Lymph Node CD4 Research Articles

Prognostic Significance of Peripheral T‐Cell Subsets in Laryngeal Squamous Cell Carcinoma

ObjectivesThe role of the immune system in head and neck squamous cell carcinoma is controversial. The aim of our study was to analyze full blood counts and distribution of T cell subsets in patients affected by laryngeal squamous cell cancer (LSCC) and their association with clinical variables and survival.Study designRetrospective study.MethodsWe analyzed the levels of platelets, lymphocytes, and neutrophils, as well as the CD4+, CD8+, and CD3+ T‐cell subpopulations by cytofluorometry in LSCC patients. A cohort of healthy patients was used as control group. The disease‐specific survival (DSS) was considered as survival outcome.ResultsSixty‐five LSCC patients and 48 controls were enrolled. In LSCC patients, neutrophils were higher than in the healthy group (P < .0001). The neutrophil‐to‐lymphocyte ratio (NLR) and the platelet‐to‐lymphocyte ratio (PLR) were both higher in LSCC patients (P < .0001). In patients treated for recurrent disease, the CD8+/CD3+ ratio was increased (P = .02), while the CD4+/CD8+ (P = .03) and CD4+/CD3+ (P = .04) ratios were lower. In patients with lymph node metastases, leukocytes (P = .03), CD3+ (P = .04), and CD4+ (P = .0098) were all higher. Among Stages III‐IV patients, low lymphocyte and low leukocyte count were associated with worse DSS.ConclusionOur data demonstrate that NLR and PLR are significantly increased in LSCC. Lower CD4+/CD8+ and CD3+/CD8+ ratios are related to recurrent disease and a higher level of CD3+ and CD4+ is associated with nodal metastasis.Level of Evidence4

251-LB: Identification of Antibodies to Hybrid Insulin Peptides (HIPs) in T1D

Hybrid insulin peptides (HIPs) are unique post-translationally modified antigens whereby derivatives of proinsulin (PI) form peptide bonds with endogenous granule peptides. HIPs have been identified in mouse β-cell extracts and human islets by mass spectrometry. Diabetogenic CD4 T cell clones in NOD mice and CD4 T cells from T1D patients are stimulated by HIP ligands and imply that HIPs may also serve as epitopes for humoral immunity. To identify and characterize antibodies (Abs) targeting HIPs among T1D individuals and evaluate their utility as biomarkers, a sensitive and specific ECL-based HIP Ab assay was adapted from the hallmark ECL-IAA protocol. The key determinant for the HIP-Ab assay was design and implementation of a conformational HIP protein probe reflecting derivatives of the HIPs capable of eliciting an antigenic response in CD4 T cell lines from T1D individuals and NOD mice. Sera from a new onset T1D cohort using a PI/IAPP HIP probe showed the presence of anti-PI/IAPP Abs (32.2%, 58/180). However unexpected prevalence was measured among control (18.8%, 18/96) and T2D (25.9%, 29/112) sera samples even when the data was stratified according to HLA genotype. These HIP-Ab epitopes are both conformation and HIP-sequence specific as they are pre-absorbed only with refolded PI/IAPP, but not alternate or random HIPs, linear PI/IAPP HIPs or cyclic PI/IAPP peptides. In sera from prediabetic subjects from the DAISY cohort, 7 of 22 (31.8%) display a peak of HIP-Abs that appears prior to the peak of IAA autoAbs in most cases (87.5%, 7/8). Large number of individuals have PI/IAPP-specific Abs in their circulation, irrespective of T1D disease state or risk. Although HIP-Abs are pervasive and emerge early in the disease process in prospectively followed at risk prediabetic individuals, they are not predictive of disease. As the appearance of PI/IAPP HIP-Abs typically precedes IAAs, it is possible that Abs to other HIPs may serve as earlier biomarkers for ensuing disease. Disclosure Y. Gu: None. J.M. Wenzlau: None. A.W. Michels: Stock/Shareholder; Self; IM Therapeutics. M. Rewers: None. K. Haskins: None. L. Yu: None.

