependent beta chemokine production was assessed following seminal plasma (SP) exposure. Purified T cells produce sufficient quantities of RANTES to result in a significant decline in CCR5bright T cell frequency following 16 hours of SP exposure (p=0.03). Meanwhile NK cells retain the capacity to produce limited amounts of MIP-1α/MIP-1β in response to anti-HIV-1 antibody- dependent stimulation (median 9.5% MIP-1α+MIP-1β+), despite the immunosuppressive nature of SP. Although these in vitro experiments suggest that SP-induced CCR5 ligand production results in the loss of surface CCR5 expression on CD4+ T cells, the in vivo implications are unclear. We therefore vaginally exposed five pigtail macaques to SP and found that such exposure resulted in an increase in CCR5+ HIV-1 target cells in three out of five animals. The in vivo data support a growing body of evidence suggesting that semen exposure recruits target cells to the vagina that are highly susceptible to HIV-1 infection, which has important implications for HIV-1 transmission and vaccine design. Importance The majority of HIV-1 vaccine studies do not take into consideration the impact that semen exposure might have on the mucosal immune system. In this study, we demonstrate that seminal plasma (SP) exposure can alter CCR5 expression on T cells. Importantly, in vitro studies of T cells in culture cannot replicate the conditions under which immune cells might be recruited to the genital mucosa in vivo, leading to potentially erroneous conclusions about the impact of semen on mucosal HIV-1 susceptibility. [ABSTRACT FROM AUTHOR]

Cryopreservation is key for delivery of cellular therapies, however the key physical and biological events during cryopreservation are poorly understood. This study explored the entire cooling range, from membrane phase transitions above 0°C to the extracellular glass transition at -123°C, including an endothermic event occurring at -47°C that we attributed to the glass transition of the intracellular compartment. An immortalised, human suspension cell line (Jurkat) was studied, using the cryoprotectant dimethyl sulfoxide. Fourier transform infrared spectroscopy was used to determine membrane phase transitions and differential scanning calorimetry to analyse glass transition events. Jurkat cells were exposed to controlled cooling followed by rapid, uncontrolled cooling to examine biological implications of the events, with post-thaw viable cell number and functionality assessed up to 72 h post-thaw. The intracellular glass transition observed at -47°C corresponded to a sharp discontinuity in biological recovery following rapid cooling. No other physical events were seen which could be related to post-thaw viability or performance significantly. Controlled cooling to at least -47°C during the cryopreservation of Jurkat cells, in the presence of dimethyl sulfoxide, will ensure an optimal post-thaw viability. Below -47°C, rapid cooling can be used. This provides an enhanced physical and biological understanding of the key events during cryopreservation and should accelerate the development of optimised cryobiological cooling protocols. [ABSTRACT FROM AUTHOR]

Antibody-derived chimeric antigen receptor (CAR) T cell therapy has achieved gratifying breakthrough in hematologic malignancies but has shown limited success in solid tumor immunotherapy. Monoclonal antibody, TAB004, specifically recognizes the aberrantly glycosylated tumor form of MUC1 (tMUC1) in all subtypes of breast cancer including 95% of triple-negative breast cancer (TNBC) while sparing recognition of normal tissue MUC1. We transduced human T cells with MUC28z, a chimeric antigen receptor comprising of the scFv of TAB004 coupled to CD28 and CD3ζ. MUC28z was well-expressed on the surface of engineered activated human T cells. MUC28z CAR T cells demonstrated significant target-specific cytotoxicity against a panel of human TNBC cells. Upon recognition of tMUC1 on TNBC cells, MUC28z CAR T cells increased production of Granzyme B, IFN-γ and other Th1 type cytokines and chemokines. A single dose of MUC28z CAR T cells significantly reduced TNBC tumor growth in a xenograft model. Thus, MUC28z CAR T cells have high therapeutic potential against tMUC1-positive TNBC tumors with minimal damage to normal breast epithelial cells. [ABSTRACT FROM AUTHOR]

• In vitro assay measures biological activity of vaccine in human PBMC. • H4-IC31 stimulates PBMC IFNγ secretion in cells from BCG-immunized individual. • Assay serves as in vitro potency test for biological activity as part of product profile. • Assay discriminates responses to intact and heat treated material. • Assay response reflects immunostimulatory effects of adjuvant. Potency assays for vaccine products are an important regulatory requirement, and are used to assess product quality and consistency prior to lot release for clinical testing. Ideally they measure an established correlate of efficacy or protection. In cases where there is no known correlate of protection, however, a functional assay that measures a biological response to a vaccine can be applied as a potency assay. Here we describe an in vitro assay which quantitatively measures human T cell activation as a biological response to the TB vaccine candidate H4-IC31. The Cytokine Secretion Assay (CSA) is based on the ability of peripheral blood mononuclear cells (PBMCs) from a Bacillus Calmette-Guérin (BCG)-vaccinated human donor to process and respond to H4-IC31. The ability of H4-IC31 to stimulate a cellular immune response is measured through the quantification of secreted IFNγ and is reported as relative stimulatory activity (RSA) compared to an in-house reference standard. The CSA is specific to the H4-IC31 vaccine, determines the RSA of H4-IC31 in the range of 50% to 150% of the reference standard, and is stability indicating as it detects differences in RSA between intact and heat treated H4-IC31. Although the CSA does not provide a link to clinical efficacy, it fulfills the critical requirements for a biological potency test to assess TB vaccine candidates and can be used along with biochemical and immunochemical assays to define a product profile during clinical development, while eliminating the use of animals for product testing. [ABSTRACT FROM AUTHOR]

Combination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used an ex vivo latency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use the ex vivo latency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishes in vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure. [ABSTRACT FROM AUTHOR]

Special AT‐rich binding protein‐1 (SATB1) is a global chromatin organizer capable of activating or repressing gene transcription in mice and humans. The role of SATB1 is pivotal for T‐cell development, with SATB1‐knockout mice being neonatally lethal, although the exact mechanism is unknown. Moreover, SATB1 is dysregulated in T‐cell lymphoma and proposed to suppress transcription of the Pdcd1 gene, encoding the immune checkpoint programmed cell death protein 1 (PD‐1). Thus, SATB1 expression in T‐cell subsets across different tissue compartments in humans is of potential importance for targeting PD‐1. Here, we comprehensively analyzed SATB1 expression across different human tissues and immune compartments by flow cytometry and correlated this with PD‐1 expression. We investigated SATB1 protein levels in pediatric and adult donors and assessed expression dynamics of this chromatin organizer across different immune cell subsets in human organs, as well as in antigen‐specific T cells directed against acute and chronic viral infections. Our data demonstrate that SATB1 expression in humans is the highest in T‐cell progenitors in the thymus, and then becomes downregulated in mature T cells in the periphery. Importantly, SATB1 expression in peripheral mature T cells is not static and follows fine‐tuned expression dynamics, which appear to be tissue‐ and antigen‐dependent. Furthermore, SATB1 expression negatively correlates with PD‐1 expression in virus‐specific CD8+ T cells. Our study has implications for understanding the role of SATB1 in human health and disease and suggests an approach for modulating PD‐1 in T cells, highly relevant to human malignancies or chronic viral infections. [ABSTRACT FROM AUTHOR]

