Total Number Of CD4 Research Articles

Myeloid-Derived Suppressor Cell Subtypes Are Responsible for the Th2 Phenotype of T Cells in CLL

In chronic lymphocytic leukemia (CLL), monoclonal B cells expand and accumulate in the blood, bone marrow, spleen and secondary lymphoid organs in response to incompletely defined cellular and molecular interactions involving multiple cell types within the tumor microenvironment (TME). Bidirectional interactions of CLL cells with components of the TME - mesenchymal stromal cells, myeloid cells, T cells, and matrix - are critical for leukemic cell survival and proliferation. Here, we focused on understanding how two non-leukemic cell types within the TME, T lymphocytes and myeloid-derived suppressor cells (MDSCs), interact to create a TME supportive of a CLL clone.In CLL, it has been suggested alterations in CD4+ and CD8+ T cell ratios and functions are responsible for a non-inflammatory and immunosuppressive cytokine TME, with expansions of Th2s and Tregs. Conversely, higher numbers of Th17 and CD8+ cells are associated with longer survival. MDSCs are not well studied in CLL, although existing reports suggest that their expansion suppresses T cell proliferation in vitro. Here we studied the effects of MDSCs and CLL B cells on T cell numbers and function.First, we identified a correlation between blood T cells and MDSCs in 56 CLL patients. Absolute numbers of MDSCs correlated with total numbers of CD3+, CD4+ and CD8+ cells (P= 0.002, < 0.001, < 0.001 and Spearman r = 0.44, 0.646, = 0.61, respectively). However, the correlation of MDSC subtypes with CD4+ and CD8+ cells differed: monocyte-like MDSCs (mMDSCs) associated with CD4+ cells (P < 0.001, Spearman r = 0.73) whereas granulocyte-like MDSCs (gMDSCs) associated with CD8+ cells (P= 0.008; Spearman r = 0.45).Moreover, in a subset of 18 CLL patients tested, MDSC numbers directly correlated with the levels of Th2 (P= 0.049, Spearman r = 0.470), Th17 (P= 0.040, Spearman r = 0.488), Tc17 (P= 0.018, Spearman r = 0.550) and Tc22 (P= 0.010, Spearman r = 0.587) cells. When dividing MDSCs into subsets, gMDSCs exhibited a similar correlation except for the absence of Th2 and the presence of a correlation with Th22 (P= 0.033, Spearman r = 0.505). Conversely, mMDSCs positively associated with Th1 (P= 0.024, Spearman r = 0.549) and Tc1 (P= 0.019, Spearman r = 0.569) cells.We next addressed the functional effect of MDSCs on naïve CD4+ cell differentiation using samples from 16 CLL patients and 10 healthy, age-matched controls (HCs). CD3+CD45RO-CD62L+ CD4+ lymphocytes were FACS-sorted and stimulated in vitro with anti-CD3/CD28 beads plus IL-2, and the percentages of CD4+ T cells producing various cytokines were quantified by intracellular flow cytometry. These results were compared with those obtained in the presence of mMDSCs (HLA-DRlo/CD11b+/CD33+/CD14+), gMDSCs (HLA-DRlo/CD11b+/CD33+/CD15+), monocytes (HLA-DRhi/CD11b+/CD14+) or CLL B lymphocytes (CD19+). On day 7, CLL T cells, cultured in the absence of other cells, generated a higher percentage of cytokine-producing cells than those from HCs (22% vs. 8%; P < 0.001). This difference was due to an increase in all cytokine-producing cells except classical Th17s producing IL-17A. When CLL T cells were co-cultured with gMDSCs, a significant expansion of Th2 (IL-4+) (P= 0.044) and a trend in growth of IL-17F+ Th17cells (P= 0.101) was found. In contrast, co-culture of CLL T cells with mMDSCs led to a reduction in Th22 (IL-22+) (P= 0.005) and Treg (FoxP3+) (P= 0.006) cells and to a trend towards expansion of Th2 (IL-4+) cells. Co-cultures of CLL T cells with monocytes, similarly to mMDSCs, led to an expansion of Th2 (IL-4+) cells (P= 0.01) and a reduction in Treg (FoxP3+) cells (P= 0.002). Finally, co-culturing naïve CD4+ lymphocytes with CLL B cells had a different and unexpected effect, promoting the expansion of Th1 (IFNγ+) cells (P= 0.033) and Th22 (IL-22+) T cells (P= 0.052).In summary, the absolute number of blood T cells, CD4+ and CD8+ subsets, and MDSCs are linked, although associations of T cell subsets differ between mMDSCs and gMDSCs. MDSCs and their subsets influence naïve T cell differentiation differently, in general leading to expansion of Th2 (IL-4+) cells. In contrast, CLL B cells had a different effect on T cell polarization, preferentially expanding Th1 (IFNγ+) cells. Thus, it appears that myeloid cells and especially MDSCs are responsible for polarization of the T cell compartment to Th2 which is said to be dominant in CLL patients. DisclosuresKolitz:Gilead, Magellan, and Novartis: Honoraria; Gilead, Novartis, and Seattle Genetics: Other: Travel Support; Boehringer Ingelheim, Cantex, Erytech, and Millennium: Research Funding; Celgene, Jazz: Equity Ownership; Gilead, Magellan, Novartis, Pharmacyclics, and Seattle Genetics: Consultancy. Barrientos:Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy; Gilead: Consultancy, Research Funding. Stamatopoulos:Novartis SA: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.

Phase II study of atezolizumab in advanced alveolar soft part sarcoma (ASPS).