CD55 Is Essential for CD103+ Dendritic Cell Tolerogenic Responses that Protect against Autoimmunity

Recent studies traced inflammatory bowel disease in some patients to deficiency of CD55 [decay-accelerating factor (DAF)], but the mechanism underlying the linkage remained unclear. Herein, we studied the importance of DAF in enabling processes that program tolerance in the gut and the eye, two immune-privileged sites where immunosuppressive responses are continuously elicited. Unlike oral feeding or ocular injection of ovalbumin in wild-type (WT) mice, which induced dominant immune tolerance, identical treatment of DAF-/- mice or DAF-/- to WT bone marrow chimeras did not. While 10% to 30% of mesenteric and submandibular lymph node CD4+ cells became robust T-regulatory cells (Tregs) in WT forkhead box P3 (Foxp3)-green fluorescent protein mice, few in either site became Tregs with little suppressor activity in DAF-/- Foxp3-green fluorescent protein mice. Phenotyping of CD103+ dendritic cells (DCs) from the ovalbumin-fed DAF-/- mice showed impaired expression of inducer of costimulation (ICOS) ligand, programmed death receptor 1-ligand 1 (PD1-L1), CxxxC chemokine receptor 1 (Cx3CR1), CCR7, and CCR9. Analyses of elicited DAF-/- Foxp3+ Tregs showed reduced expression of interferon regulatory factor 8 (IRF-8)/aldehyde dehydrogenase 1 family member A2 (Aldh1a2) and glycoprotein A repetitions predominant/latency-associated protein associated with Treg transforming growth factor-β production and presentation, as well as integrin β6/integrin β8 associated with Treg and CD103+ DC transforming growth factor-β release. Thus, DAF is required for the properties of CD103+ DCs and their naïve CD4+ cell partners that together program tolerance.

The Epigenetic Profile of CD8+ T Cells During SIV Infection

Abstract CD8+ T cells are crucial in the control of HIV infection. Chronic HIV infection results in CD8+ T cell dysfunction that is manifested in reduced cytokine production (IL-2, IFN-g, or TNF-a) and/or reduced cytotoxic function. We investigated whether epigenetic changes in CD8 T cells contribute to impaired effector mechanisms using the infant Rhesus Macaque model of SIV infection. Epigenetic changes (e.g. DNA methylation) alter the accessibility of promoter regions, thereby regulating gene expression. We analyzed the DNA methylation of the granzyme B, IL-2, IFN-g, and TNF-a promoters of matched PBMCs prior to and 12 weeks post SIV infection (n=4) and of CD8 T cells from lymph nodes (LN) (n=4) and the intestine (n=4). Bisulfite reduced DNA was analyzed by MiSeq Paired End 2×150 sequencing, and the percent methylation at classic CpG sites and at all sites was determined using Qiagen CLC Genome Workbench v.11 software. SIV-negative and -positive samples were compared applying Fisher’s Exact Test (p £ 0.05). PBMCs (p = 7.77 × 10−16), LNs (p = 0.01) and intestinal (p = 3.33 × 10−16) CD8+ T cells of SIV-infected animals exhibited increased methylation at the promoter for TNF-a. The methylation of CpG sites within the IFN-g (p = 1.11 × 10−16) and granzyme B (p = 8.88 × 10−16) promoters was only increased in LN CD8+ T cells, but not in the intestine or in PBMC. Our results suggest that immune dysfunction during SIV infection can be partially attributed to methylation changes in immune cells. Further, detailed studies of multiple epigenetic changes in larger animal cohorts are needed to explore SIV-induced tissue-, cell-, and gene-specific epigenetic modifications.

Canine tissue-associated CD4+CD8α+ double-positive T cells are an activated T cell subpopulation with heterogeneous functional potential.

Canine CD4+CD8α+ double-positive (dp) T cells of peripheral blood are a unique effector memory T cell subpopulation characterized by an increased expression of activation markers in comparison with conventional CD4+ or CD8α+ single-positive (sp) T cells. In this study, we investigated CD4+CD8α+ dp T cells in secondary lymphatic organs (i.e. mesenteric and tracheobronchial lymph nodes, spleen, Peyer’s patches) and non-lymphatic tissues (i.e. lung and epithelium of the small intestine) within a homogeneous group of healthy Beagle dogs by multi-color flow cytometry. The aim of this systematic analysis was to identify the tissue-specific localization and characteristics of this distinct T cell subpopulation. Our results revealed a mature extrathymic CD1a-CD4+CD8α+ dp T cell population in all analyzed organs, with highest frequencies within Peyer’s patches. Constitutive expression of the activation marker CD25 is a feature of many CD4+CD8α+ dp T cells independent of their localization and points to an effector phenotype. A proportion of lymph node CD4+CD8α+ dp T cells is FoxP3+ indicating regulatory potential. Within the intestinal environment, the cytotoxic marker granzyme B is expressed by CD4+CD8α+ dp intraepithelial lymphocytes. In addition, a fraction of CD4+CD8α+ dp intraepithelial lymphocytes and of mesenteric lymph node CD4+CD8α+ dp T cells is TCRγδ+. However, the main T cell receptor of all tissue-associated CD4+CD8α+ dp T cells could be identified as TCRαβ. Interestingly, the majority of the CD4+CD8α+ dp T cell subpopulation expresses the unconventional CD8αα homodimer, in contrast to CD8α+ sp T cells, and CD4+CD8α+ dp thymocytes which are mainly CD8αβ+. The presented data provide the basis for a functional analysis of tissue-specific CD4+CD8α+ dp T cells to elucidate their role in health and disease of dogs.

