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Background: Survivors of childhood cancer are at risk for therapy-related subsequent malignant neoplasms (SMN), including thyroid SMN. Telomere length (TL) is associated with cancer risk, but the relationship between TL and SMN risk among survivors is less clear. Methods: We conducted a nested, matched case–control study of radiation-exposed 15-year+ adult survivors of childhood cancer with thyroid SMN (cases) and without SMN (controls). Forty-six cases were matched to 46 controls by primary diagnosis, chemotherapy (yes/no), radiation field, and follow-up duration. Lymphocyte TL (LTL) was measured by telomere flow-FISH cytometry using blood samples banked at a mean of 38.9 years (cases), 39.2 years (controls). Genetic variation in telomere genes was assessed by whole genome sequencing. Point estimates for LTL Results: Cases had shorter median LTL than controls in three out of four leukocyte subsets. Cases were more likely to have NK cell LTL Conclusions: Long-term survivors have shorter than expected LTL, a finding that is more pronounced among survivors with thyroid SMN. Impact: The long-term impact of childhood cancer treatment on immune function is poorly understood. Our findings support immune function studies in larger survivor cohorts to assess long-term deficits in adaptive and innate immunity that may underlie SMN risk.

OBJECTIVE The effector T cell and B cell cytokine networks have been implicated in the pathogenesis of systemic autoimmune diseases, but the association of these cytokine networks with the heterogeneity of clinical manifestations and immune profiles has not been carefully examined. This study was undertaken to examine whether cytokine profiles can delineate distinct groups of patients in 4 systemic autoimmune diseases (systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, and systemic sclerosis). METHODS A total of 179 patients and 48 healthy volunteers were enrolled in the multicenter cross-sectional PRECISE Systemic Autoimmune Diseases (PRECISESADS) study. Multi-low-dimensional omics data (cytokines, autoantibodies, circulating immune cells) were examined. Coculture experiments were performed to test the impact of the cytokine microenvironment on T cell/B cell cross-talk. RESULTS A proinflammatory cytokine profile defined by high levels of CXCL10, interleukin-6 (IL-6), IL-2, and tumor necrosis factor characterized a distinct group of patients in the 4 systemic autoimmune diseases. In each disease, this proinflammatory cluster was associated with a specific circulating immune cell signature, more severe disease, and higher levels of autoantibodies, suggesting an uncontrolled proinflammatory Th1 immune response. We observed in vitro that B cells reinforce Th1 differentiation and naive T cell proliferation, leading to the induction of type 1 effector B cells and IgG production. This process was associated with an increase in CXCL10, IL-6, IL-2, and interferon-γ production. CONCLUSION This composite analysis brings new insights into human B cell functional heterogeneity based on T cell/B cell cross-talk, and proposes a better stratification of patients with systemic autoimmune diseases, suggesting that combined biomarkers would be of great value for the design of personalized treatments.

Objective To investigate the role of programmed cell death protein 1 (PD-1) and its two ligands, PD-L1 and PD-L2, in the pathogenesis of IgG4-related disease (IgG4-RD). Methods Patients with IgG4-RD (n = 43) and healthy controls (n = 34) were recruited. Expression levels of PD-1, PD-L1 and PD-L2 in plasma, submandibular gland and T cell subsets were determined by ELISA, immunohistochemistry and flow cytometry. Naïve T cells were stimulated with or without PD-L1/PD-L2 or anti-PD-L1/anti-PD-L2 for 7 days and the proportion of CD4+CD25+ Treg cells was detected by flow cytometry. Results The expression of PD-1, PD-L1 and PD-L2 in the plasma, submandibular gland and on the surface of Treg cells was increased in IgG4-RD patients. Plasma soluble (s)PD-1 was positively correlated with serum IgG, IgG1, IgG3, IgG4, IgG4-RD responder index and numbers of organs involved, and negatively correlated with serum IgM, IgA, C3 and C4. Plasma sPD-L2 was positively correlated with serum IgG1, and plasma sPD-L1 was positively correlated with sPD-L2 and negatively correlated with C3. Stimulation of PD-L1 but not PD-L2 promoted the differentiation of naïve T cells from IgG4-RD patients into CD4+CD25+ Treg cells. Conclusion Plasma concentrations of sPD-1, sPD-L1 and sPD-L2 were significantly increased in patients with IgG4-RD, and the expression of PD-1 and PD-L2 on Treg cells was upregulated. PD-1–PD-L1 can promote the differentiation of naïve T cells into Treg cells and thus participate in the pathogenesis of IgG4-RD.

Accurate recognition of antigens by specific T cells is crucial for adaptive immunity to work properly. The activation of a T-cell antigen-specific response by an antigen-presenting cell (APC) has not been clearly measured at a single T-cell level. It is also unknown whether the cell-extrinsic environment alters antigen recognition by a T cell. To measure the activation probability of a single T cell by an APC, we performed a single-cell live imaging assay and found that the activation probability changes depending not only on the antigens but also on the interactions of other T cells with the APC. We found that the specific reactivity of single naïve T cells was poor. However, their antigen-specific reactivity increased drastically when attached to an APC interacting with activated T cells. Activation of T cells was suppressed when regulatory T cells interacted with the APC. These findings suggest that although the ability of APCs to activate an antigen-specific naïve T cell is low at a single-cell level, the surrounding environment of APCs improves the specificity of the bulk response.

Background Naive T-cell-depleted grafts have been employed as an ex vivo T-cell depletion (TCD) platform to prevent graft-versus-host disease (GvHD) and improve immune reconstitution by providing rapid donor memory T-cell reconstitution after allogenic hematopoietic stem cell transplantation (allo-HSCT). CD45RA- memory T cells confer protection against viruses such as cytomegalovirus, Epstein-Barr virus, and adenovirus; however, reports have shown an unexpectedly high incidence of human herpesvirus (HHV)-6B encephalitis among pediatric allo-HSCT patients. Methods We report the first 18 consecutive allo-HSCT, 16 haplo-HSCT, and two human leukocyte antigen-matched related donors implanted with naive TCD grafts. All donors were administered three cell products: first, a CD34+ stem cell product; second, a CD45RA+ TCD graft, followed by an adoptive natural killer (NK) cell infusion within 10 days after HSCT. The study's primary endpoint was the incidence of HHV-6B encephalitis. Results Engraftment was achieved in 94.5% of cases; 2-year overall survival, event-free survival, and GvHD/relapse-free survival were 87.2% (95% CI 78.6-95.8), 67.3% (95% CI 53.1-81.5), and 64% (95% CI 50.5-78.1), respectively. HHV-6B reactivation occurred in 7 of the haplo-HSCT patients, six of who received a cell infusion with an NK/CD4 ratio Conclusions In this clinical study, we show that early adoptive NK cell infusion after a 45RA+ TCD allo-HSCT graft is safe and can prevent HHV-6B encephalitis. We recommend infusing adoptive NK cells after allo-HSCT using CD45RA+ TCD grafts.

Acute graft-versus-host disease (aGVHD) is a significant complication of allogeneic hematopoietic stem cell transplant (HSCT) and negatively affects T cell reconstitution. Extracorporeal photopheresis (ECP) reduces aGVHD, but the mechanisms remain incompletely understood. Our objective was to examine the impact of ECP on thymopoiesis in pediatric aGVHD and the mechanisms at a cellular and transcriptional level. Sixteen pediatric HSCT patients were recruited: 6 with ECP-treated aGVHD, 5 without aGVHD, and 5 with aGVHD treated with corticosteroids only. Thymopoiesis was evaluated by measuring naive T cells, TRECs, IL-7, and T cell receptor repertoire diversity. Regulatory T cell (Treg) enumeration and function and dendritic cell (DC) enumeration and phenotype were analyzed using flow cytometry. T cell transcriptome analysis was performed on ECP patients after treatment and responders pre- and post-treatment. Four ECP responders demonstrated thymic-dependent T cell recovery, and superior median naïve T cell numbers at 8 and 12 months post-HSCT compared to the aGVHD corticosteroid group. Increased Tregs and Treg suppressive function, reduced cDC/pDC and DC co-stimulatory marker expression in ECP responders suggest upregulated peripheral tolerance; these findings were not observed in partial responders. Responder post-ECP CD3+ T cell transcriptional profile demonstrated 3333 downregulated and 364 upregulated genes, with significant downregulation of ERRα and GαS pathways, and reduced expression of pro-inflammatory and adhesion proteins. Thymic function improves with successful ECP treatment. ECP reduces T cell activation and impacts peripheral tolerance via DCs and Tregs. Differences in thymic recovery, DC, and Treg cellular patterns and the T cell transcriptome were observed between ECP responders and partial responders and require further validation and investigation in additional patients. Supplementary Information The online version contains supplementary material available at 10.1007/s10875-021-00991-y.