Praziquantel (PZQ) is an anthelminthic human and veterinary drug used to treat trematode and cestode worms. Changes in immune responses have been demonstrated in humans following curative PZQ treatment of schistosome infections. These changes have been attributed to the removal of immunosupressive worms and immune responses to parasite antigens exposed from dying worms. To date, there has been no study investigating the potential direct effect of PZQ on the host immune cells. Herein, we analyzed the effect of PZQ on human CD4+ T cells classically costimulated by CD3/CD28 or costimulated by the complement regulator CD46 to induce Type 1 regulatory T cells (Tr1). Our results show that PZQ enhanced T‐cell proliferation, increased secretion of IL‐17 and IL‐10 but had no effect on secretion of GM‐CSF or IFNγ. Moreover, PZQ increased the coexpression of CD49b and LAG‐3, a hallmark of Tr1 cells, suggesting increased Tr1 differentiation. Indeed, supernatants from PZQ‐treated cells were able to decrease bystander T‐cell activation, and this was partly reduced when blocking IL‐10. Hence, our study demonstrates that PZQ directly modulates human T‐cell activation and promotes Tr1 differentiation, suggesting that PZQ may have immunomodulatory functions in parasite‐unrelated human inflammatory diseases. [ABSTRACT FROM AUTHOR]

Summary: Multiple sclerosis (MS) is the leading cause of non‐traumatic neurological disability in the United States in young adults, but current treatments are only partially effective, making it necessary to develop new, innovative therapeutic strategies. Myelin‐specific interleukin (IL)‐17‐producing T helper type 17 (Th17) cells are a major subset of CD4 T effector cells (Teff) that play a critical role in mediating the development and progression of MS and its mouse model, experimental autoimmune encephalomyelitis (EAE), while regulatory T cells (Treg) CD4 T cells are beneficial for suppressing disease. The IL‐6/signal transducer and activator of transcription 3 (STAT‐3) signaling pathway is a key regulator of Th17 and Treg cells by promoting Th17 development and suppressing Treg development. Here we show that three novel small molecule IL‐6 inhibitors, madindoline‐5 (MDL‐5), MDL‐16 and MDL‐101, significantly suppress IL‐17 production in myelin‐specific CD4 T cells in a dose‐dependent manner in vitro. MDL‐101 showed superior potency in suppressing IL‐17 production compared to MDL‐5 and MDL‐16. Treatment of myelin‐specific CD4 T cells with MDL‐101 in vitro reduced their encephalitogenic potential following their subsequent adoptive transfer. Furthermore, MDL‐101 significantly suppressed proliferation and IL‐17 production of anti‐CD3‐activated effector/memory CD45RO+CD4+ human CD4 T cells and promoted human Treg development. Together, these data demonstrate that these novel small molecule IL‐6 inhibitors have the potential to shift the Teff : Treg balance, which may provide a novel therapeutic strategy for ameliorating disease progression in MS. [ABSTRACT FROM AUTHOR]

IL-26 is a potentially important player in host defense and may be a pathogenic factor in the chronic inflammatory disorders of humans. However, the involvement of IL-26 in tuberculous pleural effusion (TPE) has not been investigated. The concentration of IL-26 was determined in pleural fluids and sera from patients with pleural effusions. Flow cytometry was performed to identify the cell origin of IL-26. The effects of tuberculosis-specific antigen (ESAT-6/CFP-10) on IL-26 expression of CD4+ T cell were explored. The impacts of IL-26 on modulating CD4+ T cell polarization were also investigated. The concentrations of IL-26 were much higher in tuberculous, malignant, and infectious PE than those in the corresponding serum. The expression of IL-26 on CD4+ T cells was much higher in tuberculous PE than those in the corresponding serum, and pleural Th1 and Th17 cells might be the major cell sources of IL-26. The addition of ESAT-6/CFP-10 to CD4+ T cells led to increasing the number of IL-26–producing CD4+ T cells and IL-26 expression on Th1 and Th17 cells. IL-26 could induce the differentiation and generation of IL-22 by memory and naive CD4+ T cells. IL-26 also upregulated the mRNA encoding CC-chemokine ligand 20 (CCL20) and CCL22 by mononuclear cells isolated from TPE. This study implies that pleural Th1 and Th17 cells are the major cell sources of IL-26, which could induce the differentiation and generation of Th22 cells by CD4+ T cells, suggesting the involvement of IL-26 in the pathogenesis of human TPE. Key messages: IL-26 is overexpressed in TPE patients and presents a higher concentration in pleural effusion than the corresponding peripheral blood. Pleural Th1 and Th17 cells might be the major cell sources of IL-26 in TPE patients. IL-26 promotes IL-22 secretion and Th22 generation by CD4+ T cells isolated from TPE patients. IL-26 may play an active role in the pathogenesis of tuberculous pleurisy. [ABSTRACT FROM AUTHOR]