11519 Background: ASPS constitutes &lt; 1% of soft tissue sarcomas and frequently presents in adolescents and young adults. There are no approved therapies for ASPS. We are currently evaluating the clinical activity of atezolizumab (atezo), an anti-PD-L1 antibody, in patients (pts) with advanced ASPS. Methods: This is a multicenter, open-label, single-arm phase II study where atezo is administered at a fixed dose of 1200 mg in adults or 15 mg/kg (1200 mg max) in pediatric pts age ≥2 once Q21 days. The primary objective is to determine the objective response rate (ORR) of atezo using RECIST 1.1. Secondary objectives include duration of response and correlating response with the immune effects of atezo in blood and paired tumor biopsies (pre- and post-treatment). Tumor specimens were analyzed with multiplex immunofluorescence immuno-oncology panels to quantify CD8+, PD-1+, and PD-L1+ cells/mm2 in the tumor microenvironment. CD8+ density was calculated as the total number of CD8+ cells divided by the entire area (mm2) of the tumor and invasive margins of the biopsy. Results: As of February 4, 2021, 44 pts have been enrolled. The median age in the study was 31 years (range, 12–70) with equal male: female distribution. 54.5% of pts were Caucasian. Baseline ECOG ≤1 was present in 97.7%. The median time on study was 11.5 months (range, 0.8–40.3 months). At data cutoff, response evaluation was available for 43 pts with an ORR of 37.2% (16/43). One pt experienced a complete response and 15 pts experienced a partial response (PR), of which 14 were confirmed. The median time to confirmed response was 3.5 months (range, 2.1–14.9 months). The median duration of confirmed response was 16.5 months (range, 4.9–38.1 months). Stable disease (SD) was present in 58.1% (25/43). One or more grade 3 adverse events potentially related to atezo were identified in 16.3% (7/43) pts. These include diarrhea, hypothyroidism, transaminitis, anemia, vertigo, extremity pain, myalgia, pneumonitis, rash, and stroke (n = 1 each). No grade 4 or 5 events have been reported. Among 8 cases with evaluable biopsy pairs, both baseline and C3D1 specimens in all cases demonstrated CD8+ T cell infiltration and PD-L1 expression. PD-1 expression was detected at baseline in 5 cases and at C3D1 in 7 cases. In 6 cases (3 SDs and 3 PRs), treatment did not change CD8+ cell density. In the other 2 cases (both PRs), CD8+ density increased &gt; 3x above baseline by C3D1. Analysis of T cell activation using pharmacodynamic response biomarkers, along with whole exome and RNA-seq to evaluate the genomic and transcriptomic landscape of ASPS, are ongoing. Conclusions: Atezo is well tolerated and demonstrates promising single agent activity with durable responses in advanced ASPS. Preliminary tumor biomarker analysis confirms the presence of multiple PD-1/PD-L1 immune checkpoint (IC) components, indicating that advanced ASPS is an ideal candidate for therapeutic IC inhibition. Funded by NCI Contract No HHSN261200800001E. Clinical trial information: NCT03141684.

Attenuated T Cell Responses Are Associated With the Blockade of Cerebral Malaria Development by YOP1-Deficient Plasmodium berghei ANKA.

Reticulon and the REEP family of proteins stabilize the high curvature of endoplasmic reticulum tubules. The REEP5 homolog in Plasmodium, Plasmodium berghei YOP1 (PbYOP1), plays an important role in the erythrocytic cycle of the P. berghei ANKA and the pathogenesis of experimental cerebral malaria (ECM), but the mechanisms are largely unknown. Here, we show that protection from ECM in Pbyop1Δ-infected mice is associated with reduced intracerebral Th1 accumulation, decreased expression of pro-inflammatory cytokines and chemokines, and attenuated pathologies in the brainstem, though the total number of CD4+ and CD8+ T cells sequestered in the brain are not reduced. Expression of adhesive molecules on brain endothelial cells, including ICAM-1, VCAM-1, and CD36, are decreased, particularly in the brainstem, where fatal pathology is always induced during ECM. Subsequently, CD8+ T cell-mediated cell apoptosis in the brain is compromised. These findings suggest that Pbyop1Δ parasites can be a useful tool for mechanistic investigation of cerebral malaria pathogenesis.

At Last the Explanation for Increased Spontaneous Miscarriages in Thyroid Autoantibody Positive Pregnant Women

In women who are pregnant, the presence of thyroid autoantibodies is associated with an increased rate of miscarriage in the first trimester, with multiple reports averaging ~17% compared with ~8% in autoantibody negative women. During pregnancy immune tolerance is altered to allow implantation of the semi-allogeneic fetus and T-regulatory cells (Tregs), which play a major role in such tolerance, have been shown to increase during pregnancy, reaching a peak in the second trimester. A deficiency in this Treg response has been widely associated with spontaneous miscarriages. While it is known that the number of Tregs in patients with autoimmune thyroid disease (AITD) is decreased there are no data on pregnant women with AITD. In this study, we examined both the number and function of Tregs in pregnant women with (n=26) and without (n=41) thyroid autoantibodies (anti-Tg and/or anti-TPO) as well as healthy non pregnant women (n=25). Tregs were measured by flow cytometry of isolated CD4+, CD25+ and FoxP3+ T-cells. We found that the total number of CD4+CD25+FoxP3+ high Tregs was significantly increased in pregnancy consistent with previous reports (from 11% to 22%, p<0.024). Furthermore, this increase in Tregs was less in pregnant women with thyroid autoantibodies (mean of 12%). In addition, studies of phosphorylated signal transducer and activator of transcription-5a (pStat-5a) as a marker of Treg function, showed that while Tregs were activated in pregnancy, their activity per cell was diminished in the pregnant antibody positive women with a frequency:MFI ratio of 201 compared to 316 in the negative group. These data demonstrate that pregnant women with thyroid antibodies have a reduced Treg response to pregnancy, both in number and function, and offer a likely explanation for the increased miscarriage rate in such patients.