Objective To perform an exploratory, descriptive pilot study of the systemic and local immune environment in patients with vesicoureteral reflux (VUR) and bladder-bowel dysfunction (BBD). Methods Consecutive children with VUR undergoing intravesical ureteral reimplantation were enrolled. Patients were assessed for presence of BBD by reported patient history and validated questionnaire. Fresh blood and bladder tissue, collected at the time of surgery, were immediately processed for analysis. Immune cell compositions were determined via flow cytometry. Immune cell activation was also defined at the time of analysis. LegendPlex assay analysis was utilized to define levels of circulating chemokines and cytokines. Results A total of 7 patients were enrolled. Although percentages of circulating immune cells in the blood of those with VUR/BBD and VUR alone were similar, within bladder tissue, VUR/BBD demonstrated increased immune infiltrates compared to VUR alone. Bladder sample analysis showed that B cells, and Effector Memory and Naive T cell percentages were significantly increased in VUR/BBD patients compared to VUR patients. T cell expression of PD1 was increased in bladder tissues of BBD/VUR. Additionally, analysis of circulating neutrophils displayed significantly increased upregulation of PDL-1 in patients with VUR/BBD vs those with VUR only. Conclusion These pilot data suggest an immune-rich microenvironment is present within VUR. Severity of inflammation appeared to correlate with presence of BBD. This implies that targeting pelvic inflammation may be a novel therapy for children with VUR- or non-VUR-related BBD. Follow-up studies are currently underway.

Following their exit from the thymus, T cells are endowed with potent effector functions but must spare host tissue from harm. The fate of these cells is dictated by a series of checkpoints that regulate the quality and magnitude of T cell-mediated immunity, known as tolerance checkpoints. In this Perspective, we discuss the mediators and networks that control the six main peripheral tolerance checkpoints throughout the life of a T cell: quiescence, ignorance, anergy, exhaustion, senescence and death. At the naive T cell stage, two intrinsic checkpoints that actively maintain tolerance are quiescence and ignorance. In the presence of co-stimulation-deficient T cell activation, anergy is a dominant hallmark that mandates T cell unresponsiveness. When T cells are successfully stimulated and reach the effector stage, exhaustion and senescence can limit excessive inflammation and prevent immunopathology. At every stage of the T cell's journey, cell death exists as a checkpoint to limit clonal expansion and to terminate unrestrained responses. Here, we compare and contrast the T cell tolerance checkpoints and discuss their specific roles, with the aim of providing an integrated view of T cell peripheral tolerance and fate regulation.

Prophylactic donor lymphocyte infusions (DLI) are used to augment post-transplant immune recovery to reduce both infectious complications and disease recurrence. Preclinical studies implicate the naive T-cell subset as the primary driver of graft-versus-host disease (GvHD). In this phase I dose escalation study, we assessed the safety of a DLI that was depleted of CD45RA+ naive T cells. Sixteen adult patients received a prophylactic DLI at a median of 113 days (range 76–280 days) following an HLA-identical, non-myeloablative allogeneic hematopoietic stem cell transplantation. Three patients each received the naive T-cell depleted DLI with a CD3+ dose of 1 × 105/kg, 1 × 106/kg, and 5 × 106/kg. The maximum dose of 1 × 107/kg was expanded to 7 patients. No dose-limiting grade III/IV acute GvHD or adverse events attributable to the DLI were observed at any dose level. One patient developed grade 2 acute GvHD of skin and upper intestines, and another developed moderate chronic GvHD of the lungs following the DLI. With a median follow-up of 2.8 years, 2-year progression-free and overall survival is 50.0% and 68.8%, respectively. In conclusion, these data suggest that a DLI that has been depleted of CD45RA+ naive T cells is feasible and carries a low risk of acute or chronic GvHD.

PD-1 expression is a hallmark of both early antigen-specific T cell activation and later chronic stimulation, suggesting key roles in both naive T cell priming and memory T cell responses. Although significant similarities exist between T cells and NK cells, there are critical differences in their biology and functions reflecting their respective adaptive and innate immune effector functions. Expression of PD-1 on NK cells is controversial despite rapid incorporation into clinical cancer trials. Our objective was to stringently and comprehensively assess expression of PD-1 on both mouse and human NK cells under multiple conditions and using a variety of readouts. We evaluated NK cells from primary human tumor samples, after ex vivo culturing, and from multiple mouse tumor and viral models using flow cytometry, quantitative reverse-transcriptase PCR (qRT-PCR), and RNA-Seq for PD-1 expression. We demonstrate that, under multiple conditions, human and mouse NK cells consistently lack PD-1 expression despite the marked upregulation of other activation/regulatory markers, such as TIGIT. This was in marked contrast to T cells, which were far more prominent within all tumors and expressed PD-1. These data have important implications when attempting to discern NK from T cell effects and to determine whether PD-1 targeting can be expected to have direct effects on NK cell functions.

Differences in immune function and responses contribute to health- and life-span disparities between sexes. However, the role of sex in immune system aging is not well understood. Here, we characterize peripheral blood mononuclear cells from 172 healthy adults 22–93 years of age using ATAC-seq, RNA-seq, and flow cytometry. These data reveal a shared epigenomic signature of aging including declining naïve T cell and increasing monocyte and cytotoxic cell functions. These changes are greater in magnitude in men and accompanied by a male-specific decline in B-cell specific loci. Age-related epigenomic changes first spike around late-thirties with similar timing and magnitude between sexes, whereas the second spike is earlier and stronger in men. Unexpectedly, genomic differences between sexes increase after age 65, with men having higher innate and pro-inflammatory activity and lower adaptive activity. Impact of age and sex on immune phenotypes can be visualized at https://immune-aging.jax.org to provide insights into future studies., Whether the immune system aging differs between men and women is barely known. Here the authors characterize gene expression, chromatin state and immune subset composition in the blood of healthy humans 22 to 93 years of age, uncovering shared as well as sex-unique alterations, and create a web resource to interactively explore the data.

Competition between antigen-specific T cells for peptide:MHC (p:MHC) complexes shapes the ensuing T cell response. Mouse model studies provided compelling evidence that competition is a highly effective mechanism controlling the activation of naïve T cells. However, assessing the effect of T cell competition in the context of a human infection requires defined pathogen kinetics and trackable naïve and memory T cell populations of defined specificity. A unique cohort of non-myeloablative hematopoietic stem cell transplant (nmHSCT) patients allowed us to assess T cell competition in response to CMV reactivation, which was documented with detailed virology data. In our cohort, HSCT donors and recipients were CMV-seronegative and -positive, respectively, thus providing genetically distinct memory and naïve T cell populations. We used single-cell transcriptomics to track donor versus recipient-derived T cell clones over the course of 90 days. We found that donor-derived T cell clones proliferated and expanded substantially following CMV-reactivation. However, for immunodominant CMV epitopes, recipient-derived memory T cells remained the overall dominant population. This dominance was maintained despite more robust clonal expansion of donor-derived T cells in response to CMV reactivation. Interestingly, the donor-derived T cells that were recruited into these immunodominant memory populations shared strikingly similar TCR properties with the recipient-derived memory T cells. This selective recruitment of identical and nearly identical clones from the naïve into the immunodominant memory T cell pool suggests that competition does not interfere with rejuvenating a memory T cell population, but results in selection of convergent clones to the memory T cell pool.SignificanceAn existing memory T cell population specific for a single epitope is sufficient to effectively curtail responses to any new antigens if the original epitope is present in a vaccination regimen or heterologous infections. We asked if T cell competition precludes recruitment of any new, naïve T cells to an existing memory T cell pool in context of CMV-specific T cell responses in a cohort of transplant patients. Our data indicate that competition does not prevent recruitment of naïve T cells into the memory T cell pool, but selects for T cells with nearly or fully congruent T cell receptor specificities. We discuss the implications of rejuvenating a memory T cell pool while preserving the T cell receptor repertoire.