• Human CD4+ T cells exposed to 1,25(OH) 2 D3 secrete α-1-antitrypsin – representing a novel cellular source of this protein. • α-1-Antitrypsin promotes IL-10 secretion by human CD4+ T cells, via direct interaction with complement C3a. • 1,25(OH) 2 D3 is unable to increase IL10 transcription in CD4+ T cells from α-1-antitrypsin-deficient individuals. • Therefore, autocrine α-1-Antitrypsin is required for 1,25(OH) 2 D3-driven IL-10 expression. Studies to identify novel immune-regulatory functions of active vitamin D (1,25(OH) 2 D3) in human CD4+ T cells revealed that 1,25(OH) 2 D3 potently induced expression of the gene SERPINA1 , encoding the anti-protease α-1-antitrypsin. We confirmed α-1-antitrypsin protein expression by 1,25(OH) 2 D3-treated CD4+ T cells, but not in CD8+ T cells or monocytes. α-1-Antitrypsin promotes anti-inflammatory IL-10 synthesis in other immune cell populations. We therefore investigated its immune-regulatory effects in CD4+ T cells. Plasma-derived α-1-antitrypsin drove IL-10 synthesis by CD4+ T cells, which was not dependent on anti-protease activity, but appeared to require a serum-binding factor, since this could not be achieved with recombinant protein. α-1-Antitrypsin is reported to bind complement components, which regulate T cell function. A role for this interaction was therefore probed. Plasma-derived, but not recombinant α-1-antitrypsin contained C3a. Surface Plasmon Resonance and Microscale Thermophoresis demonstrated α-1-antitrypsin binding to C3a. Addition of C3a to CD4+ T cells cultured with recombinant α-1-antitrypsin restored induction of IL-10, whereas neutralisation of C3a abrogated IL-10 induced by plasma-derived α-1-antitrypsin. To interrogate an endogenous role for the α-1-antitrypsin-C3a axis in 1,25(OH) 2 D3-driven CD4+ T cell IL-10 synthesis, we treated cells from healthy or α-1-antitrypsin-deficient individuals (which transcribe SERPINA1 but do not secrete protein) with 1,25(OH) 2 D3. A significant correlation was identified between SERPINA1 and IL10 gene expression in healthy donor CD4+ T cells, which was absent in cells from α-1-antitrypsin-deficient individuals. Therefore, α-1-antitrypsin is required for 1,25(OH) 2 D3-induced IL-10 expression in CD4+ T cells, interacting with C3a to drive IL-10 expression. [ABSTRACT FROM AUTHOR]

It remains unknown what causes inflammatory bowel disease (IBD), including signaling networks perpetuating chronic gastrointestinal inflammation in Crohn's disease (CD) and ulcerative colitis (UC), in humans. According to an analysis of up to 500 patients with IBD and 100 controls, we report that key transcripts of the IL-7 receptor (IL-7R) pathway are accumulated in inflamed colon tissues of severe CD and UC patients not responding to either immunosuppressive/corticosteroid, anti-TNF, or anti-α4β7 therapies. High expression of both IL7R and IL-7R signaling signature in the colon before treatment is strongly associated with nonresponsiveness to anti-TNF therapy. While in mice IL-7 is known to play a role in systemic inflammation, we found that in humans IL-7 also controlled α4β7 integrin expression and imprinted gut-homing specificity on T cells. IL-7R blockade reduced human T cell homing to the gut and colonic inflammation in vivo in humanized mouse models, and altered effector T cells in colon explants from UC patients grown ex vivo. Our findings show that failure of current treatments for CD and UC is strongly associated with an overexpressed IL-7R signaling pathway and point to IL-7R as a relevant therapeutic target and potential biomarker to fill an unmet need in clinical IBD detection and treatment. [ABSTRACT FROM AUTHOR]

• T cell receptor (TCR) gene segment representation in the T cell repertoire is not random but instead has a symmetry corresponding to the location of the gene segments along the TCR locus. • The organization of the T cell repertoire demonstrates a mathematically definable clonal hierarchy that is evident at the total T cell level, as well as within CD4+ and CD8+ T cell subsets. • Following bone marrow transplantation, the T cell repertoire demonstrates diminished T cell clonal depth and diversity, but maintains the symmetry of TCR segment use seen in health. • The T cell repertoire post-transplantation is highly dynamic, with clonal hierarchy in the recipients demonstrating significant departure from the donors in patients who develop graft-versus-host disease. Image, graphical abstract High-throughput sequencing (HTS) of human T cell receptors has revealed a high level of complexity in the T cell repertoire, which makes it difficult to correlate T cell reconstitution with clinical outcomes. The associations identified thus far are of a broadly statistical nature, precluding precise modeling of outcomes based on T cell repertoire development following bone marrow transplantation (BMT). Previous work has demonstrated an inherent, mathematically definable order observed in the T cells from a diverse group of donors, which is perturbed in recipients following BMT. In this study, T cell receptor (TCR)-β sequences from HLA-matched related donor and recipient pairs are analyzed to further develop this methodology. TCR-β sequencing from unsorted and sorted T cell subsets isolated from the peripheral blood samples of BMT donors and recipients show conservation and symmetry of VJ segment usage in the clonal frequencies, linked to the organization of the gene segments along the TCR locus. This TCR-β VJ segment translational symmetry is preserved post-transplantation and even in cases of acute graft-versus-host disease (aGVHD), suggesting that GVHD occurrence represents a polyclonal donor T cell response to recipient antigens. The complexity of the repertoire is significantly diminished after BMT, and the T cell clonal hierarchy is altered post-transplantation. Low-frequency donor clones tended to take on a higher rank in the recipients following BMT, especially in patients with aGVHD. Over time, the repertoire evolves to a more donor-like state in the recipients who did not develop GVHD as opposed to those who did. The results presented here support new methods of quantifying and characterizing post-transplantation T cell repertoire reconstitution. [ABSTRACT FROM AUTHOR]

We have reported that the major histocompatibility molecule HLA-DQ2 (DQA1*05:01/DQB1*02:01) (DQ2) is relatively resistant to HLA-DM (DM), a peptide exchange catalyst for MHC class II. In this study, we analyzed the role of DQ2/DM interaction in the generation of DQ2-restricted gliadin epitopes, relevant to celiac disease, or DQ2-restricted viral epitopes, relevant to host defense. We used paired human APC, differing in DM expression (DMnull versus DMhigh) or differing by expression of wild-type DQ2, versus a DM-susceptible, DQ2 point mutant DQ2α+53G. The APC pairs were compared for their ability to stimulate human CD4+ T cell clones. Despite higher DQ2 levels, DMhigh APC attenuated T cell responses compared with DMnull APC after intracellular generation of four tested gliadin epitopes. DMhighAPC expressing the DQ2α+53G mutant further suppressed these gliadin-mediated responses. The gliadin epitopes were found to have moderate affinity for DQ2, and even lower affinity for the DQ2 mutant, consistent with DM suppression of their presentation. In contrast, DMhigh APC significantly promoted the presentation of DQ2-restricted epitopes derived intracellularly from inactivated HSV type 2, influenza hemagglutinin, and human papillomavirus E7 protein. When extracellular peptide epitopes were used as Ag, the DQ2 surface levels and peptide affinity were the major regulators of T cell responses. The differential effect of DM on stimulation of the two groups of T cell clones implies differences in DQ2 presentation pathways associated with nonpathogen- and pathogen-derived Ags in vivo. [ABSTRACT FROM AUTHOR]

