Double-negative T-cell Research Articles

Genome-Wide Analysis Identifies Rag1 and Rag2 as Novel Notch1 Transcriptional Targets in Thymocytes.

Recombination activating genes 1 (Rag1) and Rag2 are expressed in immature lymphocytes and essential for generating the vast repertoire of antigen receptors. Yet, the mechanisms governing the transcription of Rag1 and Rag2 remain to be fully determined, particularly in thymocytes. Combining cDNA microarray and ChIP-seq analysis, we identify Rag1 and Rag2 as novel Notch1 transcriptional targets in acute T-cell lymphoblastic leukemia (T-ALL) cells. We further demonstrate that Notch1 transcriptional complexes directly bind the Rag1 and Rag2 locus in not only T-ALL but also primary double negative (DN) T-cell progenitors. Specifically, dimeric Notch1 transcriptional complexes activate Rag1 and Rag2 through a novel cis-element bearing a sequence-paired site (SPS). In T-ALL and DN cells, dimerization-defective Notch1 causes compromised Rag1 and Rag2 expression; conversely, dimerization-competent Notch1 achieves optimal upregulation of both. Collectively, these results reveal Notch1 dimerization-mediated transcription as one of the mechanisms for activating Rag1 and Rag2 expression in both primary and transformed thymocytes. Our data suggest a new role of Notch1 dimerization in compelling efficient TCRβ rearrangements in DN progenitors during T-cell development.

NKG2D Enhances Double-Negative T Cell Regulation of B Cells.

Numerous studies reported a small subpopulation of TCRαβ+CD4-CD8- (double-negative) T cells that exert regulatory functions in the peripheral lymphocyte population. However, the origin of these double-negative T (DNT) cells is controversial. Some researchers reported that DNT cells originated from the thymus, and others argued that these cells are derived from peripheral immune induction. We report a possible mechanism for the induction of nonregulatory CD4+ T cells to become regulatory double-negative T (iDNT) cells in vitro. We found that immature bone marrow dendritic cells (CD86+MHC-II- DCs), rather than mature DCs (CD86+MHC-II+), induced high levels of iDNT cells. The addition of an anti-MHC-II antibody to the CD86+MHC-II+ DC group significantly increased induction. These iDNT cells promoted B cell apoptosis and inhibited B cell proliferation and plasma cell formation. A subgroup of iDNT cells expressed NKG2D. Compared to NKG2D- iDNT cells, NKG2D+ iDNT cells released more granzyme B to enhance B cell regulation. This enhancement may function via NKG2D ligands expressed on B cells following lipopolysaccharide stimulation. These results demonstrate that MHC-II impedes induction, and iDNT cells may be MHC independent. NKG2D expression on iDNT cells enhanced the regulatory function of these cells. Our findings elucidate one possible mechanism of the induction of peripheral immune tolerance and provide a potential treatment for chronic allograft rejection in the future.

Differential alterations in peripheral lymphocyte subsets in COVID-19 patients: upregulation of double-positive and double-negative T cells.

BackgroundViral infections cause alteration in the total number of lymphocytes and their subset distribution. We aimed to study peripheral blood lymphocyte subsets in COVID-19 patients and to correlate these subsets with clinical and laboratory data, which may help in clarifying the pathogenesis to develop novel diagnostic and prognostic biomarkers for COVID-19.MethodsTwenty-six reverse-transcription polymerase chain reaction (RT-PCR) confirmed COVID-19 patients were subjected to medical history-taking and a thorough clinical examination. Laboratory tests included complete blood count, D dimer, ferritin, and C-reactive protein (CRP). Chest CT was used to diagnose COVID-19 pneumonia. Lymphocyte subsets were compared with those in 20 healthy controls using flow cytometry.ResultsLeucopenia, relative neutrophilia, lymphopenia, eosinopenia together with marked elevation in neutrophil/lymphocyte ratio were observed in our COVID-19 patients. A marked reduction was observed in T cells, including both CD4 and CD8 cells, natural killer (NK), and natural killer T cells (NKT). Double-positive T (DPT) cells, double-negative T (DNT) cells, and B cells were elevated in the patients relative to the other lymphocyte subsets.ConclusionImmune-inflammatory parameters are of utmost importance in understanding the pathogenesis and in the provisional diagnosis of COVID-19. Yet, adequate care must be taken during their interpretation because of the vast discrepancies observed between studies even in the same locality. Further studies are needed to clarify the role of B cells, DPT, and DNT cells in the pathogenesis and control of COVID-19.