REDD1 is an energy sensor and stress-induced mTOR inhibitor. Recently, its novel role in linking metabolism and inflammation/immune responses has emerged. In this study, we assessed the role of REDD1 in murine oxazolone-induced allergic contact dermatitis (ACD), a T cell–dependent model with features of human ACD. A variety of immune indices, including edema, cellular infiltration, inflammatory gene expression, and glucocorticoid response, were compared in Redd1 knockout (KO) and isogenic (C57BL/6 × 129)F1 wild-type mice after sensitization and subsequent ear challenge with oxazolone. Despite relatively normal thymic profiles and similar T cell populations in the lymph nodes of naive Redd1 KO mice, early T cell expansion and cytokine production were profoundly impaired after sensitization. Surprisingly, higher steady-state populations of CD4+ and CD8+ T cells, as well as macrophages (CD45+/Ly-6G−/CD11b+), dendritic cells (CD45+/Ly-6G−/CD11c+), neutrophils (CD45+/Ly-6G+/CD11b+), and innate lymphoid cells (CD45+/Lineage−/IL-7Ra+/ST2+/c-Kit+), were observed in the ears of naive Redd1 KO mice. Upon challenge, ear edema, T cell, macrophage, neutrophil, and dendritic cell infiltration into the ear was significantly reduced in Redd1 KO animals. Accordingly, we observed significantly lower induction of IFN-γ, IL-4, and other cytokines as well as proinflammatory factors, including TSLP, IL-33, IL-1β, IL-6, and TNF-α, in challenged ears of Redd1 KO mice. The response to glucocorticoid treatment was also diminished. Taken together, these data establish REDD1 as an essential immune modulator that influences both the initiation of ACD disease, by driving naive T cell activation, and the effector phase, by promoting immune cell trafficking in T cell–mediated skin inflammation.

Excessive activation of T cells and microglia represents a hallmark of the pathogenesis of human multiple sclerosis (MS). However, the regulatory molecules overactivating these immune cells remain to be identified. Previously, we reported that extracellular IFP35 family proteins, including IFP35 and NMI, activated macrophages as proinflammatory molecules in the periphery. Here, we investigated their functions in the process of neuroinflammation both in the central nervous system (CNS) and the periphery. Our analysis of clinical transcriptomic data showed that expression of IFP35 family proteins was up-regulated in patients with MS. Additional in vitro studies demonstrated that IFP35 and NMI were released by multiple cells. IFP35 and NMI subsequently triggered nuclear factor kappa B–dependent activation of microglia via the TLR4 pathway. Importantly, we showed that both IFP35 and NMI activated dendritic cells and promoted naïve T cell differentiation into Th1 and Th17 cells. Nmi(−/−), Ifp35(−/−), or administration of neutralizing antibodies against IFP35 alleviated the immune cells’ infiltration and demyelination in the CNS, thus reducing the severity of experimental autoimmune encephalomyelitis. Together, our findings reveal a hitherto unknown mechanism by which IFP35 family proteins facilitate overactivation of both T cells and microglia and propose avenues to study the pathogenesis of MS.

Elevated levels of circulating immune complexes are associated with autoimmunity and with worse prognoses in cancer. Here, we examined the effects of well-defined, soluble immune complexes (ICs) on human peripheral T cells. We demonstrate that IgG-ICs inhibit the proliferation and differentiation of a subset of naïve T cells but stimulate the division of another naïve-like T cell subset. Phenotypic analysis by multi-parameter flow cytometry and RNA-Seq were used to characterize the inhibited and stimulated T cells revealing that the inhibited subset presented immature features resembling those of recent thymic emigrants and non-activated naïve T cells, whereas the stimulated subset exhibited transcriptional features indicative of a more differentiated, early memory progenitor with a naïve-like phenotype. Furthermore, we show that while IgG1-ICs do not profoundly inhibit the proliferation of memory T cells, IgG1-ICs suppress the production of granzyme-β and perforin in cytotoxic memory T cells. Our findings reveal how ICs can link humoral immunity and T cell function.

Summary Ageing dramatically affects number and function of both innate and adaptive arms of immune system, particularly T cell subsets, contributing to reduced vaccination efficacy, decreased resistance to infections and increased prevalence of cancer in older people. In the present paper, we analysed the age‐related changes in the absolute number of lymphocytes in 214 Sicilian subjects, and in the percentages of T and natural killer (NK) cells in a subcohort of donors. We compared these results with the immunophenotype of the oldest living Italian supercentenarian (aged 111 years). The results were also sorted by gender. The correlation between number/percentage of cells and age in all individuals. and separately in males and females, was examined using a simple linear regression analysis. We did not record the increase in the rate of inversion of the CD4/CD8 ratio, frequently reported as being associated with ageing in literature. Our observation was the direct consequence of a flat average trend of CD4+ and CD8+ T cell percentages in ageing donors, even when gender differences were included. Our results also suggest that CD4+ and CD8+ subsets are not affected equally by age comparing females with males, and we speculated that gender may affect the response to cytomegalovirus (CMV) infection. The supercentenarian showed a unique immunophenotypic signature regarding the relative percentages of her T cell subsets, with CD4+ and CD8+ T cell percentages and CD4+ naive T cell values in line with those recorded for the octogenarian subjects. This suggests that the supercentenarian has a naive ‘younger’ T cell profile comparable to that of a >80‐year‐old female., We investigated age‐related changes in the absolute number of lymphocytes among 216 Sicilian subjects (age range 22‐111) and in the percentages of circulating lymphocyte subsets in a smaller group of donors of different ages. The data were also analyzed by gender. The supercentenarian has a naïve ‘younger’ T cell profile comparable to that of an older female.

Rapid clonal expansion of antigen specific T cells is a fundamental feature of adaptive immune responses. It enables the outgrowth of an individual T cell into thousands of clonal descendants that diversify into short-lived effectors and long-lived memory cells. Clonal expansion is thought to be programmed upon priming of a single naïve T cell and then executed by homogenously fast divisions of all of its descendants. However, the actual speed of cell divisions in such an emerging ‘T cell family’ has never been measured with single-cell resolution. Here, we utilize continuous live-cell imagingin vitroto track the division speed and genealogical connections of all descendants derived from a single naïve CD8+T cell throughout up to ten divisions of activation-induced proliferation. This comprehensive mapping of T cell family trees identifies a short burst phase, in which division speed is homogenously fast and maintained independent of external cytokine availability or continued T cell receptor stimulation. Thereafter, however, division speed diversifies and model-based computational analysis using a novel Bayesian inference framework for tree-structured data reveals a segregation into heritably fast and slow dividing branches. This diversification of division speed is preceded already during the burst phase by variable expression of the interleukin-2 receptor alpha chain. Later it is accompanied by selective expression of memory marker CD62L in slower dividing branches. Taken together, these data demonstrate that T cell clonal expansion is structured into subsequent burst and diversification phases the latter of which coincides with specification of memory vs. effector fate.SignificanceRapid clonal expansion of antigen-specific T cells is a fundamental feature of adaptive immune responses. Here, we utilize continuous live-cell imagingin vitroto track the division speed and genealogical connections of all descendants derived from a single naïve CD8+T cell throughout up to ten divisions of activation-induced proliferation. Bayesian inference of tree-structured data reveals that clonal expansion is divided into a homogenously fast burst phase encompassing two to three divisions and a subsequent diversification phase during which T cells segregate into quickly dividing effector T cells and more slowly cycling memory precursors. Our work highlights cell cycle speed as a major heritable property that is regulated in parallel to key lineage decisions of activated T cells.

Circulating T helper cells with a type 17-polarized phenotype (TH17) and expansion of aquaporin-4 (AQP4)-specific T cells are frequently observed in patients with neuromyelitis optica spectrum disorder (NMOSD). However, naive T cell populations, which give rise to T helper cells, and the primary site of T cell maturation, namely the thymus, have not been studied in these patients. Here, we report the alterations of naive CD4 T cell homeostasis and the changes in thymic characteristics in NMOSD patients. Flow cytometry was performed to investigate the naive CD4+ T cell subpopulations in 44 NMOSD patients and 21 healthy controls (HC). On immunological evaluation, NMOSD patients exhibited increased counts of CD31+thymic naive CD4+ T cells and CD31-cental naive CD4+ T cells along with significantly higher fraction and absolute counts of peripheral blood CD45RA+ CD62L+ naive CD4+ T cells. Chest computed tomography (CT) images of 60 NMOSD patients and 65 HCs were retrospectively reviewed to characterize the thymus in NMOSD. Thymus gland of NMOSD patients exhibited unique morphological characteristics with respect to size, shape, and density. NMOSD patients showed exacerbated age-dependent thymus involution than HC, which showed a significant association with disease duration. These findings broaden our understanding of the immunological mechanisms that drive severe disease in NMOSD.