Rejection is an important issue in kidney transplant. Although with adequate trough level of tacrolimus, acute rejection occurs, and we are focused on these cases. We hypothesized that the lower concentration of tacrolimus in the peripheral blood mononuclear cell would be a cause of rejection; in this regard, we describe ABCB1, which regulates intracellular concentration of tacrolimus. The effect of inflammation on the intracellular concentration of tacrolimus was evaluated, as was the association between that concentration and ABCB1 and CD44 activities. Seven kidney recipients experiencing acute rejection were prospectively enrolled. Both the whole blood concentration of tacrolimus and intracellular concentration of tacrolimus were measured at the time of enrollment and after stabilization. A human T lymphoblastoid cell line (Jurkat T cell) was treated with various concentrations of tacrolimus for 21 h and then stimulated for 3 h. The levels of mRNA interleukin-2, interleukin-8, and interferon-γ decreased dose dependently by tacrolimus. Furthermore, a fluorescence-activated cell sorter was used to count cells expressing CD44 and ABCB1; changes in intracellular concentration of tacrolimus were explored after tacrolimus treatment and stimulation. Also, B6 splenocytes were tested in the same manner as previous Jurkat T cell experiments. The tacrolimus ratio of three patients was lower at the time of acute rejection than when patients were stabilized. In vitro, the intracellular concentration of tacrolimus decreased after stimulation. Under the same conditions, CD44+ ABCB1+ cells increased in proportion on tacrolimus treatment and stimulation. This work supports the hypothesis that inflammation reduces the intracellular tacrolimus level, possibly via drug efflux mediated by CD44+ ABCB1+ and inflammation could lead to acute rejection. [ABSTRACT FROM AUTHOR]

The Tec kinases ITK (interleukin-2-inducible T-cell kinase) and RLK (resting lymphocyte kinase) are critical components of the proximal TCR/CD3 signal transduction machinery, and data in mice suggest that ITK negatively modulates regulatory T cell (TREG) differentiation. However, whether Tec kinases modulate TREG development and/or function in human T cells remains unknown. Using a novel self-delivery siRNA platform (sdRNA), we found that ITK knockdown in human primary naïve peripheral blood CD4 T cells increased Foxp3+ expression under both TREG and T helper priming conditions. TREG differentiated under ITK knockdown conditions exhibited enhanced expression of the co-inhibitory receptor PD-1 and were suppressive in a T cell proliferation assay. ITK knockdown decreased IL-17A production in T cells primed under Th17 conditions and promoted Th1 differentiation. Lastly, a dual ITK/RLK Tec kinase inhibitor did not induce Foxp3 in CD4 T cells, but conversely abrogated Foxp3 expression induced by ITK knockdown. Our data suggest that targeting ITK in human T cells may be an effective approach to boost TREG in the context of autoimmune diseases, but concomitant inhibition of other Tec family kinases may negate this effect. [ABSTRACT FROM AUTHOR]

Abstract Zinc (Zn) is an essential trace element that modulate innate and acquired immune responses, and its deficiency triggers lymphopenia. However, the precise mechanisms underlying zinc-mediated lymphocyte maintenance have not been well clarified. Here, we have successfully generated a zip6 -null mutant zebrafish line using TALENs. The Zip6 -null mutant zebrafish developed normally during gastrulation. Loss of zip6 in zebrafish resulted in significant T lymphocyte reduction and a decrease in intracellular Zn levels. And the zip6 deficiency increases caspase-related cell apoptosis in both zebrafish cells and human T cells. Our results suggest that ZIP6 plays a critical part in T cell development, and enhance our understanding of Zn homeostasis and immune system maintenance. Highlights • Generate a zip6 -null mutant zebrafish line using TALENs. • Loss of zip6 in zebrafish results in significant T lymphocyte reduction. • Zip6 deficiency increases caspase-related cell apoptosis. [ABSTRACT FROM AUTHOR]

Regulatory T cell (Treg) therapy is a potential curative approach for a variety of immune-mediated conditions, including autoimmunity and transplantation, in which there is pathological tissue damage. In mice, IL-33R (ST2)--expressing Tregs mediate tissue repair by producing the growth factor amphiregulin, but whether similar tissue-reparative Tregs exist in humans remains unclear. We show that human Tregs in blood and multiple tissue types produced amphiregulin, but this was neither a unique feature of Tregs nor selectively upregulated in tissues. Human Tregs in blood, tonsil, synovial fluid, colon, and lung tissues did not express ST2, so ST2+ Tregs were engineered via lentiviral-mediated overexpression, and their therapeutic potential for cell therapy was examined. Engineered ST2+ Tregs exhibited TCR-independent, IL-33--stimulated amphiregulin expression and a heightened ability to induce M2-like macrophages. The finding that amphiregulin-producing Tregs have a noneffector phenotype and are progressively lost upon TCR-induced proliferation and differentiation suggests that the tissue repair capacity of human Tregs may be an innate function that operates independently from their classical suppressive function. [ABSTRACT FROM AUTHOR]

T-cell receptors (TCR) have considerable potential as therapeutics and antibody-like reagents to monitor disease progression and vaccine efficacy. Whereas antibodies recognize only secreted and surface-bound proteins, TCRs recognize otherwise inaccessible disease-associated intracellular proteins when they are presented as processed peptides bound to major histocompatibility complexes (pMHC). TCRs have been primarily explored for cancer therapy applications but could also target infectious diseases such as cytomegalovirus (CMV). However, TCRs are more difficult to express and engineer than antibodies, and advanced methods are needed to enable their widespread use. Here, we engineered the human CMV-specific TCR RA14 for high-affinity and robust soluble expression. To achieve this, we adapted our previously reported mammalian display system to present TCR extracellular domains and used this to screen CDR3 libraries for clones with increased pMHC affinity. After three rounds of selection, characterized clones retained peptide specificity and activation when expressed on the surface of human Jurkat T cells. We obtained high yields of soluble, monomeric protein by fusing the TCR extracellular domains to antibody hinge and Fc constant regions, adding a stabilizing disulfide bond between the constant domains and disrupting predicted glycosylation sites. One variant exhibited 50 nM affinity for its cognate pMHC, as measured by surface plasmon resonance, and specifically stained cells presenting this pMHC. Our work has identified a human TCR with high affinity for the immunodominant CMV peptide and offers a new strategy to rapidly engineer soluble TCRs for biomedical applications. [ABSTRACT FROM AUTHOR]