Regulation of cyclin T1 during HIV replication and latency establishment in human memory CD4 T cells

BackgroundThe regulatory cyclin, Cyclin T1 (CycT1), is a host factor essential for HIV-1 replication in CD4 T cells and macrophages. The importance of CycT1 and the Positive Transcription Elongation Factor b (P-TEFb) complex for HIV replication is well-established, but regulation of CycT1 expression and protein levels during HIV replication and latency establishment in CD4 T cells is less characterized.MethodsTo better define the regulation of CycT1 levels during HIV replication in CD4 T cells, multiparameter flow cytometry was utilized to study the interaction between HIV replication (intracellular p24) and CycT1 of human peripheral blood memory CD4 T cells infected with HIV in vitro. CycT1 was further examined in CD4 T cells of human lymph nodes.ResultsIn activated (CD3+CD28 costimulation) uninfected blood memory CD4 T cells, CycT1 was most significantly upregulated in maximally activated (CD69+CD25+ and HLA.DR+CD38+) cells. In memory CD4 T cells infected with HIV in vitro, two distinct infected populations of p24+CycT1+ and p24+CycT1- cells were observed during 7 days infection, suggestive of different phases of productive HIV replication and subsequent latency establishment. Intriguingly, p24+CycT1- cells were the predominant infected population in activated CD4 T cells, raising the possibility that productively infected cells may transition into latency subsequent to CycT1 downregulation. Additionally, when comparing infected p24+ cells to bystander uninfected p24- cells (after bulk HIV infections), HIV replication significantly increased T cell activation (CD69, CD25, HLA.DR, CD38, and Ki67) without concomitantly increasing CycT1 protein levels, possibly due to hijacking of P-TEFb by the viral Tat protein. Lastly, CycT1 was constitutively expressed at higher levels in lymph node CD4 T cells compared to blood T cells, potentially enhancing latency generation in lymphoid tissues.ConclusionsCycT1 is most highly upregulated in maximally activated memory CD4 T cells as expected, but may become less associated with T cell activation during HIV replication. The progression into latency may further be predicated by substantial generation of p24+CycT1- cells during HIV replication.

Mesenteric lymph node CD4+ T lymphocytes migrate to liver and contribute to non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is characterized by altered intestinal microbiota and intestinal immune disorder. Here we investigated the role of mesenteric lymph node (MLN) CD4+ T lymphocytes in NAFLD. In high fat diet (HFD)-fed mice, the percentage ratios of Th1 to Th2 cells and Th17 to Treg cells were imbalanced in MLNs. Co-culture assays showed MLN CD4+ T lymphocytes from HFD-fed mice tended to migrate to the liver and promoted hepatic inflammation. Adoptive transfer of MLN CD4+ T lymphocytes from NAFLD mice to HFD-fed mice resulted in higher transaminase, worse hepatic inflammation and lipid accumulation. Antibiotics and probiotics were administrated to regulate intestinal microbiota, and the restoration of MLN Th1/Th2 and Th17/Treg cells in alleviated NAFLD were found. In summary, MLNs CD4+ T subtype cells may involve in NAFLD, and the restoration of MLN CD4+ T subtype cells ratio by regulating intestinal bacteria could be the new strategies.