Ataxia Telangiectasia (AT) is a rare inherited disorder characterized by progressive cerebellar ataxia, chromosomal instability, cancer susceptibility and immunodeficiency. AT is caused by mutations in the ATM gene, which is involved in multiple processes linked to DNA double strand break repair. Immunologically, ATM mutations lead to hampered V(D)J recombination and consequently reduced numbers of naive B and T cells. In addition, class switch recombination is disturbed resulting in antibody deficiency causing common, mostly sinopulmonary, bacterial infections. Yet, AT patients in general have no clinical T cell associated infections and numbers of memory T cells are usually normal. In this study we investigated the naive and memory T cell compartment in five patients with classical AT and compared them with five healthy controls using a 24-color antibody panel and spectral flow cytometry. Multidimensional analysis of CD4 and CD8 TCRαβ+ cells revealed that early naive T cell populations, i.e. CD4+CD31+ recent thymic emigrants and CD8+CCR7++CD45RA++ T cells, were strongly reduced in AT patients. However, we identified normal numbers of stem cell memory T cells expressing CD95, which are antigen-experienced T cells that can persist for decades because of their self-renewal capacity. We hypothesize that the presence of stem cell memory T cells explains why AT patients have an intact memory T cell compartment. In line with this novel finding, memory T cells of AT patients were normal in number and expressed chemokine receptors, activating and inhibitory receptors in comparable percentages as controls. Comparing memory T cell phenotypes by Boolean gating revealed similar diversity indices in AT compared to controls. We conclude that AT patients have a fully developed memory T cell compartment despite strongly reduced naive T cells. This could be explained by the presence of normal numbers of stem cell memory T cells in the naive T cell compartment, which support the maintenance of the memory T cells. The identification of stem cell memory T cells via our spectral flow cytometric approach is highly relevant for better understanding of T cell immunity in AT. Moreover, it provides possibilities for further research on this recently identified T cell population in other inborn errors of immunity.

Naive T cells are critical for protection against emerging viral and bacterial infections. However, the ability of these cells to elicit effective long-term immune responses declines with age and contributes to increased disease susceptibility in older individuals. This decline has been linked with the breakdown of cellular quiescence that causes partial differentiation of naive T cells with age, but the underlying mediators of this breakdown are unclear. Comparisons to stem cell quiescence in mice and man offer insight into naive T cells and aging. However, the utilization of single cell technologies in combination with advances in the biology of human tissue aging is needed to provide further understanding of naive T cell complexity and quiescence breakdown with age.

The kynurenine pathway (KP) is a key regulator of many important physiological processes and plays a harmful role in cancer, many neurologic conditions, and chronic viral infections. In HIV infection, KP activity is consistently associated with reduced CD4 T cell counts and elevated levels of T cell activation and viral load; it also independently predicts mortality and morbidity from non-AIDS events. Kynurenine 3-monooxygenase (KMO) is a therapeutically important target in the KP. Using the nonhuman primate model of SIV infection in rhesus macaques, we investigated whether KMO inhibition could slow the course of disease progression. We used a KMO inhibitor, CHDI-340246, to perturb the KP during early acute infection and followed the animals for 1 y to assess clinical outcomes and immune phenotype and function during pre–combination antiretroviral therapy acute infection and combination antiretroviral therapy–treated chronic infection. Inhibition of KMO in acute SIV infection disrupted the KP and prevented SIV-induced increases in downstream metabolites, improving clinical outcome as measured by both increased CD4+ T cell counts and body weight. KMO inhibition increased naive T cell frequency and lowered PD-1 expression in naive and memory T cell subsets. Importantly, early PD-1 expression during acute SIV infection predicted clinical outcomes of body weight and CD4+ T cell counts. Our data indicate that KMO inhibition in early acute SIV infection provides clinical benefit and suggest a rationale for testing KMO inhibition as an adjunctive treatment in SIV/HIV infection to slow the progression of the disease and improve immune reconstitution.

Maternal human immunodeficiency virus (HIV) infection has been shown to leave profound and lasting impacts on the HIV-exposed uninfected (HEU) infant, including increased mortality and morbidity, immunological changes, and developmental delays compared to their HIV-unexposed (HU) counterparts. Exposure to HIV or antiretroviral therapy may influence immune development, which could increase morbidity and mortality. However, a direct link between the increased mortality and morbidity and the infant’s immune system has not been identified. To provide a global picture of the neonatal T cell repertoire in HEU versus HU infants, the diversity of the T cell receptor beta chain (TRB) expressed in cord blood samples from HEU infants was determined using next-generation sequencing and compared to healthy (HU) infants collected from the same community. While the TRB repertoire of HU infants was broadly diverse, in line with the expected idea of a naïve T cell repertoire, samples of HEU infants showed a significantly reduced TRB diversity. This study is the first to demonstrate differences in TRB diversity between HEU and HU cord blood samples and provides evidence that maternal HIV, in the absence of transmission, influences the adaptive immune system of the unborn child.

The human adaptive immune response is known to weaken in advanced age, resulting in increased severity of pathogen-born illness, poor vaccine efficacy, and a higher prevalence of cancer in the elderly. Age-related erosion of the T cell compartment has been implicated as a likely cause, but the underlying mechanisms driving this immunosenescence have not been quantitatively modeled and systematically analyzed. T cell receptor diversity, or the extent of pathogen-derived antigen responsiveness of the T cell pool, is known to diminish with age, but inherent experimental difficulties preclude accurate analysis on the full organismal level. In this paper, we formulate a mechanistic mathematical model of T cell population dynamics on the immunoclonal subpopulation level, which provides quantitative estimates of diversity. We define different estimates for diversity that depend on the individual number of cells in a specific immunoclone. We show that diversity decreases with age primarily due to diminished thymic output of new T cells and the resulting overall loss of small immunoclones.

SummaryBackgroundImmunological alterations may led to the reduction in capacity and endurance levels in elite athletes by e.g. increased susceptibility to infections. There is a need to explain the impact of intensive physical effort on the CD4+memory T cell subsets.MethodsFourteen participants median aged 19 years old (range 17–21 years) were recruited form Pogoń Szczecin S.A., soccer club. They performed progressive efficiency test on mechanical treadmill until exhaustion twice: during preparatory phases to spring and autumn competition rounds. We examined the influence of exhaustive effort on the selected CD45+, especially CD4+memory T cell subsets and inflammation markers determined before, just after the test and during recovery time.ResultsSignificant changes in total CD45+cells and decrease in T lymphocytes percentage after the run was observed. Significant fluctuations in T cells’ distribution were related not only to the changes in Th or Tc subsets but also to increase in naïve T cell percentage during recovery. Increase in TNF-α and IL-8 post-exercise, IL-6 and IL-10 plasma levels in recovery was also found.ConclusionsThe novel finding of our study is that the run performed on mechanical treadmill caused a significant release of CD4+T naïve cells into circulation. Post-exercise increase in circulating NK cells is related with fast biological response to maximal effort. However, at the same time an alternative mechanism enhancing inflammation is involved.

Background HIV-1-specific CD8+ T cells are required for immune suppression of HIV-1 replication and elimination of the associated viral reservoirs. However, effective induction of functional HIV-1-specific CD8+ T cells from naive cells remains problematic in the setting of human vaccine trials. In this study, we investigated priming of functional HIV-1-specific CD8+ T cells from naive cells. Methods HIV-1-specific CD8+ T cells were primed from naive T cells of HIV-1-seronegative individuals using TLR4 ligand LPS or STING ligand 3′3′-cGAMP in vitro. We established HIV-1-specific CD8+ T cell lines from primed T cells and then investigated functional properties of these cells. Findings HIV-1-specific CD8+ T cells primed with LPS failed to suppress HIV-1. In contrast, 3′3′-cGAMP effectively primed HIV-1-specific CD8+ T cells with strong ability to suppress HIV-1. 3′3′-cGAMP-primed T cells had higher expression levels of perforin and granzyme B than LPS-primed ones. The expression levels of granzyme B and perforin and viral suppression ability of 3′3′-cGAMP-primed T cells were positively correlated with the production level of type I IFN from PBMCs stimulated with 3′3′-cGAMP. Interpretation The present study demonstrates the potential of 3′3′-cGAMP to induce HIV-1-specific CD8+ T cells with strong effector function from naive cells via a strong type I IFN production and suggests that this STING ligand may be useful for AIDS vaccine and cure treatment.

T cell lymphoproliferative disorders that arise in the skin are mainly derived from post thymic T cells most commonly of CD4 subset. Human CD4 positive T cells are dynamic exhibiting phenotypic and functional malleability. For example, with repetitive antigen exposure most commonly associated with age, CD4 positive T cells acquire a cytotoxic phenotype. The authors present four cases characterized by cutaneous infiltrates of cytotoxic CD30 negative CD4 positive T cells in the skin; three cases were forms of malignant lymphoma other than mycosis fungoides and one case was a reactive lymphomatoid photodermatosis associated with underlying collagen vascular disease. The three patients with lymphoma were adult men, two above 50 years of age and all with disseminated cutaneous disease. One of these patients whose biopsy showed a large cell morphology succumbed to the disease while one patient with localized disease responded to local radiation. In all three cases there was a nodular and diffuse pan-dermal infiltrate which was predominated by non-cerebriform atypical lymphocytes ranging from small to intermediate sized cells in two cases to a large cell dominant morphology in one case. The biopsies showed some degree of epidermotropism, and in one case it was striking. Neoplastic cells were positive for CD4, and at least one cytotoxic protein (i.e. granzyme and/or TIA). CD56, CD57 or CD30 were negative. In addition, CD28, the naive T cell marker, was negative. Based on the few cases reported herein, one might suggest that the prognosis mirrors that seen in other forms of cutaneous T cell lymphoma with mature small cell dominant infiltrates exhibiting an indolent pattern while a CD30 negative large cell T cell lymphoma would be expected to demonstrate an aggressive clinical course.