CD8αα expression increases during demyelinating disease progression

Abstract Experimental autoimmune encephalomyelitis (EAE) is a well-described mouse model for multiple sclerosis (MS), a demyelinating autoimmune disorder of the central nervous system (CNS). While the role of CD4 T cells is well established, other immune cell types, particularly CD8 T cells, are key contributors to disease pathogenesis. Our study aims to categorize the functional contributions of CD8 T cell subsets to EAE kinetics and disease outcomes. The use of myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide alongside an adjuvant to inoculate mice, provides a reproducible disease model. Demyelinating disease severity was analyzed at defined timepoints including, onset (d10–14), peak (d17–21), early chronic (d28–35) and late chronic (d80–120) stages. The total number of CD3+ T cells in the CNS remained consistent during EAE. However, it goes from a CD4 T cell dominated response at peak EAE to an equalized 1:1 ratio of CD4 and CD8 T cells at late chronic stage. Furthermore, differentiation of CD8 T cell subsets based on CD8α and CD8β protein expression revealed that CD8αα T cells numbers increase to comprise ~40% of the CD8+ population at late chronic EAE. In addition, CD8βLO cells become a prominent subset during late chronic stage, with a concomitant decrease in CD8αβHI T cells. Moreover, CD8αα T cells are activated and exhibit a central memory phenotype along with downregulation of exhaustion markers by FACS analysis, and yet have significantly reduced cytokine expression. These results are consistent with single cell RNA sequencing data obtained at the peak and late chronic time points. Thus, CD8αα T cells constitute a phenotypically distinct class of CD8 T cell subsets, with a blunted effector role in demyelinating disease outcome.

Proinflammatory CD8+ T‐Cells Contribute to Age‐Related Glucose Intolerance

Previously, we have found that T Cells play a role in age-related glucose intolerance. However, the specific T cell subtype that causes this impairment is unknown. Therefore, the purpose of this study was to test the hypothesis that 1) CD8+ T cells that accumulate in metabolically active tissues of old mice exhibit a proinflammatory phenotype 2) depletion of CD8+ T-cells will improve glucose tolerance in old mice. This study was approved by the institutional animal care and use committee at the University of Texas at Arlington. First, we assessed CD8+ cell infiltration and phenotype in epididymal white adipose tissue (eWAT) and skeletal muscle from young (4-6mo; n=9) and old (22-24mo; n=12) sedentary male mice. CD8+, Interferon-γ (IFN-γ) producing T cells, and Tumor Necrosis Factor-α (TNF-α) producing T cells were assessed by flow cytometry. Data are expressed as mean ± standard error. Group differences were assessed with an independent samples or paired samples T cells depending on experimental design. In eWAT there were significantly more total CD8+ (6232.1±2616.0, 713.5±215.2 cells per gram tissue, p=0.05) and TNF-α producing CD8+ T cells (2296.6±1167.3 cell/g, 206.6±74.7 cells per gram tissue, p=0.04) in the old compared to the young mice. There were no differences between old and young in IFN-γ producing T cells in eWAT. In the gastrocnemius there were no differences in the number of total CD8+ or IFN-γ producing CD8+ cells. However, the proportion of IFN-γ producing T cells (37.2±6.1%, 24.5±4.7% p=0.05) was greater in the old gastrocnemius compared to the young. Numbers of TNF-α producing T cells (268.6±144.5, 33.5±13.2 cells per gram tissue, p=0.07) tended to be greater in the old gastrocnemius compared to the young. To determine whether CD8+ T cells mediate age-related glucose intolerance, an additional cohort of old sedentary male and female mice (22-24mo) were injected (100μg, i.p. every 5 days for 28 days) with either an anti-CD8 (n=9) or control antibody (n=8). Glucose tolerance was assessed by i.p. injection of glucose (2g/kg) and blood glucose was measured over 120min using a handheld glucometer before and after the antibody treatment period. Anti-CD8 treatment significantly blunted the number of CD8+ cells in the eWAT (3420.2±1473.8, 17693.2±4745.8 cells per gram, p=0.03) compared to controls. There was also a lower number of CD8+ cells the quadriceps of the anti-CD8 group (116.0±48.5cell/g, 695.3±82 cells per gram, p<0.001) group compared to controls. There were no alterations in fasting glucose from pretreatment to posttreatment in the anti-CD8 (139.5±9.8, 142.1±7.4mg/dl, p=.40) or control group (128.9±5.8, 139±8.3 mg/dl, p=0.16). There was a significant reduction in the glucose tolerance test area under the curve (AUC) from pretreatment to posttreatment in the anti-CD8 group (30647.5±2282.1, 26077.5±2102.6 arbitrary units, p=0.04), suggesting an improvement in glucose tolerance. There were no alterations in the control group (27055.3±2488.5, 27666.6±2987.9 arbitrary units, p=0.4). In conclusion, these data suggest that increased infiltration of proinflammatory CD8+ cells in metabolically active tissues with age play an important role in age-related impairments in glucose tolerance.

Clinical and diagnostic findings in COVID-19 patients: an original research from SG Moscati Hospital in Taranto Italy.