Objective: Mirabegron is widely considered as an effective and safe drug for patients with overactive bladder (OAB). However, there is no evidence regarding the efficacy of mirabegron in human T cell lymphotropic virus‐1 (HTLV‐1)‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients with OAB symptoms. The aim of the present study was to clarify the efficacy of mirabegron in HAM/TSP patients with OAB symptoms. Methods: The present study evaluated the efficacy of mirabegron treatment (50 mg, once daily) in nineteen HAM/TSP patients with OAB symptoms by assessing subjective symptoms using the overactive bladder symptom score (OABSS) and International Prostate Symptom Score (IPSS) before and 12 weeks after administration. Voided volume (VV), maximum flow rate (Qmax), and post‐void residual (PVR) urine volume were evaluated as objective symptoms. Results: Mirabegron treatment improved OABSS in terms of night‐time frequency, urgency, and total score (P < .001). In addition, on the IPSS, mirabegron therapy improved urgency, nocturia, storage symptoms (Questions 2, 4 and 7 on the IPSS), as well as the total score (P < .001). The quality of life (QoL) on the IPSS also improved after treatment (P < .001). However, there were no significant changes in objective symptoms, as measured by VV, Qmax, and PVR, after treatment. One patient (5.3%) complained of dry mouth; because this adverse effect was very mild, the patient did not discontinue mirabegron. Conclusions: Mirabegron administration improved subjective symptoms in HAM/TSP patients with neurogenic OAB. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9-mediated programmed cell death protein 1 (PD-1) disruption in chimeric antigen receptor (CAR) T cells could be an appealing choice to improve the therapeutic efficacy of CAR T cells in an immunosuppressive tumor microenvironment. In most of the reported cases, Cas9 was delivered into T cells by way of electroporation with RNA or protein. However, transient expression of Cas9 by transfection with a plasmid encoding its gene is apparently simpler, as it avoids the steps of in vitro transcription of DNA or protein production. This study tried nucleofection into human primary T cells of plasmids encoding both CRISPR/Cas9 for disrupting the PD-1 gene and the piggyBac transposon system for expressing CD133-specific CAR in one reaction. Based on drug selection, CD133-specific CAR T cells were obtained in which, on average, 91.5% of the PD-1 gene sites were disrupted, but almost no Cas9 gene expression was found in the final engineered CAR T cells. The PD-1-deficient CD133-specific CAR T cells showed similar levels of cytokine secretion and improved proliferation and cytotoxicity in vitro, and enhanced inhibition of tumor growth in an orthotopic mouse model of glioma, compared to conventional CD133-CAR T cells. The described method could be useful for the production of PD-1-deficient CAR T cells for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

T helper (Th)2 polarization plays an important role in the pathogenesis of allergic diseases; the underlying mechanism remains to be further investigated. B cell lymphoma protein‐2 like protein‐12 (Bcl2L12) has the anti‐apoptotic function. This study aims to elucidate the contribution of Bcl2L12 to Th2 polarization in patients with allergic rhinitis (AR). In this study, human CD4+ T cells were isolated from blood samples collected from AR patients and healthy control (HC) subjects. The immune response profiles of CD4+ T cells were analyzed by immunologic approaches. The results showed that AR CD4+ T cells (CD4+ T cells collected from AR patients) showed defects of apoptosis. The expression of FasL in AR CD4+ T cells was lower than that of HC CD4+ T cells. Serum IL‐5 levels were negatively correlated with the expression of FasL in AR CD4+ T cells. Exposure of CD4+ T cells to IL‐5 in the culture suppressed the expression of FasL and increased the expression of Bcl2L12. IL‐5 increased the levels of Bcl2L12 in CD4+ T cells, the latter bound to the FasL promoter to prevent FasL gene transcription. Inhibition of Bcl2L12 restored the apoptosis machinery in AR CD4+ T cells. In conclusion, overexpression of Bcl2L12 in CD4+ T cells compromises the apoptosis machinery; the latter can be restored by inhibition of Bcl2L12. BcL2L12 in CD4+ T cells may be a novel target for the treatment of AR and other allergic disorders. CD4+ T cells express Bcl2L12, which restricts FasL expression and induces apoptotic defects in CD4+ T cells. [ABSTRACT FROM AUTHOR]

Regulatory T-cells (Tregs) are a very important subtype of lymphocytes when it comes to self-control in the human immunological system. Tregs are decisive not only in the protection against destruction of own tissues by autoimmune immunocompetent cells but also in the immunological answer to developing cancers. On the other hand, Tregs could be responsible for the progression of acute and chronic leukemias. In our study, we review publications available in the PUMED database concerning acute leukemia, with a particular emphasis on child's leukemias. The percentage of regulatory T-lymphocytes in peripheral blood and bone marrow was elevated compared to those in healthy individuals and correlated with progressive disease. Regulatory T-cells taken from children diagnosed with leukemia showed a higher suppressive capability, which was confirmed by detecting elevated levels of secreted IL-10 and TGF-beta. The possibility of pharmacological intervention in the self-control of the immunological system is now under extensive investigation in many human cancers. Presumably, Treg cells could be a vital part of targeted therapies. Routine Treg determination could be used to assess the severity of disease and prognosis in children with acute lymphoblastic leukemia. This proposition results from the fact that in some studies, higher percentage of Treg cells in peripheral blood was demonstrated. However, observations confirming these facts are scarce; thus, extrapolating them to the population of children with hematological malignancies needs to be verified in additional studies. [ABSTRACT FROM AUTHOR]

Summary: Extracorporeal photopheresis (ECP) is a widely used immunomodulatory therapy for the treatment of various T cell‐mediated disorders such as cutaneous T cell lymphoma (CTCL), graft‐versus‐host disease (GvHD) or systemic sclerosis. Although clinical benefits of ECP are already well described, the underlying mechanism of action of ECP is not yet fully understood. Knowledge on the fate of CD14+ monocytes in the context of ECP is particularly limited and controversial. Here, we investigated the immunoregulatory function of ECP treated monocytes on T cells in an in‐vitro ECP model. We show that ECP‐treated monocytes significantly induce proinflammatory T cell types in co‐cultured T cells, while anti‐inflammatory T cells remain unaffected. Furthermore, we found significantly reduced proliferation rates of T cells after co‐culture with ECP‐treated monocytes. Both changes in interleukin secretion and proliferation were dependent on cell‐contact between monocytes and T cells. Interestingly, blocking interactions of programmed death ligand 1 (PD‐L1) to programmed death 1 (PD‐1) in the in‐vitro model led to a significant recovery of T cell proliferation. These results set the base for further studies on the mechanism of ECP, especially the regulatory role of ECP‐treated monocytes. Although clinical benefits of ECP are already well described, the underlying mechanism of action of ECP, which is commonly used for the treatment of various T cell‐mediated disorders, is not yet fully understood. In this study, we established an in‐vitro ECP model focusing on ECP‐treated monocytes in co‐culture with CD4+ T cells as knowledge on the role of monocytes in ECP is particularly controversial and should be further investigated. We found that in‐vitro ECP treatment of monocytes (1) significantly induced proinflammatory T cell types dependent on cell‐contact, whereas anti‐inflammatory T cells were unaffected, (2) significantly reduced proliferation rates of co‐cultured T cells in a cell‐contact‐dependent manner partially due to PD‐L1–PD‐1 interactions as (3) blockade of this interaction by antibodies led to a slight but significant recovery of T cell proliferation. [ABSTRACT FROM AUTHOR]