Autoimmune Lymphoproliferative Syndrome in Children with Nonmalignant Organomegaly, Chronic Immune Cytopenia, and Newly Diagnosed Lymphoma

This study investigated the frequency of and predictive factors for autoimmune lymphoproliferative syndrome (ALPS) in children with lymphoma, chronic immune cytopenia, and nonmalignant organomegaly. Thirty-four children with suspected ALPS (n=13, lymphoma; n=12, immune cytopenia; n=9, nonmalignant organomegaly) were included. Double-negative T-cells, lymphocyte apoptosis, and genetic findings were analyzed. Patients were stratified into two groups as proven/probable ALPS and clinically suspected patients according to the ALPS diagnostic criteria. Of the 34 patients, 18 (53%) were diagnosed with proven/probable ALPS. One patient had a mutation (c.652-2A>C) in the FAS gene. The remaining 16 (47%) patients were defined as clinically suspected patients. Predictive factors for ALPS were anemia and thrombocytopenia in patients with lymphoma, splenomegaly and lymphadenopathy in patients with immune cytopenia, and young age in patients with nonmalignant organomegaly. ALPS may not be rare in certain risk groups. Our study indicates that screening for ALPS may be useful in children having lymphoma with cytopenia at diagnosis, in those having nonmalignant organomegaly with immune cytopenia, and in those having chronic immune thrombocytopenic purpura or autoimmune hemolytic anemia with organomegaly developing during follow-up.

Next Generation Sequencing for Detecting Somatic FAS Mutations in Patients With Autoimmune Lymphoproliferative Syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a primary immune regulatory disorder clinically defined by chronic and benign lymphoproliferation, autoimmunity and an increased risk of lymphoma due to a genetic defect in the FAS-FASL apoptotic pathway. Genetic defects associated with ALPS are germinal and somatic mutations in FAS gene, in addition to germinal mutations in FASLG, FADD, CASP8 and CASP10 genes. The accumulation of CD3+TCRαβ+CD4-CD8- double negative T-cells (DNT) is a hallmark of the disease and 20-25% of ALPS patients show heterozygous somatic mutations restricted to DNT in the FAS gene (ALPS-sFAS patients). Nowadays, somatic mutations in the FAS gene are detected through Sanger sequencing in isolated DNT. In this study, we report an ALPS-sFAS patient fulfilling clinical and laboratory ALPS criteria, who was diagnosed through NGS with a targeted gene panel using DNA from whole blood. Data analysis was carried out with Torrent Suite Software and variant detection was performed by both germinal and somatic variant caller plugin. The somatic variant caller correctly detected other six ALPS-sFAS patients previously diagnosed in the authors’ laboratories. In summary, this approach allows the detection of both germline and somatic mutations related to ALPS by NGS, avoiding the isolation of DNT as the first step. The reads of the somatic variants could be detected even in patients with DNT in the cut off limit. Thus, custom-designed NGS panel testing may be a faster and more reliable method for the diagnosis of new ALPS patients, including those with somatic FAS mutations (ALPS-sFAS).