The coronavirus disease 2019 (COVID-19) pandemic is a worldwide medical challenge due to the scarcity of proper information and remedial resources. The ability to efficiently avoid a further SARS-CoV-2 pandemic will, therefore, depend on understanding several factors which include host immunity, virus behavior, prevention measures, and new therapies. This is a multi-phase observatory study conducted in the SG Moscati Hospital of Taranto in Italy that was converted into COVID-19 Special Care Unit for SARS-Co-V2 risk management. Patients were admitted to the 118 Emergency Pre-Hospital and Emergency Department based on two diagnostic criteria, the nasopharyngeal swab assessed by reverse-transcriptase-polymerase-chain-reaction (RT-PCR) and CT-scan image characterized by ground glass opacity. Patients were divided into four groups, positive-positive (ER-PP), negative-positive (ER-NP), negative-negative (ER-NN) and a group admitted to the ICU (ER-IC). A further control group was added when the T and B lymphocyte subsets were analyzed. Data included gender, age, vital signs, arterial blood gas analysis (ABG), extensive laboratory results with microbiology and bronchoalveolar lavage fluid (BALF) which were analyzed and compared. Fundamental differences were reported among the groups. Males were significantly higher in PP, ICU, and NP groups, from 2 to 4-fold higher than females, while in the NN group, the number of females was mildly higher than males; the PP patients showed a marked alkalotic, hypoxic, hypocapnia ABG profile with hyperventilation at the time of admission; finally, the laboratory and microbiology results showed lymphopenia, fibrinogen, ESR, CRP, and eGFR were markedly anomalous. The total number of CD4+ and CD8+ T cells was dramatically reduced in COVID-19 patients with levels lower than the normal range delimited by 400/μL and 800/μL, respectively, and were negatively correlated with blood inflammatory responses.

Probiotic supplementation in marathonists and its impact on lymphocyte population and function after a marathon: a randomized placebo-controlled double-blind study

Probiotic supplementation arises as playing an immune-stimulatory role. High-intensity and -volume exercise can inhibit immune cell function, which threatens athletic performance and recovery. We hypothesized that 30 days of probiotic supplementation could stabilize the immune system of athletes preventing immune suppression after a marathon race. Twenty-seven male marathonists were double-blinded randomly into probiotic (Bifidobacterium-animalis-subsp.-Lactis (10 × 109) and Lactobacillus-Acidophilus (10 × 109) + 5 g of maltodextrin) and placebo (5 g of maltodextrin) group. They received 30 sachets and supplemented 1 portion/day during 30 days before the race. Blood were collected 30 days before (rest), 1 day before (pre), 1 h after (post) and 5 days after the race (recovery). Both chronic and acute exercise modulated a different T lymphocyte population (CD3+CD4−CD8− T-cells), increasing pre-race, decreasing post and returning to rest values at the recovery. The total number of CD8 T cell and the memory subsets statistically decreased only in the placebo group post-race. Pro-inflammatory cytokine production by stimulated lymphocytes decreased in the probiotic group after the supplementation period. 30 days of probiotic supplementation maintained CD8 T cell and effector memory cell population and played an immunomodulatory role in stimulated lymphocytes. Both, training and marathon modulated a non-classical lymphocyte population regardless of probiotic supplementation.

Abstract 391: Lrg1 blockade modulates the tumor immune microenvironment and improves the efficacy of cancer therapeutics

Abstract Although the use of checkpoint inhibitors and adoptive T cell therapy have been reported in multiple cancers resulting in a plethora of clinical trials, the efficacy of these therapeutic strategies is often limited by the presence of impaired vasculature in the tumour mass. Indeed, in solid tumors, blood vessels are dysfunctional, leaky and poorly perfused, and thwart not only the efficacy of systemically derived drugs but also the extravasation of T cells, promoting an immunosuppressive microenvironment. Leucine-rich alpha-2-glycoprotein 1 (LRG1) is a secreted glycoprotein that is associated with pathogenic neovascularization, autoimmune diseases, chronic inflammation, and is significantly up-regulated in many solid cancers. We have observed that treatment with a LRG1 function-blocking antibody (15C4) significantly limits growth of solid tumors inducing vessel normalization and functionality. In the present study, we aim to investigate if LRG1 blockade enhances an immunostimulatory milieu, making tumors more responsive to current immunotherapies. Murine melanoma cells harbouring the internal influenza nucleoprotein antigen, NP68, were grafted subcutaneously before antigen-specific F5B6 CD8+ T cells were also transferred. Interestingly, the combination with 15C4 promoted a greater reduction in tumor growth rate. Moreover, not only was the number of donor CD8+ T cells significantly higher when delivered with 15C4, but also the total number of CD3+ T cells was increased following combination therapy, suggesting a more robust host-derived lymphocyte trafficking. As blocking LRG1 normalizes the tumor vasculature and enhances T cell extravasation, we then evaluated whether 15C4 could improve delivery and efficacy of an anti-PD1 checkpoint inhibitor. As monotherapies, both 15C4 and anti-PD1 antibodies induced a marked reduction in tumor volume but when the two were combined, a significant additive effect was also observed. Histological analysis revealed that the combination therapy was associated with a significantly higher number of activated CD8+ T cells, together with enhanced expression of Granzyme B. Current studies are investigating whether the observed boost in cytotoxic T cell density and functionality is a direct consequence of vessel normalization or whether LRG1 blockade is able to modulate the tumour immune microenvironment at different levels: for example, FACS analysis of tumour-infiltrating leukocytes revealed a reduction in the percentage of regulatory T cells after dual therapy, while histological analysis suggested an increase in the number of M1 macrophages. These data show that LRG1 blockade orchestrates a shift in the tumour microenvironment from being immune silent to immune active and, therefore, might represent a novel tool to enhance the efficacy of cancer therapies. Citation Format: Carlotta Camilli, Camilla Pilotti, Marie N. O'Connor, David Kallenberg, Markella Alatsatianos, Angharad H. Watson, Julia Ohme, Athina Dritsoula, Ann Ager, Stephen E. Moss, John Greenwood. Lrg1 blockade modulates the tumor immune microenvironment and improves the efficacy of cancer therapeutics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 391.