Abstract CD3+CD20+ T cells are a population of CD3+ T cells co-expressing CD20 that make up to ∼3–5% of the CD3+ T-cell compartment in the peripheral blood of human beings. In healthy individuals, CD3+CD20+ T cells are heterogeneous for containing a lower proportion of CD4+ cells, but produce higher levels of IL-17A and/or IFN-γ than those of CD3+CD20− T cells. Recently, emerging studies have shown a pathogenic behavior of CD3+CD20+ T cells in autoimmune diseases and CD20+ T-cell malignancies, and patients with the diseases may benefit from anti-CD20 immunotherapy to deplete these cells. However, CD3+CD20+ T cells may also play a protective role in ovarian cancer and HIV infection for their strong propensity to IFN-γ production. In this review, we will describe the current knowledge about CD3+CD20+ T-cell biology, and discuss their functional roles in autoimmune diseases as well as cancer and infectious diseases. [ABSTRACT FROM AUTHOR]

Highlights • Frequent clonotypes differ from tissue to tissue in some patients with graft-versus-host disease (GVHD). • Frequent clonotypes in certain GVHD tissues differ from patient to patient. • A few T cell clones account for almost all of human GVHD tissue-infiltrating Tcells. ABSTRACT Owing to the difficulty in isolating T cells from human biopsy samples, the characteristics of T cells that are infiltratinghuman acute graft-versus-host disease (GVHD) tissues remain largely uninvestigated. In the present study, TCR-β deep sequencing of various GVHD tissue samples and concurrent peripheral blood obtained from transplant recipients was performed in combination with functional assays of tissue-infiltrating T cell clones. The T cell repertoire was more skewed in GVHD tissues than in the peripheral blood. The frequent clonotypes differed from tissue to tissue in the same patient, and the frequent clonotypes in the same tissue differed from patient to patient. Two T cell clones were successfully isolated from GVHD skin of a patient. In a cytotoxicity assay, both Tcell clones lysed patient peripheral blood mononuclear cells, but not donor-derived Epstein-Barr virus-transformed lymphoblastoid cells. Their clonotypes were identical to the most and second most frequent T cell clonotypes in the original GVHD skin and accounted for almost all of the skin-infiltrating T cells. These results suggest that human acute GVHD may result from only a few different alloreactive cytotoxic T cell clones, which differ from tissue to tissue and from patient to patient. The characterization of T cells infiltrating human GVHD tissues should be further investigated. [ABSTRACT FROM AUTHOR]

Abstract We investigated the effect of aging on the multi-dimensional characteristics and heterogeneity of human peripheral CD8+ T cells defined by the expression of a set of molecules at the single cell level using the recently developed mass cytometry or Cytometry by Time-Of-Flight (CyTOF) and computational algorithms. CD8+ T cells of young and older adults had differential expression of molecules, especially those related to cell activation and migration, permitting the clustering of young and older adults through an unbiased approach. The changes in the expression of individual molecules were collectively reflected in the altered high-dimensional profiles of CD8+ T cells in older adults as visualized by the dimensionality reduction analysis tools principal component analysis (PCA) and t -distributed stochastic neighbor embedding (t -SNE). A combination of PhenoGraph clustering and t -SNE analysis revealed heterogeneous subsets of CD8+ T cells that altered with aging. Furthermore, intermolecular quantitative relationships in CD8+ T cells appeared to change with age as determined by the computational algorithm conditional-Density Resampled Estimate of Mutual Information (DREMI). The results of our study showed that heterogeneity, multidimensional characteristics, and intermolecular quantitative relationships in human CD8+ T cells altered with age, distinctively clustering young and older adults through an unbiased approach. Highlights • High-dimensional profiles of CD8+ T cells alter with age. • Intermolecular quantitative relationships in CD8+ T cells change with age. • Such changes permit clustering of young and older adults by an unbiased approach. [ABSTRACT FROM AUTHOR]

In chronic lymphocytic leukemia (CLL), signaling through several prosurvival B cell surface receptors activates the PI3K signaling pathway. Idelalisib is a highly selective PI3K (PI3Kδ) isoform-specific inhibitor effective in relapsed/refractory CLL and follicular lymphoma. However, severe autoimmune adverse effects in association with the use of idelalisib in the treatment of CLL, particularly as a first-line therapy, gave indications that idelalisib may preferentially target the suppressive function of regulatory T cells (Tregs). On this background, we examined the effect of idelalisib on the function of human Tregs ex vivo with respect to proliferation, TCR signaling, phenotype, and suppressive function. Our results show that human Tregs are highly susceptible to PI3Kδ inactivation using idelalisib compared with CD4+ and CD8+ effector T cells (Teffs) as evident from effects on anti-CD3/CD28/CD2-induced proliferation (order of susceptibility [IC50]: Treg [.5 μM] > CD4+ Teff [2.0 μM] > CD8+ Teff [6.5 μM]) and acting at the level of AKT and NF-κB phosphorylation. Moreover, idelalisib treatment of Tregs altered their phenotype and reduced their suppressive function against CD4+ and CD8+ Teffs. Phenotyping Tregs from CLL patients treated with idelalisib supported our in vitro findings. Collectively, our data show that human Tregs are more dependent on PI3Kδ-mediated signaling compared with CD4+ and CD8+ Teffs. This Treg-preferential effect could explain why idelalisib produces adverse autoimmune effects by breaking Treg-mediated tolerance. However, balancing effects on Treg sensitivity versus CD8+ Teff insensitivity to idelalisib could still potentially be exploited to enhance inherent antitumor immune responses in patients. [ABSTRACT FROM AUTHOR]