The central nervous system (CNS) is considered to be immune privileged, owing in part to the absence of major histocompatibility (MHC) class II+ cells in the healthy brain parenchyma. However, systemic inflammation can activate microglia to express MHC class II, suggesting that systemic inflammation may be sufficient to mature microglia into functional antigen presenting cells (APCs). We examined the effects of systemic lipopolysaccharide (LPS)-induced inflammation on the phenotype and function of putative APCs within the mouse brain parenchyma, as well as its supporting tissues-the choroid plexus and meninges. Microglia isolated from different regions of the brain demonstrated significant heterogeneity in their ability to present antigen to naive OT-II CD4+ T cells following exposure to systemic LPS. Olfactory bulb microglia (but not cortical microglia) intimately interacted with T cells in vivo and stimulated T cell proliferation in vitro, albeit in the absence of co-stimulation. In contrast, myeloid cells within the choroid plexus and meninges were immunogenic and upregulated the co-stimulatory molecule CD80 following systemic inflammation. Dural APCs, which clustered around LYVE-1+ lymphatics, were more efficient at stimulating naive T cell proliferation than choroid plexus APCs, suggesting that the dura may be an under-appreciated site for immune interactions. This study has highlighted the functional diversity of myeloid cells within the sub-compartments of the CNS and its supporting tissues. Furthermore, these findings demonstrate that systemic inflammation can mature selected microglia populations and choroid plexus/meningeal myeloid cells into functional APCs, which may contribute to the pathogenesis of neuroinflammation and neurodegenerative diseases.

Significance T cell-mediated immune responses are compromised in aged individuals, leading to increased morbidity and reduced response to vaccination. Finding new ways to boost T cell immunity in the elderly is key for enhancing their immune competence. In this work, we performed a systematic analysis of proteins and metabolites in young versus aged T cells. Metabolic rewiring occurs in young T cells following stimulation but is dampened in aged T cells. Moreover, we show that aged T cell functions can be enhanced by metabolite addition., T cell-mediated immune responses are compromised in aged individuals, leading to increased morbidity and reduced response to vaccination. While cellular metabolism tightly regulates T cell activation and function, metabolic reprogramming in aged T cells has not been thoroughly studied. Here, we report a systematic analysis of metabolism during young versus aged naïve T cell activation. We observed a decrease in the number and activation of naïve T cells isolated from aged mice. While young T cells demonstrated robust mitochondrial biogenesis and respiration upon activation, aged T cells generated smaller mitochondria with lower respiratory capacity. Using quantitative proteomics, we defined the aged T cell proteome and discovered a specific deficit in the induction of enzymes of one-carbon metabolism. The activation of aged naïve T cells was enhanced by addition of products of one-carbon metabolism (formate and glycine). These studies define mechanisms of skewed metabolic remodeling in aged T cells and provide evidence that modulation of metabolism has the potential to promote immune function in aged individuals.

The disruption of adaptive immune response has adverse effects on the establishment and maintenance of pregnancy. The adaptive immune system is regulated by several types of immune cells. However, there is limited information about cell hierarchy in the adaptive immune response to the establishment and maintenance of pregnancy in women. The assessment of the outcome of pregnancy in primary immunodeficiency diseases could help in understanding the cell hierarchy in the adaptive immune system during pregnancy. Common variable immunodeficiency (CVID) is a heterogeneous adaptive immune system disorder characterized by primary hypogammaglobulinemia. A few studies have previously reported the assessment of the T and B cell subpopulations in CVID patients. However, an assessment of the subpopulations of T and B cells and the outcome of pregnancy in women with CVID has not been reported till date. Most CVID patients show a general decrease in the expression of CD27 in B cells. The assessment of pregnancy and the subpopulations of T and B cells in CVID women with severe reduction in the naive T and switched B cells could help understand whether these cells are essential for the establishment and maintenance of pregnancy in women.

Background: Atopic dermatitis (AD) is an inflammatory cutaneous disorder. The advancements in the understanding of AD immunological pathogenesis have caused the development of therapies that suppress the dysregulated immune response. We aimed to evaluate the immunomodulatory effect of dental stem cells (dental follicle-mesenchymal stem cells [DF-MSCs]) on AD patients. Materials & methods: We investigated the immunoregulatory potential of DF-MSCs on T cell response in AD and compared them with psoriasis and healthy individuals and the underlying mechanisms. Results: DF-MSCs significantly reduced Fas, FasL and TNFR II frequency in T cells, increased naive T cell population while reducing memory T cell, decreased inflammatory cytokine levels and promoted Tregs frequency in the AD population. Conclusion: These results imply that DF-MSCs are modulating inflammation through decreasing T cell apoptosis, inducing Treg expansion and stabilizing cytokine levels.

International audience; The amino-acid catabolizing enzyme Interleukin-4 induced gene 1 (IL4I1) remains poorly characterized despite it is emerging as a pertinent therapeutic target for cancer. IL4I1 is secreted in the synaptic cleft by antigen-presenting cells. It inhibits TCR signaling, modulates naïve T cell differentiation and limits effector T cell proliferation. IL4I1 expression in tumors shapes the tumor microenvironment and impairs the antitumor cytotoxic T cell response, thereby facilitating cancer immune escape. Several mechanisms participate in these effects. Recent data suggest a role of new IL4I1 metabolites in activation of the aryl-hydrocarbon receptor (AHR). Here, we observe that expression of IL4I1 is poorly correlated with that of validated targets of AHR in human cancers. Moreover, dendritic cells do not upregulate AHR target genes in relation with IL4I1 expression in vivo. Finally, IL4I1 activity towards tryptophan leading to production of AHR-activating products is very low, and should be negligible when tryptophan-degrading enzymes of higher affinity compete for the substrate. We recently showed that IL4I1 expression by dendritic cells directly regulates immune synapse formation and modulates the repertoire and memory differentiation of responding CD8 T cells after viral infection. Thus, IL4I1 may restrain tumor control through regulating the priming of tumor-specific CD8 T cells, independently of AHR activation.

The authors show that autophagy in lymphatic endothelial cells (LECs) dampens collagen-induced arthritis by regulating two distinct pathways: the promotion of T reg cells in LNs and the inhibition of S1P-dependent migration of pathogenic Th17 cells in inflamed organs., Lymphatic endothelial cells (LECs) present peripheral tissue antigens to induce T cell tolerance. In addition, LECs are the main source of sphingosine-1-phosphate (S1P), promoting naive T cell survival and effector T cell exit from lymph nodes (LNs). Autophagy is a physiological process essential for cellular homeostasis. We investigated whether autophagy in LECs modulates T cell activation in experimental arthritis. Whereas genetic abrogation of autophagy in LECs does not alter immune homeostasis, it induces alterations of the regulatory T cell (T reg cell) population in LNs from arthritic mice, which might be linked to MHCII-mediated antigen presentation by LECs. Furthermore, inflammation-induced autophagy in LECs promotes the degradation of Sphingosine kinase 1 (SphK1), resulting in decreased S1P production. Consequently, in arthritic mice lacking autophagy in LECs, pathogenic Th17 cell migration toward LEC-derived S1P gradients and egress from LNs are enhanced, as well as infiltration of inflamed joints, resulting in exacerbated arthritis. Our results highlight the autophagy pathway as an important regulator of LEC immunomodulatory functions in inflammatory conditions.