Reduced early life mucosal integrity decreases thymic cell counts and increases local, but not thymic regulatory, T cell recruitment: Gut mucosal integrity breach and thymic T cells

Early life immune gut microbiota contact is critical for regulatory T cell–mediated oral tolerance induction. We induced a mucosal integrity breach with low dextran sulfate sodium dose right after weaning in BALB/c mice along with a standard high dose to study the impact of increased gut microbiota lymphatic tissue contact on the thymus. Both doses increased gut permeability, which caused a short-term generalized thymic involution and regulatory T cell induction in the mesenteric lymph nodes, even in the absence of clinically apparent inflammation in the low-dose group. The thymic regulatory T cells resisted thymic involution. In the low-dose group, we found acutely altered gut mobilization patterns characterized by changed gut-homing marker CD103 expression on mesenteric lymph node CD4+T cells as well as on mature CD8+T cells and developing CD4−/CD8−thymocytes. Furthermore, CD218a (IL-18-receptor-a) expression was acutely decreased on both mature CD8+T cells and regulatory T cells, while increased on the mesenteric lymph node CD8+T cells, indicating a direct link between the thymus and the mesenteric lymph nodes with CD218a in a functional role in thymic involution. Acute and non-persisting regulatory responses in the mesenteric lymph nodes were induced in the form of a relative regulatory T cell increase. We saw no changes in total thymic regulatory T cells and thus the thymus does not seem to play a major role of in the regulatory immunity induced by increased gut microbiota lymphatic tissue contact around weaning, which in our study primarily was located to the gut.

Prevention of SIVmac251 reservoir seeding in rhesus monkeys by early antiretroviral therapy

The precise time when the viral reservoir is seeded during acute HIV-1 infection remains unclear. We previously demonstrated that the viral reservoir was seeded by day 3 following SIVmac251 infection in rhesus monkeys. Here we report the impact of initiating ART on day 0 (6 h), 1, 2, or 3 following intrarectal SIVmac251 infection in 20 rhesus monkeys (N = 5/group). After 6 months of daily suppressive ART, antiretroviral drugs were discontinued, and viral rebound was monitored. 0% (0 of 5), 20% (1 of 5), 60% (3 of 5), and 100% (5 of 5) of animals that initiated ART on days 0 (6 h), 1, 2, or 3, respectively, showed viral rebound following ART discontinuation and correlated with integrated viral DNA in lymph node CD4+ T cells. These data demonstrate that the viral reservoir is seeded within the first few days of infection and that early ART initiation limits the viral reservoir.

Impact of one versus two doses of mesenchymal stromal cells on lung and cardiovascular repair in experimental emphysema

BackgroundA single administration of mesenchymal stromal cells (MSCs) has been shown to reduce lung inflammation in experimental elastase-induced emphysema; however, effects were limited in terms of lung-tissue repair and cardiac function improvement. We hypothesized that two doses of MSCs could induce further lung and cardiovascular repair by mitigating inflammation and remodeling in a model of emphysema induced by multiple elastase instillations. We aimed to comparatively investigate the effects of one versus two doses of MSCs, administered 1 week apart, in a murine model of elastase-induced emphysema.MethodsC57BL/6 mice were randomly divided into control (CTRL) and emphysema (E) groups. Mice in the E group received porcine pancreatic elastase (0.2 IU, 50 μL) intratracheally once weekly for four consecutive weeks; the CTRL animals received sterile saline (50 μL) using the same protocol. Three hours after the last instillation, the E group was further randomized to receive either saline (SAL) or murine MSCs (105 cells) intratracheally, in one or two doses (1 week apart). Fourteen days later, mice were euthanized, and all data analyzed.ResultsBoth one and two doses of MSCs improved lung mechanics, reducing keratinocyte-derived chemokine and transforming growth factor-β levels in lung homogenates, total cell and macrophage counts in bronchoalveolar lavage fluid (BALF), and collagen fiber content in airways and blood vessels, as well as increasing vascular endothelial growth factor in lung homogenates and elastic fiber content in lung parenchyma. However, only the two-dose group exhibited reductions in tumor necrosis factor-α in lung tissue, BALF neutrophil and lymphocyte count, thymus weight, and total cellularity, as well as CD8+ cell counts and cervical lymph node CD4+ and CD8+ T cell counts, as well as further increased elastic fiber content in the lung parenchyma and reduced severity of pulmonary arterial hypertension.ConclusionsTwo doses of MSCs enhanced lung repair and improvement in cardiac function, while inducing T cell immunosuppression, mainly of CD8+ cells, in elastase-induced emphysema.

GITR Agonism Enhances Cellular Metabolism to Support CD8+ T-cell Proliferation and Effector Cytokine Production in a Mouse Tumor Model.