CRISPR screen identifies genes that sensitize AML cells to double-negative T-cell therapy

AbstractAcute myeloid leukemia (AML) remains a devastating disease in need of new therapies to improve patient survival. Targeted adoptive T-cell therapies have achieved impressive clinical outcomes in some B-cell leukemias and lymphomas but not in AML. Double-negative T cells (DNTs) effectively kill blast cells from the majority of AML patients and are now being tested in clinical trials. However, AML blasts obtained from ∼30% of patients show resistance to DNT-mediated cytotoxicity; the markers or mechanisms underlying this resistance have not been elucidated. Here, we used a targeted clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) screen to identify genes that cause susceptibility of AML cells to DNT therapy. Inactivation of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitinating complex components sensitized AML cells to DNT-mediated cytotoxicity. In contrast, CD64 inactivation resulted in resistance to DNT-mediated cytotoxicity. Importantly, the level of CD64 expression correlated strongly with the sensitivity of AML cells to DNT treatment. Furthermore, the ectopic expression of CD64 overcame AML resistance to DNTs in vitro and in vivo. Altogether, our data demonstrate the utility of CRISPR/Cas9 screens to uncover mechanisms underlying the sensitivity to DNT therapy and suggest CD64 as a predictive marker for response in AML patients.

γδ T Cells in Merkel Cell Carcinomas Have a Proinflammatory Profile Prognostic of Patient Survival.

Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using multiplex-IHC/immunofluorescence (m-IHC/IF). CD4+ or CD8+ T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double-negative (DN) T-cell infiltrates (>20% DN T cells), and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2- γδ T cells. In the context of γδ T-cell inflammation, these cells expressed PD-1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes, and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene γδ T-cell signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores, and complete responses to anti-PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions.See related Spotlight on p. 600.

Idiopathic splenomegaly in childhood and the spectrum of RAS-associated lymphoproliferative disease: a case report

BackgroundKRAS (KRAS proto-oncogene, GTPase; OMIM: 190,070) encodes one of three small guanosine triphosphatase proteins belonging to the RAS family. This group of proteins is responsible for cell proliferation, differentiation and inhibition of apoptosis. Gain-of-function variants in KRAS are commonly found in human cancers. Non-malignant somatic KRAS variants underlie a subset of RAS-associated autoimmune leukoproliferative disorders (RALD). RALD is characterized by splenomegaly, persistent monocytosis, hypergammaglobulinemia and cytopenia, but can also include autoimmune features and lymphadenopathy. In this report, we describe a non-malignant somatic variant in KRAS with prominent clinical features of massive splenomegaly, thrombocytopenia and lymphopenia.Case presentationA now-11-year-old girl presented in early childhood with easy bruising and bleeding, but had an otherwise unremarkable medical history. After consulting for the first time at 5 years of age, she was discovered to have massive splenomegaly. Clinical follow-up revealed thrombocytopenia, lymphopenia and increased polyclonal immunoglobulins and C-reactive protein. The patient had an unremarkable bone marrow biopsy, flow cytometry showed no indication of expanded double negative T-cells, while malignancy and storage disorders were also excluded. When the patient was 8 years old, whole exome sequencing performed on DNA derived from whole blood revealed a heterozygous gain-of-function variant in KRAS (NM_004985.5:c.37G > T; (p.G13C)). The variant was absent from DNA derived from a buccal swab and was thus determined to be somatic.ConclusionsThis case of idiopathic splenomegaly in childhood due to a somatic variant in KRAS expands our understanding of the clinical spectrum of RAS-associated autoimmune leukoproliferative disorder and emphasizes the value of securing a molecular diagnosis in children with unusual early-onset presentations with a suspected monogenic origin.

Phenotypical and Functional Characterization of Cytotoxic Unconventional T-Cells.

During the last two decades, the immunology field has been focused on the study of conventional T-cells, leading to important advances in identification of specific subsets involved in promoting and suppressing immune response in patients with cancer, autoimmune disease or transplanted patients. In these recent years, the research on unconventional subset of CD4-CD8- double-negative T-cells is growing. DNTs are a unique subset of T-cells characterized by being CD4-CD8-CD3+ and express either ß or α T-cell receptors (TCR). In this chapter, we describe the methods used to phenotypically characterize and isolate these cells in order to study their functional profile.

Double-negative T cells in autoimmune diseases.