By characterizing the poorly defined T cell composition in developing human fetal skin, Reitermaier et al. identify a unique population of TCR αβγδ+ T cells. These cells may contribute to early skin development and immune defense against invading pathogens in utero., T cells in human skin play an important role in the immune defense against pathogens and tumors. T cells are present already in fetal skin, where little is known about their cellular phenotype and biological function. Using single-cell analyses, we identified a naive T cell population expressing αβ and γδ T cell receptors (TCRs) that was enriched in fetal skin and intestine but not detected in other fetal organs and peripheral blood. TCR sequencing data revealed that double-positive (DP) αβγδ T cells displayed little overlap of CDR3 sequences with single-positive αβ T cells. Gene signatures, cytokine profiles and in silico receptor–ligand interaction studies indicate their contribution to early skin development. DP αβγδ T cells were phosphoantigen responsive, suggesting their participation in the protection of the fetus against pathogens in intrauterine infections. Together, our analyses unveil a unique cutaneous T cell type within the native skin microenvironment and point to fundamental differences in the immune surveillance between fetal and adult human skin., Graphical Abstract

Inflammatory bowel diseases (IBDs) are characterized by chronic, inflammatory gastrointestinal lesions and often require life-long treatment with immunosuppressants and repetitive surgical interventions. Despite progress in respect to the characterization of molecular mechanisms e.g. exerted by TNF-alpha, currently clinically approved therapeutics fail to provide long-term disease control for most patients. The transcription factor interferon regulatory factor 4 (IRF4) has been shown to play important developmental as well as functional roles within multiple immune cells. In the context of colitis, a T cell-intrinsic role of IRF4 in driving immune-mediated gut pathology is established. Here, we conversely addressed the impact of IRF4 inactivation in non-T cells on T cell driven colitis in vivo. Employing the CD4+CD25− naïve T cell transfer model, we found that T cells fail to elicit colitis in IRF4-deficient compared to IRF4-proficient Rag1−/− mice. Reduced colitis activity in the absence of IRF4 was accompanied by hampered T cell expansion both within the mesenteric lymph node (MLN) and colonic lamina propria (cLP). Furthermore, the influx of various myeloids, presumably inflammation-promoting cells was abrogated overall leading to a less disrupted intestinal barrier. Mechanistically, gene profiling experiments revealed a Th17 response dominated molecular expression signature in colon tissues of IRF4-proficient, colitic Rag1−/− but not in colitis-protected Rag1−/−Irf4−/− mice. Colitis mitigation in Rag1−/−Irf4−/− T cell recipients resulted in reduced frequencies and absolute numbers of IL-17a-producing T cell subsets in MLN and cLP possibly due to a regulation of conventional dendritic cell subset 2 (cDC2) known to impact Th17 differentiation. Together, extending the T cell-intrinsic role for IRF4 in the context of Th17 cell driven colitis, the provided data demonstrate a Th17-inducing and thereby colitis-promoting role of IRF4 through a T cell-extrinsic mechanism highlighting IRF4 as a putative molecular master switch among transcriptional regulators driving immune-mediated intestinal inflammation through both T cell-intrinsic and T cell-extrinsic mechanisms. Future studies need to further dissect IRF4 controlled pathways within distinct IRF4-expressing myeloid cell types, especially cDC2s, to elucidate the precise mechanisms accounting for hampered Th17 formation and, according to our data, the predominant mechanism of colitis protection in Rag1−/−Irf4−/− T cell receiving mice.

T cells are predominantly produced by the thymus and play a significant role in maintaining our adaptive immune system. Physiological involution of the thymus occurs gradually with age, compromising naive T cell output, which can have severe clinical complications. Also, T cells are utilized as therapeutic agents in cancer immunotherapies. Therefore, there is an increasing need for strategies aimed at generating naive T cells. The majority of in vitro T cell generation studies are performed in two-dimensional (2D) cultures, which ignore the physiological thymic microenvironment and are not scalable; therefore, we applied a new three-dimensional (3D) approach. Here, we use a gelatin-based 3D microgel system for T lineage induction by co-culturing OP9-DL4 cells and mouse fetal-liver-derived hematopoietic stem cells (HSCs). Flow cytometric analysis revealed that microgel co-cultures supported T lineage induction similar to 2D cultures while providing a 3D environment. We also encapsulated mouse embryonic thymic epithelial cells (TECs) within the microgels to provide a defined 3D culture platform. The microgel system supported TEC maintenance and retained their phenotype. Together, these data show that our microgel system has the capacity for TEC maintenance and induction of in vitro T lineage differentiation with potential for scalability.

Contains fulltext : 235331.pdf (Publisher’s version ) (Open Access) The dendritic cell (DC)-derived cytokine profile contributes to naive T cell differentiation, thereby directing the immune response. IL-37 is a cytokine with anti-inflammatory characteristics that has been demonstrated to induce tolerogenic properties in DC. In this study we aimed to evaluate the influence of IL-37 on DC-T cell interaction, with a special focus on the role of the chemokine CXCL1. DC were cultured from bone marrow of human IL-37 transgenic (hIL-37Tg) or WT mice. The phenotype of unstimulated and LPS-stimulated DC was analyzed (co-stimulatory molecules and MHCII by flow cytometry, cytokine profile by RT-PCR and ELISA), and T cell stimulatory capacity was assessed in mixed lymphocyte reaction. The role of CXCL1 in T cell activation was analyzed in T cell stimulation assays with anti-CD3 or allogeneic DC. The expression of the co-stimulatory molecules CD40, CD80 and CD86, and of MHCII in LPS-stimulated DC was not affected by endogenous expression of IL-37, whereas LPS-stimulated hIL-37Tg DC produced less CXCL1 compared to LPS-stimulated WT DC. T cell stimulatory capacity of LPS-matured hIL-37Tg DC was comparable to that of WT DC. Recombinant mouse CXCL1 did not increase T cell proliferation either alone or in combination with anti-CD3 or allogeneic DC, nor did CXCL1 affect the T cell production of interferon-γ and IL-17. Endogenous IL-37 expression does not affect mouse DC phenotype or subsequent T cell stimulatory capacity, despite a reduced CXCL1 production. In addition, we did not observe an effect of CXCL1 in T cell proliferation or differentiation.

BackgroundAging is accompanied by alterations in immune response which leads to increased susceptibility to infectious diseases, cancer, autoimmunity, and inflammatory disorders. This decline in immune function is termed as immunosenescence; however, the mechanisms are not fully elucidated. Experimental approaches of adaptive immunity, particularly for T cells, have been the main focus of immunosenescence research. This systematic review evaluates and discusses T cell markers implicated in immunosenescence.ObjectiveTo determine the best flow cytometry markers of circulating T cells associated with immunosenescence.MethodsWe systematically queried PubMed, MEDLINE, EBSCO, and BVS databases for original articles focused on two age groups of healthy humans: 18–44 (young adults) and >60 (older adults) years. In accordance with the Cochrane methodology, we synthesized data through qualitative descriptions and quantitative random effects meta-analysis due to extensive heterogeneity.ResultsA total of 36 studies conducted in the last 20 years were included for the qualitative analysis and four out of these studies were used to perform the meta-analysis. A significant decrease in naïve T cell subset was observed in older adults compared to young adults. Primary markers used to identify senescent cells were loss of CD28 and increased expression of CD57 and KLRG1 in terminally-differentiated memory T cell subset in older adults. Moreover, we observed an increase in proinflammatory cytokines and decrease in telomere length in old adult T cells. It was not possible to perform quantitative synthesis on cell markers, cytokines, and telomere length because of the significant variations between the groups, which is attributed to differences in protocols and unreported measurements, thus generating a high risk of bias.ConclusionsHeterogeneity among studies in terms of data report, measurement techniques and high risk of bias were major impediments for performing a robust statistical analysis that could aid the identification of eligible flow cytometry markers of immunosenescence phenotype in T cells.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with an overall 5-year survival rate of 10%. Disease lethality is due to late diagnosis, early metastasis and resistance to therapy, including immunotherapy. PDA creates a robust fibroinflammatory tumor microenvironment that contributes to immunotherapy resistance. While previously considered an immune privileged site, evidence demonstrates that in some cases tumor antigen-specific T cells infiltrate and preferentially accumulate in PDA and are central to tumor cell clearance and long-term remission. Nonetheless, PDA can rapidly evade an adaptive immune response using a myriad of mechanisms. Mounting evidence indicates PDA interferes with T cell differentiation into potent cytolytic effector T cells via deficiencies in naive T cell priming, inducing T cell suppression or promoting T cell exhaustion. Mechanistic research indicates that immunotherapy combinations that change the suppressive tumor microenvironment while engaging antigen-specific T cells is required for treatment of advanced disease. This review focuses on recent advances in understanding mechanisms limiting T cell function and current strategies to overcome immunotherapy resistance in PDA.

The need to maintain cell populations at near-constant sizes – that is, to maintain homeostasis – is ubiquitous in living organisms. Cell populations are usually dynamic, with production through replenishment from a source and division being countered by cell death or differentiation. In the context of T lymphocytes, measuring the relative contributions of these processes and how each of them responds to physiological perturbations is important for understanding how vertebrates maintain large and diverse immune repertoires.