The human G-protein-coupled bitter taste receptor T2R38 has recently been demonstrated to be expressed on peripheral blood neutrophils, monocytes and lymphocytes. To further define a potential contribution of the T2R38 receptor in adaptive immune response, the objective of this study was to analyze its expression in resting and activated lymphocytes and T cell subpopulations. Freshly isolated PBMC from healthy donors were used for expression analysis by flow cytometry. Quantum™ MESF beads were applied for quantification in absolute fluorescence units. Activation methods of T cells were anti-CD3/CD28, phytohaemagglutinin (PHA) or phorbol 12-myristate 13-acetate (PMA) together with ionomycin. Lymphocytes from young donors expressed higher levels of T2R38 compared to the elderly. CD3+ T cells expressed higher levels that CD19+ B cells. Receptor expression followed T cell activation with an upregulation within 24 h and a peak at 72 h. Higher levels of T2R38 were produced in lymphocytes by stimulation with anti-CD3/CD28 compared to PHA or PMA/ionomycin. Both subpopulations of CD4+ as well as CD8+ T cells were found to express the T2R38 receptor; this was higher in CD4+ than CD8+ cells; the amount of T2R38 in central and effector memory cells was higher as compared to naïve cells, although this was not statistically significant for CD8+ cells without prior activation by anti-CD3/CD28. Upon treatment of PBMC with the natural T2R38 agonist goitrin Calcium flux was activated in the lymphocyte population with functional T2R38 receptor at >20 μM which was completely blocked by phospholipase Cβ-2 inhibitor U73211. Further, goitrin selectively inhibited TNF-alpha secretion in PBMC with functional T2R38. This quantitative analysis of T2R38 expression in distinct PBMC subsets may provide a basis for understanding the significance of bitter compounds in immune modulation. Whether these findings can have implications for the treatment of inflammatory and immunologic disorders by bitter tasting pharmaceuticals or foods needs further investigation.

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease resulting from abnormal interactions between T and B cells. The acquisition of SLE is linked to genetic susceptibility, and diverse environmental agents can trigger disease onset in genetically susceptible individuals. However, the strongest risk factor for developing SLE is being female (9:1 female to male ratio). The female sex steroid, estradiol, working through its receptors, contributes to the gender bias in SLE although the mechanisms remain enigmatic. In a small clinical trial, monthly administration of the estrogen receptor (ERα) antagonist, ICI182,780 (fulvestrant), significantly reduced disease indicators in SLE patients. In order to identify changes that could account for improved disease status, the present study utilized fulvestrant (Faslodex) to block ERα action in cultured SLE T cells that were purified from blood samples collected from SLE patients (n = 18, median age 42 years) and healthy control females (n = 25, median age 46 years). The effects of ERα antagonism on estradiol-dependent gene expression and canonical signaling pathways were analyzed. Pathways that were significantly altered by addition of Faslodex included T helper (Th) cell differentiation, steroid receptor signaling [glucocorticoid receptor (GR), ESR1 (ERα)], ubiquitination, and sumoylation pathways. ERα protein expression was significantly lower (p 0.05) between SLE patients and controls. A previously undetected interaction between GR and ERα signaling pathways suggests posttranslational modification of steroid receptors in SLE T cells may alter ERα/GR actions and contribute to the strong gender bias of this autoimmune disorder.

Robust, long-lasting immune responses are elicited by memory T cells that possess properties of stem cells, enabling them to persist long-term and to permanently replenish the effector pools. Thus, stem cell-like memory T (TSCM) cells are of key therapeutic value and efforts are underway to characterize TSCM cells and to identify means for their targeted induction. Here, we show that inhibition of mechanistic/mammalian Target of Rapamycin (mTOR) complex 1 (mTORC1) by rapamycin or the Wnt-β-catenin signalling activator TWS119 in activated human naive T cells leads to the induction of TSCM cells. We show that these compounds switch T cell metabolism to fatty acid oxidation as favoured metabolic programme for TSCM cell generation. Of note, pharmacologically induced TSCM cells possess superior functional features as a long-term repopulation capacity after adoptive transfer. Furthermore, we provide insights into the transcriptome of TSCM cells. Our data identify a mechanism of pharmacological mTORC1 inhibitors, allowing us to confer stemness to human naive T cells which may be significantly relevant for the design of innovative T cell-based cancer immunotherapies.

Controllable protein–protein interactions modify the behavior of a yeast cell and a human T cell. [ABSTRACT FROM AUTHOR]

Mosquito-transmitted Plasmodium falciparum infection can cause human cerebral malaria (HCM) with high mortality rates. The abundance of infected red blood cells that accumulate in the cerebral vasculature of patients has led to the belief that these brain-sequestered cells solely cause pathogenesis. However, animal models suggest that CD8+ T cells migrate to and accumulate in the brain, directly contributing to experimental cerebral malaria (ECM) mortality. In this issue of the JCI, Riggle et al. explored the brain vasculature from 34 children who died from HCM or other causes and frequently found CD3+ CD8+ T cells in contact with endothelial cells. Further, the authors show that coinfection with HIV enhanced such CD3+ CD8+ T cell luminal distribution. These findings suggest that the mouse model for cerebral malaria may accurately reflect human disease pathology. This study sheds new light on the mechanisms behind blood-brain barrier breakdown in this complicated neurological disease and opens up alternative approaches for treatment. [ABSTRACT FROM AUTHOR]

Abstract Background Multiple sclerosis is a chronic incapacitating disease of the central nervous system, it has been reported that the disturbance in the development and function of Treg subpopulations is associated with the disability status in the RRMS. Accordingly, in the current study, the objective was to specify nanocurcumin effects on Treg cells frequency, and function in patients with RRMS. Methods and materials 50 patients with RRMS were enrolled in this study in which 25 were treated for at least six months with nanocurcumin capsules while the other half received placebo capsules as the control group. The blood sample was collected prior to the administration of nanocurcumin and placebo capsules and following six months. At baseline and after a six-month treatment, the frequency of Treg lymphocytes, the expression of transcription factor related to these cells and the secretion levels of cytokines were assessed by flowcytometry, real-time PCR and ELISA, respectively. Results A significant reduction was observed in the proportion of peripheral Treg cell frequency, and the levels of TGF-β, IL-10 and FoxP3 expression in patients with RRMS. Our data revealed that the frequency of Treg cells (p =.0027), the expression of FoxP3 (p =.0005), TGF-β (p =.0005), and IL-10 (p =.0002) and the secretion levels of the TGF-β (p =.033), and IL-10 (p =.029) in cultured PBMCs are increased in nanocurcumin-treated group compared to placebo group. Conclusion The results of the current work indicated that nanocurcumin is capable of restoring the frequency and function of Treg cells in MS patients. Highlights • Multiple sclerosis is an autoimmune disease with disturbance in function of Tregs. • Nanocurcumin effects on Treg cells frequency, and function in patients with RRMS. • Nanocurcumin increases frequency of Tregs (p =.0027) and FoxP3 (p =.0005). • Nanocurcumin decreases the EDSS in compared to the baseline levels (p =.039). [ABSTRACT FROM AUTHOR]