Aberrant hyperactivation of cytotoxic T-cell as a potential determinant of COVID-19 severity

ObjectivesWe hypothesized that immune response may contribute to progression of coronavirus disease-19 (COVID-19) at the second week of illness. Therefore, we compared cell-mediated immune (CMI) responses between severe and mild COVID-19 cases. MethodsWe examined peripheral blood mononuclear cells of laboratory-confirmed COVID-19 patients from their first and third weeks of illness. Severe pneumonia was defined as an oxygen saturation ≤93% at room air. Expressions of molecules related to T-cell activation and functions were analyzed by flow cytometry. ResultsThe population dynamics of T cells at the first week were not different between the two groups. However, total numbers of CD4+ and CD8+ T cells tended to be lower in the severe group at the third week of illness. Expressions of Ki-67, PD-1, perforin, and granzyme B in CD4+ or CD8+ T cells were significantly higher in the severe group than in the mild group at the third week. In contrast to the mild group, the levels of their expression did not decrease in the severe group. ConclusionsSevere COVID-19 had a higher degree of proliferation, activation, and cytotoxicity of T-cells at the late phase of illness without cytotoxic T-cell contraction, which might contribute to the development of severe COVID-19.

TREM-1 and TREM-2 Expression on CD14+ Cells in Bronchoalveolar Lavage Fluid in Pulmonary Sarcoidosis and Hypersensitivity Pneumonitis in the Context of T Cell Immune Response.

Background Sarcoidosis and hypersensitivity pneumonitis (HP) are immunologically mediated processes caused by hypersensitivity reaction accompanied by similar features including lymphocytic alveolitis and granuloma formation. Recent studies describe the role of TREM receptors in T cell activation, differentiation, and granuloma formation. Alveolar macrophages activation via TREM receptors may be the key factor mediating subsequent immune response. The aim of the study was to analyse TREM-1 and TREM-2 expression to identify further molecular mechanisms participating in the immunopathogenesis of sarcoidosis and HP. Methods Flow cytometry was performed to analyse TREM-1 and TREM-2 expression on CD14+ cells in bronchoalveolar lavage fluid from patients having sarcoidosis or HP and a control group. Results The study proved increased TREM-1 expression on alveolar macrophages in pulmonary sarcoidosis and diminished TREM-1 expression in HP-Sarcoidosis: median: 76.7; HP: median: 29.9; control: median: 53.3, (sarcoidosis versus HP: p < 0.001; sarcoidosis versus control: p < 0.05). TREM-2 expression was increased in both, sarcoidosis and HP-sarcoidosis: median: 34.79; HP: median: 36.00; control: median: 12.98, (sarcoidosis versus control: p < 0.05; HP versus control: p < 0.05). Correlation analysis showed negative correlation between TREM-1 and total number of CD8+ cytotoxic T cells. In sarcoidosis TREM-1 expression decreased with changes of HRCT image, decrease in CD4/CD8 ratio and decrease in DLCO. Conclusions Differences in TREM receptor expression in sarcoidosis (increase in TREM-1 and TREM-2) and HP (increase in TREM-2) and correlation analysis suggests that activation via TREM may participate in typical immunological characteristics of sarcoidosis and HP.

Memory CD8 T cell responses are impaired during Zika virus infection in mice

Abstract Nearly half the world’s population is at risk for infection by Zika virus (ZIKV), the causative agent of congenital Zika syndrome in infants and Guillain-Barre syndrome in adults. ZIKV vaccination strategies are currently focused on neutralizing antibody (Ab) generation, yet recent evidence suggests that Abs may contribute to pathogenesis under certain conditions. In contrast, increasing evidence supports a key role for T cells in protecting against ZIKV. We have begun to evaluate the generation of memory T cell responses to ZIKV infection in unmanipulated immunocompetent mice. Four-week-old wild-type C57BL/6 mice were infected with ZIKV for 30 days, followed by challenge with ZIKV and evaluation of memory CD4 and CD8 T cell responses at day 3 after secondary ZIKV infection. As expected, total numbers of CD4 and CD8 T cells expanded during secondary ZIKV challenge when compared to primary ZIKV responses at 7 days post-infection. However, only antigen-experienced CD4 T cells (CD11a+CD49d+) and not antigen-experienced CD8 T cells (CD11a+CD8lo) were similarly expanded in secondary infection. These CD8 T cell responses were comprised of short-lived effector cells and not memory precursors, indicating that CD8 T cells memory responses are impaired after primary ZIKV infection. These data suggest potential mechanisms of immune-evasion by ZIKV, and ongoing studies are focused on defining the mechanisms by which ZIKV inhibits the generation of memory CD8 T cell responses. Our findings should offer new strategies for developing ZIKV vaccines that enhance protective T cell responses and avoid pathogenic Ab responses.

T lymphocyte subpopulations and intestinal helminthes profile among tuberculosis patients co-infected with HIV before and after anti tubercular treatment at University of Gondar Hospital, Northwest Ethiopia