GITR is a costimulatory receptor currently undergoing phase I clinical trials. Efficacy of anti-GITR therapy in syngeneic mouse models requires regulatory T-cell depletion and CD8+ T-cell costimulation. It is increasingly appreciated that immune cell proliferation and function are dependent on cellular metabolism. Enhancement of diverse metabolic pathways leads to different immune cell fates. Little is known about the metabolic effects of GITR agonism; thus, we investigated whether costimulation via GITR altered CD8+ T-cell metabolism. We found activated, GITR-treated CD8+ T cells upregulated nutrient uptake, lipid stores, glycolysis, and oxygen consumption rate (OCR) in vitro Using MEK, PI3Kδ, and metabolic inhibitors, we show increased metabolism is required, but not sufficient, for GITR antibody (DTA-1)-induced cellular proliferation and IFNγ production. In an in vitro model of PD-L1-induced CD8+ T-cell suppression, GITR agonism alone rescued cellular metabolism and proliferation, but not IFNγ production; however, DTA-1 in combination with anti-PD-1 treatment increased IFNγ production. In the MC38 mouse tumor model, GITR agonism significantly increased OCR and IFNγ and granzyme gene expression in both tumor and draining lymph node (DLN) CD8+ T cells ex vivo, as well as basal glycolysis in DLN and spare glycolytic capacity in tumor CD8+ T cells. DLN in GITR-treated mice showed significant upregulation of proliferative gene expression compared with controls. These data show that GITR agonism increases metabolism to support CD8+ T-cell proliferation and effector function in vivo, and that understanding the mechanism of action of agonistic GITR antibodies is crucial to devising effective combination therapies. Cancer Immunol Res; 6(10); 1199-211. ©2018 AACR.

CD32+ and PD-1+ Lymph Node CD4 T Cells Support Persistent HIV-1 Transcription in Treated Aviremic Individuals.

A recent study conducted in blood has proposed CD32 as the marker identifying the "elusive" HIV reservoir. We have investigated the distribution of CD32+ CD4 T cells in blood and lymph nodes (LNs) of HIV-1-uninfected subjects and viremic untreated and long-term-treated HIV-1-infected individuals and their relationship with PD-1+ CD4 T cells. The frequency of CD32+ CD4 T cells was increased in viremic compared to treated individuals in LNs, and a large proportion (up to 50%) of CD32+ cells coexpressed PD-1 and were enriched within T follicular helper (Tfh) cells. We next investigated the role of LN CD32+ CD4 T cells in the HIV reservoir. Total HIV DNA was enriched in CD32+ and PD-1+ CD4 T cells compared to CD32- and PD-1- cells in both viremic and treated individuals, but there was no difference between CD32+ and PD-1+ cells. There was no enrichment of latently infected cells with inducible HIV-1 in CD32+ versus PD-1+ cells in antiretroviral therapy (ART)-treated individuals. HIV-1 transcription was then analyzed in LN memory CD4 T cell populations sorted on the basis of CD32 and PD-1 expression. CD32+ PD-1+ CD4 T cells were significantly enriched in cell-associated HIV RNA compared to CD32- PD-1- (averages of 5.2-fold in treated individuals and 86.6-fold in viremics), CD32+ PD-1- (2.2-fold in treated individuals and 4.3-fold in viremics), and CD32- PD-1+ (2.2-fold in ART-treated individuals and 4.6-fold in viremics) cell populations. Similar levels of HIV-1 transcription were found in CD32+ PD-1- and CD32- PD-1+ CD4 T cells. Interestingly, the proportion of CD32+ and PD-1+ CD4 T cells negatively correlated with CD4 T cell counts and length of therapy. Therefore, the expression of CD32 identifies, independently of PD-1, a CD4 T cell population with persistent HIV-1 transcription and coexpression of CD32 and PD-1, the CD4 T cell population with the highest levels of HIV-1 transcription in both viremic and treated individuals.IMPORTANCE The existence of long-lived latently infected resting memory CD4 T cells represents a major obstacle to the eradication of HIV infection. Identifying cell markers defining latently infected cells containing replication-competent virus is important in order to determine the mechanisms of HIV persistence and to develop novel therapeutic strategies to cure HIV infection. We provide evidence that PD-1 and CD32 may have a complementary role in better defining CD4 T cell populations infected with HIV-1. Furthermore, CD4 T cells coexpressing CD32 and PD-1 identify a CD4 T cell population with high levels of persistent HIV-1 transcription.