Objective: The aim of study was to conduct immunohistochemical quantification of CD3+ and CD8+ decidual lymphocytes in preeclampsia.Methods: A study group included 30 cases of preeclampsia and a control group included 20 healthy pregnant women, all delivered by Cesarean section. Samples of placental bed were analyzed after immunohistochemical staining of CD45+, CD3+ and CD8+ cells.Results: The group with preeclampsia included a significantly higher number of CD3+ (p < 0.01) and CD8+ (p < 0.05) T lymphocytes.Conclusion: It is certain that thebalance dysregulation of T cell of the immune milieu of deciduais of importance in etiopathogenesis and manifestations of preeclampsia. [ABSTRACT FROM AUTHOR]

The aromatic hydrocarbons receptor (AhR) is a ligand‐dependent transcription factor that plays a role in mediating toxicity to xenobiotics. Its key role in immune regulation has been recently demonstrated. Recent data pointed to the efficacy of ITE (2‐(1′H‐indole‐3′‐carbonyl)‐thiazole‐4‐carboxylic acid methyl ester), a nontoxic ligand of AhR, in experimental models of inflammatory diseases. Such effect was mainly through the expansion of regulatory T cells (Tregs). Similarly, TCDD (2,3,7,8‐tetrachlorodibenzo‐p‐dioxin), a toxic ligand of AhR, has been shown to exert comparable effects on Tregs in mice. Herein, we showed that ITE has no effects on natural Tregs. However, it supports the de novo generation of Tregs in humans while promoting their suppressive functions. Our data bring new elements supporting the use of ITE in human therapy of inflammatory diseases. ITE, a non‐toxic endogenous aryl hydrocarbon receptor ligand, enhances de novo generation of regulatory T cells in humans and promotes their suppressive functions. [ABSTRACT FROM AUTHOR]

Background: Although the role of Th17 and regulatory T cells in the progression of atherosclerosis has been highlighted in recent years, their molecular mediators remain elusive. We aimed to evaluate the association between the CD69 receptor, a regulator of Th17/regulatory T cell immunity, and atherosclerosis development in animal models and in patients with subclinical disease.Methods: Low-density lipoprotein receptor-deficient chimeric mice expressing or not expressing CD69 on either myeloid or lymphoid cells were subjected to a high fat diet. In vitro functional assays with human T cells were performed to decipher the mechanism of the observed phenotypes. Expression of CD69 and NR4A nuclear receptors was evaluated by reverse transcription-polymerase chain reaction in 305 male participants of the PESA study (Progression of Early Subclinical Atherosclerosis) with extensive (n=128) or focal (n=55) subclinical atherosclerosis and without disease (n=122).Results: After a high fat diet, mice lacking CD69 on lymphoid cells developed large atheroma plaque along with an increased Th17/regulatory T cell ratio in blood. Oxidized low-density lipoprotein was shown to bind specifically and functionally to CD69 on human T lymphocytes, inhibiting the development of Th17 cells through the activation of NR4A nuclear receptors. Participants of the PESA study with evidence of subclinical atherosclerosis displayed a significant CD69 and NR4A1 mRNA downregulation in peripheral blood leukocytes compared with participants without disease. The expression of CD69 remained associated with the risk of subclinical atherosclerosis in an adjusted multivariable logistic regression model (odds ratio, 0.62; 95% CI, 0.40-0.94; P=0.006) after adjustment for traditional risk factors, the expression of NR4A1, the level of oxidized low-density lipoprotein, and the counts of different leucocyte subsets.Conclusions: CD69 depletion from the lymphoid compartment promotes a Th17/regulatory T cell imbalance and exacerbates the development of atherosclerosis. CD69 binding to oxidized low-density lipoprotein on T cells induces the expression of anti-inflammatory transcription factors. Data from a cohort of the PESA study with subclinical atherosclerosis indicate that CD69 expression in PBLs inversely correlates with the presence of disease. The expression of CD69 remained an independent predictor of subclinical atherosclerosis after adjustment for traditional risk factors. [ABSTRACT FROM AUTHOR]

Introduction: Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are involved in anti-bacterial immunity. MAIT cells are found in the intestines, but their role and distribution within the large intestine have not been fully elucidated. Therefore, we investigated the distribution of MAIT cells within the cecum and colon. Material and methods: Surgically resected tissues of the cecum and colon were obtained from 4 patients with cecal appendix cancer and 8 patients with colorectal cancer, respectively. Lymphocytes were isolated from the intestinal epithelium (intraepithelial lymphocytes - IELs) and the underlying lamina propria (lamina propria lymphocytes - LPLs), and then, MAIT cells were analyzed by flow cytometry. Results: Compared with the colon, the cecum showed a significantly increased frequency of MAIT cells among IELs (p < 0.01). CD69 expression on MAIT cells was significantly increased in the cecum and colon compared with that in the blood, and the frequency of natural killer group 2, member A+ cells among MAIT cells was significantly increased in the cecum. Conclusions: These results suggest that the distribution of MAIT cells was different between the cecum and colon and that MAIT cells were more likely to be activated, especially in the intestinal epithelium of the cecum than in the colon and blood. [ABSTRACT FROM AUTHOR]

Transforming growth factor–b1 (TGF-b1) is one of very few cytokines produced in a latent form, requiring activation to exert any of its vastly diverse effects on development, immunity,and cancer. Regulatory T cells (Tregs) suppress immune cells within close proximity by activating latent TGF-b1 presented by GARP (glycoprotein A repetitions predominant) to integrin aVb8 on their surface. We solved the crystal structure of GARP: latent TGF-b1 bound to an antibody that stabilizes the complex and blocks release of active TGF-b1. This Finding reveals how GARP exploits an unusual medley of interactions, including fold complementation by the amino terminus of TGF-b1, to chaperone and orient the cytokine for binding and activation by aVb8. Thus, this work further elucidates the mechanism of antibody-mediated blockade of TGF-b1 activation and immunosuppression by Tregs. [ABSTRACT FROM AUTHOR]