The detection and functional characterization of antigen-reactive T helper (Th) cells has been challenging due to their low frequency and functional heterogeneity. Antigen-reactive T cell enrichment (ARTE) allows the in-depth characterization of antigen-specific Th lymphocytes as a prerequisite for better understanding the role of adaptive immune responses in health and disease. ARTE is based on detection of the activation markers CD154 (CD40L) (expressed on all conventional Th cell subsets, Tcons) and CD137 (4-1BB) (expressed on regulatory T cells, Tregs), which are upregulated on the surface of CD4+ T cells upon short-term (7 h) in vitro stimulation with antigens in the presence of antigen-presenting cells (APCs). To substantially increase the sensitivity for the detection of antigen-specific Th cells, ARTE combines magnetic pre-enrichment of rare antigen-reactive T cells with multiparameter flow cytometry. Using CD154 and CD137 in combination allows the parallel detection of reactive Tcons and Tregs, after stimulation with the antigen. Thus, the ARTE technology now enables to characterize antigen-specific T cells with increased sensitivity of detection allowing even the investigation of antigen-specific Th cells in the naive T cell repertoire and regardless of prior knowledge of MHC alleles or antigenic epitopes.

Summary The therapeutic applications of regulatory T cells (Tregs) include treating autoimmune diseases, graft-versus-host disease and induction of transplantation tolerance. For ex-vivo expanded Tregs to be used in deceased donor transplantation, they must be able to suppress T cell responses to a broad range of human leukocyte antigen (HLA). Here, we present a novel approach for the expansion of polyspecific Tregs in cynomolgus macaques that was adapted from a good manufacturing practice-compliant protocol. Tregs were isolated by fluorescence-activated cell sorting (FACS) and expanded in the presence of a panel of CD40L-stimulated B cells (CD40L-sBc). Prior to Treg culture, CD40L-sBc were expanded in vitro from multiple major histocompatibility complex (MHC)-disparate macaques. Expanded Tregs expressed high levels of forkhead box protein 3 (FoxP3) and Helios, a high percentage of Treg-specific demethylated region (TSDR) demethylation and strong suppression of naïve T cell responses in vitro. In addition, these Tregs produced low levels of inflammatory cytokines and were able to expand post-cryopreservation. Specificity assays confirmed that these Tregs were suppressive upon activation by any antigen-presenting cells (APCs) whose MHC was shared by CD40L-sBc used during expansion, proving that they are polyspecific. We developed an approach for the expansion of highly suppressive cynomolgus macaque polyspecific Tregs through the use of a combination of CD40L-engineered B cells with the potential to be translated to clinical studies. To our knowledge, this is the first report that uses a pool of MHC-mismatched CD40L-sBc to create polyspecific Tregs suitable for use in deceased-donor transplants.

Paediatric heart transplantation recipients suffer an increased incidence of infectious, autoimmune and allergic problems. The relative roles of thymus excision and immunosuppressive treatments in contributing to these sequelae are not clear. We compared the immunological phenotypes of 25 heart transplant recipients (Tx), 10 children who underwent thymus excision during non-transplantation cardiac surgery (TE) and 25 age range-matched controls, in two age bands: 1-9 and 10-16 years. Significant differences from controls were seen mainly in the younger age band with Tx showing lower CD3 and CD4 cell counts whilst TE showed lower CD8 cell counts. Naïve T cell and recent thymic emigrant proportions and counts were significantly lower than controls in both groups in the lower age band. T cell recombination excision circle (TREC) levels were lower than controls in both groups in both age bands. There were no differences in regulatory T cells, but in those undergoing thymus excision in infancy, their proportions were higher in TE than Tx, a possible direct effect of immunosuppression. T cell receptor V beta spectratyping showed fewer peaks in both groups than in controls (predominantly in the older age band). Thymus excision in infancy was associated with lower CD8 cell counts and higher proportions of Tregs in TE compared to Tx. These data are consistent with thymus excision, particularly in infancy, being the most important influence on immunological phenotype after heart transplantation.

CD8+ T cells play a pivotal role in clearing intracellular pathogens and combatting tumours. Upon infection, naïve CD8+ T cells differentiate into effector and memory cells, and this program is underscored by large-scale and coordinated changes in the chromatin architecture and gene expression. Importantly, recent evidence demonstrates that the epigenetic mechanisms that regulate the capacity for rapid effector function of memory T cells are shared by innate immune cells such as natural killer (NK) cells. Thus, it appears that the crucial difference between innate and adaptive immunity is the presence of the naïve state. This important distinction raises an intriguing new hypothesis, that the naïve state was evolutionary installed to restrain a default program of effector and memory differentiation in response to antigen recognition. We argue that the hallmark of adaptive T immunity is therefore the naïve program, which actively maintains CD8+ T cell quiescence until receipt of appropriate activation signals. In this review, we examine the mechanistic control of naïve CD8+ T cell quiescence and summarise the multiple levels of restraint imposed in naïve cells in to limit spontaneous and inappropriate activation. This includes epigenetic mechanisms and transcription factor (TF) regulation of gene expression, in addition to novel inhibitory receptors, abundance of RNA, and protein degradation.

Healthy immune aging is in part determined by how well the sizes of naïve T cell compartments are being maintained with advancing age. Throughout adult life, replenishment largely derives from homeostatic proliferation of existing naïve and memory T cell populations. However, while the subpopulation composition of CD4 T cells is relatively stable, the CD8 T cell compartment undergoes more drastic changes with loss of naïve CD8 T cells and accumulation of effector T cells, suggesting that CD4 T cells are more resilient to resist age-associated changes. To determine the epigenetic basis for these differences in behaviors, we compared chromatin accessibility maps of CD4 and CD8 T cell subsets from young and old individuals and related the results to the expressed transcriptome. The dominant age-associated signatures resembled hallmarks of differentiation, which were more pronounced for CD8 naïve and memory than the corresponding CD4 T cell subsets, indicating that CD8 T cells are less able to keep cellular quiescence upon homeostatic proliferation. In parallel, CD8 T cells from old adults, irrespective of their differentiation state, displayed greater reduced accessibility to genes of basic cell biological function, including genes encoding ribosomal proteins. One possible mechanism is the reduced expression of the transcription factors YY1 and NRF1. Our data suggest that chromatin accessibility signatures can be identified that distinguish CD4 and CD8 T cells from old adults and that may confer the higher resilience of CD4 T cells to aging.

Background Sipuleucel-T (Provenge) is the first therapeutic vaccination approved by the FDA so far, indicated for advanced metastatic prostate cancer patients. Despite an improvement of the overall survival, the benefits of the therapy are still short-term so increasing the duration of the efficacy is necessary. Specifically, T-cell anergy is one of the challenges that we need to overcome to improve the overall efficacy. IL-7 is known to promote the naive T cell activation and to increase the proliferation and activation of the T cell memory subsets. Therefore, in this phase II clinical trial, we tested the therapeutic potential of a human recombinant glycosylated IL-7 after completion of the Provenge therapy on asymptomatic advanced prostate cancer patients. Methods To get a comprehensive analysis of the immune landscape in these patients, we performed CyTOF analysis on PBMC samples obtained at week 1 (baseline) and week 6 after the beginning of the IL-7 therapy. After stimulation with PMA/Ionomycin, we proceeded to surface and intracellular cytokine staining before acquisition on the CyTOF. The data were then analyzed by expert gating on Cytobank. Results At 6 weeks post therapy, our data showed an increase in the number of circulating T lymphocytes in the IL-7 cohort, especially CD8 T cells, in accordance with previous literature. Even though of the frequency of CD4 T cells did not increase, the cells showed greater functionalities, with increased expression of IL-2, TNFα and IL-6 upon stimulation by PMA-Ionomycine. Cytotoxic subsets were also positively affected, with increased expression of IFNγ in CD8 T cells, TNFα in NK cells and IL-2 in γδ T cells. Moreover, PD-1 expression was decreased on CD4, CD8 and γδ T cells while CD137 increased on CD4, CD8 and NK cells. In addition, despite a reduction in the pool of circulating monocytes, we observed higher TNFα expression in these cells. Conclusions Altogether, our data revealed multiple effects of IL-7 in these patients, highlighting a complex set of in vivo mechanisms. In the future, knowledge of these effects may help in choosing the best agents to use in combination with IL-7 and/or the best patients to benefit from IL-7 as part of their therapeutic approach. Trial Registration NCT01881867 Ethics Approval The study was last approved by Fred Hutchinson Cancer Research Center Institutional Review Board, IR file 8037, on January 23, 2020