Inhibition of p38 MAPK in combination with ART reduces SIV-induced immune activation and provides additional protection from immune system deterioration.

Differences in immune activation were identified as the most significant difference between AIDS-susceptible and resistant species. p38 MAPK, activated in HIV infection, is key to induction of interferon-stimulated genes and cytokine-mediated inflammation and is associated with some of the pathology produced by HIV or SIV infection in AIDS-susceptible primates. As small molecule p38 MAPK inhibitors are being tested in human trials for inflammatory diseases, we evaluated the effects of treating SIV-infected macaques with the p38 MAPK inhibitor PH-797804 in conjunction with ART. PH-797804 had no side effects, did not impact negatively the antiviral immune response and, used alone, had no significant effect on levels of immune activation and did not reduced the viremia. When administered with ART, it significantly reduced numerous immune activation markers compared to ART alone. CD38+/HLA-DR+ and Ki-67+ T-cell percentages in blood, lymph node and rectal CD4+ and CD8+ T cells, PD-1 expression in CD8+ T cells and plasma levels of IFNα, IFNγ, TNFα, IL-6, IP-10, sCD163 and C-reactive protein were all significantly reduced. Significant preservation of CD4+, CD4+ central memory, CD4+/IL-22+ and CD4+/IL-17+ T-cell percentages and improvement of Th17/Treg ratio in blood and rectal mucosa were also observed. Importantly, the addition of PH-797804 to ART initiated during chronic SIV infection reduced immune activation and restored immune system parameters to the levels observed when ART was initiated on week 1 after infection. After ART interruption, viremia rebounded in a similar fashion in all groups, regardless of when ART was initiated. We concluded that the inhibitor PH-797804 significantly reduced, even if did not normalized, the immune activation parameters evaluated during ART treatment, improved preservation of critical populations of the immune system targeted by SIV, and increased the efficacy of ART treatment initiated in chronic infection to levels similar to those observed when initiated in acute infection but did not affect positively or negatively viral reservoirs.

Therapeutic control of leishmaniasis by inhibitors of the mammalian target of rapamycin

Leishmaniasis is a serious global health problem affecting many people worldwide. While patients with leishmaniasis can be treated with several agents, drug toxicicty and the emergence of resistant strains render available treatments ineffective in the long run. Inhibitors of the mammalian target of rapamycin (mTOR) have been demonstrated to exert anti-pathogen properties. In this study, we tested the therapeutic efficacy of several mTOR inhibitors in controlling infection with Leishmania major. Rapamycin, GSK-2126458 and KU-0063794 were administered to BALB/c mice, which had received an intrafootpad injection of the parasite. Footpad swelling and parasite burden were assessed, and cytokine production by mouse splenocytes and phenotypic changes in draining lymph node cells were evaluated. Treatment with a clinically relevant dose of rapamycin or with GSK-2126458, but not with KU-0063794, dramatically lowered both the footpad swelling and the parasite load in the draining lymph node. Importantly, the employed dose of rapamycin did not kill the promastigotes in vitro as judged by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and electron microscopy. Moreover, the IL-4 production capacity of splenocytes harvested from infected mice that were treated with rapamycin was significantly reduced. Consequently, the IFN-γ:IL-4 production ratio was elevated, suggesting a T helper-type 1 (Th1)-skewed cytokine profile. Finally, the expression level of CD69, an early activation marker, on splenic and lymph node CD4+ and CD8+ T cells was enhanced in rapamycin-treated mice. Taken together, our findings suggest that select mTOR inhibitors may be used in therapeutic settings for the management of leishmaniasis. We propose that the beneficial effects of such inhibitors stem from their immunomodulatory properties. Therefore, the adjuvanticity of mTOR inhibitors may also be considered in vaccination strategies against Leishmania species.

Total body CD4+ T cell dynamics in treated and untreated SIV infection revealed by in vivo imaging

The peripheral blood represents only a small fraction of the total number of lymphocytes in the body. To develop a more thorough understanding of T cell dynamics, including the effects of SIV/SHIV/HIV infection on immune cell depletion and immune reconstitution following combination antiretroviral therapy (cART), one needs to utilize approaches that allow direct visualization of lymphoid tissues. In the present study, noninvasive in vivo imaging of the CD4+ T cell pool has revealed that the timing of the CD4+ T cell pool reconstitution following initiation of ART in SIV-infected nonhuman primates (NHPs) appears seemingly stochastic among clusters of lymph nodes within the same host. At 4 weeks following initiation or interruption of cART, the changes observed in peripheral blood (PB) are primarily related to changes in the whole-body CD4 pool rather than changes in lymphocyte trafficking. Lymph node CD4 pools in long-term antiretroviral-treated and plasma viral load-suppressed hosts appear suboptimally reconstituted compared with healthy controls, while splenic CD4 pools appear similar between the 2 groups.