BackgroundTuberculosis continues to be a health problem of both developed and developing countries, and its incidence has currently increased due to HIV induced immune suppression. HIV-co-infection decreases the total number of CD4+ T cells since the virus preferentially replicates with in activated CD4+ T cells and macrophages, resulting in the disruption of granuloma to contain M. tuberculosis. In this study, we investigated the change in T lymphocyte subpopulations before and after anti-tubercular treatment and the effect of intestinal parasites on the cell populations of tuberculosis patients before the initiation of anti TB treatment.MethodA prospective cohort study was conducted in the outpatient TB Clinic, University of Gondar hospital between January 2014 and August 2015. Blood samples were collected from 80 newly diagnosed TB patients with and without HIV co-infection. The mean CD4+ and CD8+ T lymphocyte counts of the patients were assessed before and after the course of anti-TB treatment. The mean values of T lymphocytes of TB, TB/HIV co-infected patients and of the control groups were compared. Data was analyzed by SPSS version 16 and the graph pad prism software.ResultsA total of 80 tuberculosis patients 40 of whom were co-infected with HIV participated in our study. The mean CD4 + T lymphocytes counts of the TB/HIV cohort were 354.45 ± 138cell/μl, and the mean CD8+ cell counts were 926.82 ± 384cell/μl. There were significant changes in the mean CD4+ and CD8+ T cell counts after the course of anti-TB treatment in both groups of patients(p < 0.05). However, no statistically significant differences were observed in the mean CD4 + and CD8+ T cell counts of helminthes infected and non-infected patients (P > 0.05).ConclusionWe found significantly lower CD4+ T cell counts among TB infected HIV negative patients compared with controls who showed that TB was the cause of non–HIV-associated declination of circulating CD4 counts, and the reduction was reversible with anti-tubercular treatment in both HIV-negative and ART naïve TB-HIV co-infected patients.

Microbiota Suppresses Innate Type 2 Immune Responses in the lungs in Mice

Microbiota has been known to promote generation of regulatory T cells and prevent development of autoimmune diseases. However, our knowledge is limited regarding the role for microbiota in allergic airway diseases.The goal of this project was to address this question directly by using a mouse model. C57BL/6 mice were housed in a germ-free (GF) condition or specific pathogen-free (SPF) condition during pregnancy and after birth. The steady-state immune status was analyzed, and innate type 2 immune responses in the lungs were compared by exposing them intranasally (i.n.) to extract of fungus Alternaria. At steady-state, the total numbers of CD3+T cells and B220+B cells in the lungs were lower in GF mice as compared to SPF mice. On the other hand, plasma levels of total IgE and IgG1 were elevated in GF mice as compared to SPF mice. When naïve mice were exposed i.n, to Alternaria extract, GF mice produced significantly higher amounts of type 2 cytokines in the lungs. No differences were observed in Alternaria-induced IL-33 release or IL-33-induced innate type 2 response in two conditions. In contrast, lung levels of IL-10 were significantly lower at steady-state and after Alternaria exposure in GF mice as compared to SPF mice. Microbiota affects the steady-state levels of lung lymphocytes and suppresses IgE antibody production and innate type 2 responses to inhaled allergen(s). Lung IL-10, but not IL-33, is likely involved in regulating these responses and protecting animals from developing allergic airway diseases.

Chemotherapy markedly reduces B cells but not T cells and NK cells in patients with cancer.

Chemotherapy is still the backbone of systemic treatment in the majority of cancers. However, immunotherapies, especially those based on checkpoint inhibition, are additional therapy options for many. For this, functional T cells are a mandatory requirement. The aim of this prospective study was to investigate the influence of chemotherapy on the cellular immune status of individual patients. Peripheral blood samples of 26 patients with solid malignancies undergoing chemotherapy were analyzed for lymphocyte populations and their subsets in a longitudinal approach. Chemotherapy decreased total B lymphocyte counts [median value (25-75 percentile): before chemotherapy 76/µl (39-160) vs. after chemotherapy 49/µl (24-106); p = 0.001]. Among B cells, specific subsets decreased particularly [naïve B cells (49/µl (21-111) vs. 25/µl (13-56); p = 0.001], memory B cells [3/µl (2-8) vs. 2/µl (1-4); p = 0.001], and class-switched B cells [11/µl (6-20) vs. 6/µl (3-12); p = 0.011]. In contrast, chemotherapy had no influence on the total numbers of CD4 + and CD8 + T lymphocytes or on their subsets (T helper cells 1, 2, and 17 as well as cytotoxic T cells in early, intermediate, late, terminal effector and exhausted status as well as both T-cell types with naïve, center memory, effector memory, activated, or regulatory phenotype). Furthermore, the count of natural killer (NK) lymphocytes showed no significant change before and after chemotherapy. In summary, this study shows a decrease of B lymphocytes during systemic chemotherapy, but no relevant effect on T lymphocytes, NK lymphocytes and their subsets. This could support the idea of an effective additive T-cell-dependent immunotherapy to chemotherapy.

Immunosuppressive effect of hispidulin in allergic contact dermatitis

BackgroundLong-term use of most immunosuppressants to treat allergic contact dermatitis (ACD) generates unavoidable severe side effects, warranting discovery or development of new immunosuppressants with good efficacy and low toxicity is urgently needed to treat this condition. Hispidulin, a flavonoid compound that can be delivered topically due to its favorable skin penetrability properties, has recently been reported to possess anti-inflammatory and immunosuppressive properties. However, no studies have investigated the effect of hispidulin on Th1 cell activities in an ACD setting.MethodsA contact hypersensitivity (CHS) mouse model was designed to simulate human ACD. The immunosuppressive effect of hispidulin was investigated via ear thickness, histologic changes (i.e., edema and spongiosis), and interferon-gamma (IFN-γ) gene expression in 1-fluoro-2,4-dinitrobenzene (DNFB)-sensitized mice. Cytotoxicity, total number of CD4+ T cells, and percentage of IFN-γ-producing CD4+ T cells were also investigated in vitro using isolated CD4+ T cells from murine spleens.ResultsTopically applied hispidulin effectively inhibited ear swelling (as measured by reduction in ear thickness), and reduced spongiosis, IFN-γ gene expression, and the number of infiltrated immune cells. The inhibitory effect of hispidulin was observed within 6 h after the challenge, and the observed effects were similar to those effectuated after dexamethasone administration. Hispidulin at a concentration up to 50 μM also suppressed IFN-γ-producing CD4+ T cells in a dose-dependent manner without inducing cell death, and without a change in total frequencies of CD4+ T cells among different concentration groups.ConclusionThe results of this study, therefore, suggest hispidulin as a novel compound for the treatment of ACD via the suppression of IFN-γ production in Th1 cells.