Background Dendritic cells (DC) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8+ T cell mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141+ DC, the human cDC1 equivalent. CD141+ DC exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8+ T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 to human CD141+ DC. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1 specific naive and memory CD8+ T cells was examined and compared to a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DC. Methods Human anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1- epitope specific CD8+ T cells and reactivity of T cell responses in melanoma patients was assessed by IFNγ production following incubation of CD141+ DC and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naive NY-ESO-1-specific CD8+ T cells were used to investigate naive T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1β, TNF and CD107a and by lysis of target tumor cells. Results CLEC9A-NY-ESO-1 Ab were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141+ DC for activation of NY-ESO-1-specific CD8+ T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in melanoma patients. Moreover, CLEC9A-NY-ESO-1 induced priming of naive NY-ESO-1-specific CD8+ T cells with polyclonal effector function and potent tumor killing capacity in vitro. Conclusions These data advocate human CLEC9A-NY-ESO-1 antibody as an attractive strategy for specific targeting of CD141+ DC to enhance tumour immunogenicity in NY-ESO-1-expressing malignancies. Ethics Approval Written informed consent was obtained for human sample acquisition in line with standards established by the Declaration of Helsinki. Study approval was granted by the Mater Human Research Ethics Committee (HREC13/MHS/83 and HREC13/MHS/86) and The U.S. Army Medical Research and Materiel Command (USAMRMC) Office of Research Protections, Human Research Protection Office (HRPO; A-18738.1, A-18738.2, A-18738.3). All animal experiments were approved by the University of Queensland Animal Ethics Committee and conducted in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes in addition to the laws of the United States and regulations of the Department of Agriculture.

Background Immuno-Oncology research relies heavily on murine syngeneic tumor models. However, whilst the median age for a cancer diagnosis is 65 years or older, for practical purposes the majority of preclinical studies are conducted in young mice, despite the fact that ageing has been shown to have a significant impact on the immune response. Materials and Methods Using aged mice bearing CT26 tumors, we analysed how aging impacts the immune composition of the tumor, spleen and tumor-draining lymph nodes by flow cytometry. Results We found many age-related changes between aged (60–72 weeks old) and young (6–8 weeks old) mice, such as a reduction in the naive T cell population and a decreased CD8/Treg ratio in aged animals. Profiling of co-inhibitory and co-stimulatory receptor expression levels on immune cells in aged versus young mice also identified altered expression profiles in both the periphery and tumor. We hypothesised that these differences may contribute to impaired anti-cancer immune responses in aged mice. To investigate this, we compared the anti-tumor efficacy of immune checkpoint blockade (PD-L1 and CTLA-4) and T-cell costimulation (OX-40) in aged versus young mice. Our data demonstrate that aged mice retained their capacity to generate effective anti-tumor immune responses, albeit often attenuated when compared to the responses observed in young mice. Conclusions These differences highlight the potential importance of age-related immunological changes in assessing and refining the translational insights gained from preclinical mouse models. Disclosure Information S. Sitnikova: A. Employment (full or part-time); Significant; AstraZeneca. M. Morrow: A. Employment (full or part-time); Significant; AstraZeneca. V. Valge-Archer: A. Employment (full or part-time); Significant; AstraZeneca. R.W. Wilkinson: A. Employment (full or part-time); Significant; AstraZeneca. M.J. Robinson: A. Employment (full or part-time); Significant; AstraZeneca. S.J. Dovedi: A. Employment (full or part-time); Significant; AstraZeneca.

MR1-restricted T (MR1T) cells are a T cell subset that recognizes and mediates host immune responses to a broad array of microbial pathogens, including Mycobacterium tuberculosis . Here, we sought to characterize development of circulating human MR1T cells defined by MR1-5-OP-RU tetramer labelling and expression of TRAV1-2/CD26/CD161. We analysed postnatal expansion, maturation and functionality of peripheral blood MR1T cells in cohorts from three different geographic settings with different tuberculosis (TB) vaccination practices, levels of exposure to and infection with M. tuberculosis. Early after birth, frequencies of MR1-5-OP-RU tetramer-defined MR1T cells increased rapidly by several fold. This coincided with marked phenotypic changes, from a predominantly CD4 + and TRAV1-2 − phenotype in neonates, to predominantly TRAV1-2 + CD8 + MR1T cells that also expressed CD26 and CD161. We also observed that tetramer + MR1T cells that expressed TNF upon mycobacterial stimulation were very low in neonates, but increased ~10-fold in the first year of life. These functional MR1T cells in all age groups were MR1-5-OP-RU tetramer + , TRAV1-2 + cells and expressed CD26 and CD161, markers that appeared to signal phenotypic and functional maturation of this cell subset. This age-associated maturation was also marked by the loss of naïve T cell markers on tetramer + TRAV1-2 + MR1T cells more rapidly than tetramer + TRAV1-2 − MR1T cells and non-MR1T cells. These data suggest that neonates have infrequent populations of MR1T cells with diverse phenotypic attributes; and that exposure to the environment rapidly and preferentially expands the MR1-5-OP-RU tetramer + TRAV1-2 + population of MR1T cells, which becomes the predominant population of functional MR1T cells early during childhood. Author Summary MR1-restricted T (MR1T) cells defend against many microbial infections that cause illness and death in young children, including Mycobacterium tuberculosis, which causes tuberculosis (TB). Here, we characterized the development of human MR1T cells from after birth to adulthood. We analysed expansion, maturation and functionality of MR1T cells in populations from three different geographic settings with different tuberculosis vaccination practices and levels of exposure to and infection with M. tuberculosis. We found that early after birth, MR1T cells were rare but then expanded rapidly coinciding with marked phenotypic changes, from heterogeneous phenotypes in neonates, to one predominant type of MR1T cells in adults. We also observed that MR1T cells that defend against mycobacteria were rare in neonates but then increased ~10-fold in the first year of life. We also observed that MR1T cells matured more rapidly than other types of T cells. Our data suggest that neonates have rare populations of MR1T cells with diverse phenotypes and that exposure to the environment rapidly and preferentially expands a dominant population of MR1T cells early during childhood. Our data improves our understanding of the human development of MR1T cells relevant to important childhood pathogens, which could aid in the improvement of infant vaccines.

Background: Triple-negative breast cancer (TNBC) represents the most aggressive breast cancer subtype, which recently attracts great interest for immune therapeutic development. In this context, in-depth understanding of TNBC immune landscape is highly demanded.Results: Here we report full-length single-cell RNA sequencing results of 9683 tumor-infiltrated immune cells isolated from 14 treatment naïve TNBC tumors, where 22 immune cell subsets, including T cells, macrophages, B cells, and DCs have been characterized. We identify a new T cell subset, CD8+CXCL8+ T cell, which associates with poor survival, and a subset of “pre-exhaustion” T cell cluster, which is predictive of favorable prognosis. A novel immune cell subset comprised of TCR+ macrophages, is found to be widely distributed in TNBC tumors. Further analyses reveal an up-regulation of molecules associated with TCR signaling and cytotoxicity in these immune cells.Conclusions: Altogether, our study provides a valuable resource to understand the immune ecosystem of TNBC. The novel immune cell subsets reported herein might be functionally important in cancer immunity. These data will be helpful for the immunotherapeutic strategy design of this disease.

New strategies for immune modulation have shown real promise in regenerative medicine as well as the fight against autoimmune diseases, allergies, and cancer. Dendritic cells (DCs) are gatekeepers of the immune system and their ability in shaping the adaptive immune responses makes DCs ideal targets for immune modulation. Carbohydrates are abundant in different biological systems and are known to modulate DC phenotype and function. However, how simple monosaccharides instruct DC function is less well understood. In this study, we used a combinatorial array of immobilized monosaccharides to investigate how they modulate DC phenotype and function and crucially the impact of such changes on downstream adaptive immune responses. Our data show that a selection of monosaccharides significantly suppress lipopolysaccharide-induced DC activation as evidenced by a reduction in CD40 expression, IL-12 production, and indoleamine 2,3-dioxygenase activity, while inducing a significant increase in IL-10 production. These changes are indicative of the induction of an anti-inflammatory or regulatory phenotype in DCs, which was further confirmed in DC–T cell co-cultures where DCs cultured on the ‘regulatory’ monosaccharide-coated surfaces were shown to induce naïve T cell polarization toward regulatory phenotype. Our data also highlighted a selection of monosaccharides that are able to promote mixed Treg and Th17 cell differentiation, a T cell phenotype expected to be highly immune suppressive. These data show the potential immunomodulatory effects of immobilized monosaccharides in priming DCs and skewing T cell differentiation toward an immune-regulatory phenotype. The ability to fine-tune immune responses using these simple carbohydrate combinations (e.g. as coatings for existing materials) can be utilized as novel tools for immune modulation with potential applications in regenerative medicine, implantable medical devices, and wound healing where reduction of inflammatory responses and maintaining immune homeostasis are desirable., Graphical abstract Image 1