OX40 ligand is inhibitory during the effector phase of crescentic glomerulonephritis

The functional relevance of OX40 ligand (OX40L) in the effector phase of crescentic glomerulonephritis (GN) is unknown. These studies defined the role of endogenous OX40L during the effector stage of murine crescentic GN. GN was induced by immunization with sheep globulin/adjuvant on Day 0 and injection of sheep anti-mouse glomerular basement membrane immunoglobulin (Ig) on Day 10. Rat IgG or neutralizing anti-OX40L antibody was administered on Days 10-18 and immune responses and renal injury assessed on Day 20. Compared with naïve animals, OX40L was upregulated in the lymph nodes (LNs) and on leucocytes and resident non-immune cells in the kidneys of mice with GN. Inhibition of OX40L in GN augmented renal injury, as indicated by increased crescent formation, proteinuria and glomerular leucocyte accumulation. In line with increased injury, anti-OX40L treatment increased proliferation and decreased apoptosis of CD4 T cells in the LNs, without affecting LN CD4 cytokine production and CD8 T-cell responses. Blockade of OX40L decreased LN regulatory T-cell (Treg) proliferation, transforming growth factor β production and foxp3 expression. OX40L inhibition did not affect B cell expansion or circulating antibody levels. In the kidney, neutralization of OX40L augmented interferon γ (IFNγ) expression by CD4 and CD8 T cells and shifted macrophage polarization towards the pro-inflammatory M1 phenotype. OX40L is protective during the effector phase of murine crescentic GN by reducing the expansion of CD4 T cells and enhancing Treg responses in the LNs, and by locally inhibiting T-cell IFNγ production and pro-inflammatory macrophage phenotype in the kidney.

Generation of a mouse model of extrathymically-derived regulatory T cells in type 1 diabetes

Abstract Regulatory T cells (Tregs) are an immunosuppressive population that were initially identified as uniformly and stably expressing the transcription factor Foxp3. Tregs have now been further distinguished based on whether they developed in the thymus (thymically-derived Tregs, or tTregs) or in the periphery (peripherally-derived Tregs, or pTregs). Previous studies have shown that deletion of CNS1, a conserved non-coding DNA response element in the Foxp3 locus, leads to selective impairment of pTreg generation without disrupting tTreg generation in the B6 background. However, the role of pTregs versus tTregs in autoimmunity is unclear. Using CRISPR-Cas9, we removed the CNS1 region in the NOD mouse model of spontaneous type 1 diabetes. Based on in vitro Treg generation assays, removal of the CNS1 region impairs Foxp3 induction in naïve NOD CD4+ T cells. However, it did not lead to statistically significant differences in the percent of Tregs found in various tissues, including the spleen, thymus, gut, lymph nodes, and pancreatic islets of non-diabetic mice aged 8–13 weeks. Single-cell RNA seq comparing a 12-week old NOD mouse to a CNS1 KO NOD mouse also did not reveal strong differences in gene expression in the pancreatic islet, gut, or pancreatic lymph node, although flow cytometry analysis does reveal a small but significant decrease in RORyt+ Tregs and an increase in Helios+ Tregs in the gut of CNS1 KO NOD mice. CNS1 KO NOD mice are also being compared to normal NOD mice for type 1 diabetes onset and incidence. These preliminary data suggest that extrathymically-derived Tregs do not play a large role in this mouse model of type 1 diabetes.