THE IMMUNOCOMPETENCE OF THE LYMPHOCYTIC POOL OF PERIPHERAL BLOOD IN ACUTE PURULENT LYMPHADENITIS IN CHILDREN

The substantial prevalence of different nosological forms of lymphadenitis in children is due to morpho-functional immaturity and age-related features of the structural components of the lymph node. The lymphatic system in children under 6 years of age is well developed, however, their immune competence is not perfect. Therefore, most often lymphadenitis of neodontogenic origin occurs at this age when the immune response of the organism to a significant number of bacterial and viral stimuli is insufficient for its reliable protection.&#x0D; The purpose of the work was to study the immunocompetence of the lymphocytic pool of peripheral blood in acute purulent lymphadenitis in children with overburden heredity.&#x0D; Materials and methods of research. A clinical examination and evaluation of the expression of surface markers of peripheral blood lymphocytes in 22 children with acute purulent neonotogenic submandibular lymphadenitis aged from 3 to 7 years was performed. The patients were divided into 2 groups. The first group included 17 persons with this disease, and the second one involved 5 children who have been diagnosed with a history of heredity in their families. The appearance of acute lymphadenitis in children was preceded by acute respiratory viral infection.&#x0D; The general clinical methods of examination included an assessment of the state of the somatic status, the oral cavity and the immediate inflammation cell. Incubation of peripheral blood cells was performed with monoclonal antibodies to CD3+, CD4+, CD4/25+, CD8+, CD16+, and a mechanism for incubation with FITS-conjugated goat antibodies to mouse immunoglobulins was used to detect them. The level of expression of surface markers of lymphocytes was determined as a percentage by the method of indirect immunofluorescence. Blood collection was performed at the time of hospitalization and for recovery. Prior to conducting surgical intervention, an ultrasound examination was required.&#x0D; Results of the research and their discussion. It was found out the fact that acute lymphadenitis in children of the second group occurred in the childhood of two grandparents and one grandmother, and later in their daughters, and their 4 sons. In 2 patients (a boy and a girl) it was traced only through the father or mother. In one mother, inflammation is diagnosed twice in the submandibular areas. Her sons had one-sided inflammation of the lymph nodes (right).&#x0D; The ultrasound biolocation, which was conducted with the aim of clarifying the diagnosis, determined the presence of single hypoechoic formations of ordinal form with clear contours of different sizes in all patients. At the time of hospitalization, in the group of children with hereditary imitation in the peripheral blood, it has been determined the fact that a decrease in the total number of CD3+ was in 2,2 times, CD4+ in 1,6 times, CD16+ in 2,5 times, CD4+, CD25+ in 2,1 times, immunoregulatory index in 2,5 times, the number of CD8+ in 1,6 times than in the group of healthy children.&#x0D; This clearly indicates to the presence of more pronounced immunological disorders, namely, in this group of children. At the end of treatment of patients of the second group it was followed a tendency to improve all indexes, however, the number of CD3+, CD4+ and immunoregulatory index values were lower than the control ones in 1,3, 1,4, and 1,6 times, respectively. At that time, the content of CD8+, CD16+, CD4+, CD25+ came close to healthy children.&#x0D; Thus, in acute purulent neodontogenic submandibular lymphadenitis, a decrease in the level of major lymphocyte populations in the immunoregulatory index at the T-suppressor direction of the immune response is observed, as the number of lymphocytes of this type increased by 1,3 times. More significantly, these changes were inherent for a group of children with hereditary imitation, which requires a more careful study of this phenomenon in the corporate participation of clinicians, immunologists, morphologists.

Abstract P4-06-21: MEK inhibitor cobimetinib induces immunogenic cell death and immune-modulatory effects in triple negative breast cancer

Abstract Background Triple-negative breast cancer (TNBC) has been associated with a robust tumor immune infiltrate. Tumor-infiltrating lymphocytes (TILs) in TNBC have been demonstrated a prognostic value. The mitogen-activated protein kinase (MAPK) signaling pathway have been shown to regulate the immune response with the production of immunomodulatory cytokines, such as TNFα, interleukin (IL)-1, IL-10, and IL-12. Clinical studies have shown that the high expression level of Extracellular signal–related kinase (ERK), a member of the MAPK pathway, correlates with shorter survival in TNBC patients. Accordingly, ERK is a potential target for anti-tumor and cancer immunotherapy. In this study, we aimed to investigate a MEK inhibitor cobimetinib and elucidate whether the MEK/ERK pathway is implicated in immunogenic cell death (ICD) and/or other immunomodulatory effects. Methods Mouse TNBC cell line 4T1 was treated with cobimetinib. The cell viability and cell apoptosis were determined by MTT assay and flow cytometric analysis. Cobimeinib-induced damage-associated molecular patterns (DAMPs), such as such as cell-surface translocation of calreticulin (CRT), extracellular release of ATP, and increase in high-mobility group box protein B1 (HMGB1) release from dying tumor cells, were examined by immunoblotting and flow cytometric analysis. Further, the molecular mechanisms involving cobimetinib-induced cell death were examined by Immunoblotting. Last but not least, establishment of 4T1 animal model in immunocompetent and immunodeficient mice was conducted to investigate the efficacy of cobimetinib in restoration of immunosurveillance and cancer metastasis in vivo. Results The results showed that cobimetinib impaired cell proliferation and induced cell apoptosis in a dose-dependent manner in 4T1 cells. Importantly, cobimetinib treated 4T1 cells elicited ICD, by increasing HMGB1 and ATP release, and by promoting membrane exposure of CRT. The molecular mechanisms involved in cobimetinib-induced DAMPs were partially through the downregulation of p-ERK expression. Moreover, cobimetinib induced processing of procaspase-3 and -8 in 4T1 cells, thereby resulting in caspase activation. In addition, 4T1 tumor bearing immunocompetent mice treated with cobimetinib showed reduced size of primary tumors, fewer lung metastases and increased survival. In addition, we noticed that the tumor-suppressive effects of cobimetinib were much stronger in immunocompetent mice than in immunodeficient mice, evident by distinct mice survival in drug-treated mice. Flow cytometric analysis of murine splenocytes also revealed that cobimetinib treatment increased total number of CD8+ T cells, dendritic cell maturation, and suppressed the number of Myeloid-derived suppressor cells (MDSCs) in immunocompetent mice. Conclusions Our findings suggested that the MEK/ERK inhibitor cobimetinib induces ICD in vitro and exerts additional immune-modulatory effects in immunocompetent TNBC mice model. Our study highlights the possibility for the use of cobimetinib as an ICD inducer and immunomodulator for therapeutic intervention in TNBC. Keywords Cobimetinib, immunogenic cell death, MAPK, ERK phosphorylation, immune regulation Citation Format: Liu C-Y, Lau K-Y, Chen J-L, Chu P-Y, Huang C-T, Wang W-L, Lien P-J, Tseng L-M. MEK inhibitor cobimetinib induces immunogenic cell death and immune-modulatory effects in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-21.