Allograft transplantation provokes miRNA 297–669 cluster that reduces T regulatory cell generation through targeting TGF-β2

Abstract T regulatory cells (Tregs) are anti-inflammatory cells capable of quelling the harshest immune responses. Accordingly, Treg therapy is rapidly becoming a viable treatment option for patients stricken with autoimmune diseases or those in need of an organ transplant. In a model of allotransplantation, via microarray, our work identified miRNA cluster 297–669 is induced in CD4+ cells in the lymph nodes draining the allograft in C57BL/6 mice given a C3H skin transplant. One member of this cluster, miR 466a-3p, is induced in a co-culture of lymph node cells stimulated with alloantigen in vitro. We have shown that this miRNA specifically targets transforming growth factor beta 2 (TGF-β2). The reduction in TGF-β2 levels in draining lymph node CD4+ T cells caused a resultant decrease in the generation of FoxP3+ Tregs, both within the graft and in the draining lymph node. Furthermore, blocking the effect of this miRNA using a specific locked nucleic acid (LNA) reversed this effect. Generation of Tregs using transforming growth factor beta1 (TGF-β1), interleukin-2 and CD3/CD28 is the most common method of induction. Here we show that replacing TGF-β1 with TGF-β2 has no detrimental effect on Treg induction. Indeed, Tregs induced with TGF-β2 have equal in vivo suppressive capability as TGF-β1 Tregs in a skin graft model when adoptively transferred. Moreover, these TGF-β2 Tregs have increased inducible T-cell costimulatory (ICOS) expression. The ability of TGF-β2 to induce Tregs was reversed when a TGF-β2 neutralizing antibody was used, while this antibody had no effect on TGF-β1 induced Tregs. The work herein describes, for the first time, a novel isoform of TGF-β that may be involved in the generation of Tregs with implications in all areas of immunology.

Blood CXCR3+ CD4 T Cells Are Enriched in Inducible Replication Competent HIV in Aviremic Antiretroviral Therapy-Treated Individuals

We recently demonstrated that lymph nodes (LNs) PD-1+/T follicular helper (Tfh) cells from antiretroviral therapy (ART)-treated HIV-infected individuals were enriched in cells containing replication competent virus. However, the distribution of cells containing inducible replication competent virus has been only partially elucidated in blood memory CD4 T-cell populations including the Tfh cell counterpart circulating in blood (cTfh). In this context, we have investigated the distribution of (1) total HIV-infected cells and (2) cells containing replication competent and infectious virus within various blood and LN memory CD4 T-cell populations of conventional antiretroviral therapy (cART)-treated HIV-infected individuals. In the present study, we show that blood CXCR3-expressing memory CD4 T cells are enriched in cells containing inducible replication competent virus and contributed the most to the total pool of cells containing replication competent and infectious virus in blood. Interestingly, subsequent proviral sequence analysis did not indicate virus compartmentalization between blood and LN CD4 T-cell populations, suggesting dynamic interchanges between the two compartments. We then investigated whether the composition of blood HIV reservoir may reflect the polarization of LN CD4 T cells at the time of reservoir seeding and showed that LN PD-1+ CD4 T cells of viremic untreated HIV-infected individuals expressed significantly higher levels of CXCR3 as compared to CCR4 and/or CCR6, suggesting that blood CXCR3-expressing CD4 T cells may originate from LN PD-1+ CD4 T cells. Taken together, these results indicate that blood CXCR3-expressing CD4 T cells represent the major blood compartment containing inducible replication competent virus in treated aviremic HIV-infected individuals.

Regulation of T helper cell responses during antigen presentation by norepinephrine-exposed endothelial cells.

Dermal blood vessels and regional lymph nodes are innervated by sympathetic nerves and, under stress, sympathetic nerves release norepinephrine (NE). Exposure of primary murine dermal microvascular endothelial cells (pDMECs) to NE followed by co-culture with Langerhans cells (LCs), responsive CD4+ T-cells and antigen resulted in modulation of CD4+ T-cell responses. NE-treatment of pDMECs induced increased production of interleukin (IL)-6 and IL-17A while down-regulating interferon (IFN)-γ and IL-22 release. This effect did not require contact between pDMECs and LCs or T-cells and depended upon pDMEC production of IL-6. The presence of NE-treated pDMECs increased the proportion of CD4+ T-cells expressing intracellular IL-17A and increased IL-17A mRNA while decreasing the proportion of IFN-γ- or IL-22-expressing CD4+ T-cells and mRNA levels for those cytokines. Retinoic acid receptor-related orphan receptor gamma (ROR-γt) mRNA was significantly increased in CD4+ T-cells while T-box transcription factor (T-bet) mRNA was decreased. Intradermal administration of NE prior to hapten immunization at the injection site produced a similar bias in draining lymph node CD4+ T-cells towards IL-17A and away from IFN-γ and IL-22 production. Under stress, release of NE may have significant regulatory effects on the outcome of antigen presentation through actions on ECs with enhancement of inflammatory skin disorders involving IL-17/T helper type 17 (Th17) cells.