The feasibility and efficacy of subcutaneous plerixafor for mobilization of peripheral blood stem cells in allogeneic HLA-identical sibling donors: results of the HOVON-107 study.

ABSTRACTBackgroundPlerixafor (PFX) mobilizes CD34+ cells into circulation by disrupting the CXCR4 binding of the hematopoietic stem cell in its bone marrow niche.Study design and Methodsin the prospective HOVON‐107 study (www.hovon.nl) 23 allogeneic HLA–identical sibling donors received one or two subcutaneous (sc) injections of plerixafor 0.320 mg/kg.The primary endpoint, was defined as feasibility to mobilize a minimum of 2.0 x106 CD34+ cells/kg recipient weight obtained by leukopheresis in at least 90% of the donors.Resultsmedian 3.3 x 106 CD34+ cells/kg (1.9‐6.5) were collected after 1 (n=12) or 2 (n=10) sc injections of PFX. Side effects occurred in 15/23 (65%) donors: most were grade 1‐2; in 5 donors grade 3 and all resolved. All grafts were directly transplanted. Compared to 10 grafts obtained with G‐CSF the number of CD34+ cells was 2.4 fold lower but the percentage of phenotypically most immature CD34+ subset was higher (31% vs 15%). The total number of CD3+ cells in the graft seemed higher after PFX‐mobilization, but CD4/CD 8 ratios, and frequencies of Th2, Th17 and regulatory T‐cells or NK cells were comparable. All patients engrafted and no increase in incidence or severity of acute or chronic graft versus host disease was observed.Conclusionstem cell mobilization with sc PFX 0.320 mg/kg in allogeneic sibling donors is feasible with limited toxicity for donors. 14 allogeneic donors were mobilized with PFX 0.320 mg intravenously according to the same protocol. Due to the limited numbers, these results are in the supplementary section.

Epigallocatechin-3 Gallate Inhibits STAT-1/JAK2/IRF-1/HLA-DR/HLA-B and Reduces CD8 MKG2D Lymphocytes of Alopecia Areata Patients.

Background: Alopecia areata (AA) is associated with Interferon- γ (IFN-γ) mediated T-lymphocyte dysfunction and increased circulating Interleukine-17 (IL-17) levels. Epigallocatechin-3-gallate (EGCG) specifically inhibits IFN-γ pathways and unlike Janus Kinase 1 and 2 (JAK1/JAK2) inhibitors (tofacitinib, ruxolitinib), EGCG is safer, more cost-effective, and is a topically active agent. Our objective is to test the mode of action of EGCG in vitro and ex vivo using HaCat, Jurkat cell lines, and peripheral blood mononuclear cells (PBMCs) of AA patients and healthy controls (HCs), respectively. Methods: distribution of T helper cells (Th1, Th17), and cytotoxic cells (CD8) in PBMCs isolated from 30 AA patients and 30 HCs was investigated by flowcytomterty. In vitro treatment of HaCat and Jurkat cells with 40 μm EGCG for 48 h was performed to measure the level of phosphorylation of signal transducer and activator of transcription protein STAT1, and replicated in ex vivo model using PBMCs of AA patients. Results: Interestingly, 40 μm EGCG is capable of completely inhibiting phosphorylation of STAT1 after 48 h in HaCat and Jurkat cells and ex vivo in PBMCs of AA patients. Based on QPCR data, the action of EGCG on p-STAT1 seems to be mediated via downregulation of the expression of JAK2 but not JAK1 leading to the inhibition of human leukocyte antigens (HLA-DR and HLA-B) expression probably via IRF-1. On the other hand, AA patients have significantly increased levels of Th1, Th17, and CD8 cells and the production of IFN-γ and IL-17 by PBMCs in AA patients was significantly higher compared to HC; p = 0.008 and p = 0.006, respectively. Total numbers of CD8+ cells were not significantly different between treated and untreated samples. However, CD8+ cells with positive Natural killer group 2 member D (NKG2D) transmembrane receptor (CD8+ NKG2D+ subset) was significantly reduced when PBMCs were treated with 20 μm EGCG for 48 h. Conclusion: These results suggest that EGCG has a synergistic action that inhibits expression of HLA-DR and HLA-B molecules via the IFN-γ pathway to maintain immune privilege in HF; also it reduces CD8+ NKG2D+ subset.