TCRαβ+CD4-CD8- double-negative T (DNT) cells, a principal subset of mature T lymphocytes, have been closely linked with autoimmune/inflammatory conditions. However, controversy persists regarding their ontogeny and function. Here, we present an overview on DNT cells in different autoimmune diseases to advance a deeper understanding of the contribution of this population to disease pathogenesis. DNT cells have been characterized in various chronic inflammatory diseases and they have been proposed to display pathogenic or regulatory function. The tissue location of DNT cells and the effector cytokines they produce bespeak to their active involvement in chronic inflammatory diseases. By producing various cytokines, expanded DNT cells in inflamed tissues contribute to the pathogenesis of a variety of autoimmune inflammatory diseases. However, it is unclear whether this population represents a stable lineage consisting of different subsets similar to CD4+ T helper cell subset. Better understanding of the possible heterogeneity and plasticity of DNT cells is needed to reveal interventional therapeutic opportunities.

Oligoclonality and subpopulation structure of bone marrow T-cells in patients with aplastic anaemia

Introduction. The main pathogenetic mechanism of the development of aplastic anemia (AA) is a violation of the immune regulation of hematopoiesis.Aim: to study of the subpopulation composition of T-cells and the repertoire of the T-cell receptor in AA patients.Patients and Methods. The study included AA patients (n = 40) without prior immunosuppressive therapy in 2018–2020. The T-cell subpopulation structure and T-cell receptor Vβ-family (TCR-Vβ) oligoclonality were studied in samples of bone marrow using flow cytometry.Results. We report characteristic properties of T-cell subpopulations of bone marrow in all AA patients: elevated counts of cytotoxic T-cells, effector CD4+ and CD8+ cells, CD4+ memory cells, which may suggest a long-term antigenic stimulation with subsequent activation of these cell subpopulations resulting in hyperexpression of pro-inflammatory cytokines. Diminishing of naive CD4+ and CD8+ cells, regulatory and double negative T-cells may indicate a relaxing control of cytokine-producing T-cells. A relationship has been established between the AA severity and counts of effector, regulatory, double negative and PD-1 positive T-cells. A highest count of potentially cytokine-producing T-cells and lowest count of cells involved in T-cell activity regulation were observed in very severe AA patients. Studies of the TCR-Vβ repertoire revealed oligoclonal expansion in the cytotoxic T-cell subpopulation.Conclusion. Enrichment in selected Vβ families suggests autoreactive T-cell clonality and attests to the immune nature of AA. A dynamic TCR-Vβ repertoire assay may be recommended in the disease monitoring. Flow cytometry helps identify valuable biomarkers for T-cell clone monitoring in AA and a better assessment of the disease progression.

Key diagnostic markers for autoimmune lymphoproliferative syndrome with molecular genetic diagnosis

AbstractAutoimmune lymphoproliferative syndrome (ALPS) is a rare immunodeficiency caused by mutations in genes affecting the extrinsic apoptotic pathway (FAS, FASL, CASP10). This study evaluated the clinical manifestations, laboratory findings, and molecular genetic results of 215 patients referred as possibly having ALPS. Double-negative T-cell (DNT) percentage and in vitro apoptosis functional tests were evaluated by fluorescence-activated cell sorting; interleukin 10 (IL-10) and IL-18 and soluble FAS ligand (sFASL) were measured by enzyme-linked immunosorbent assay. Genetic analysis was performed by next-generation sequencing. Clinical background data were collected from patients' records. Patients were categorized into definite, suspected, or unlikely ALPS groups, and laboratory parameters were compared among these groups. Of 215 patients, 38 met the criteria for definite ALPS and 17 for suspected ALPS. The definite and suspected ALPS patient populations showed higher DNT percentages than unlikely ALPS and had higher rates of lymphoproliferation. Definite ALPS patients had a significantly more abnormal in vitro apoptosis function, with lower annexin, than patients with suspected ALPS (P = .002) and patients not meeting ALPS criteria (P < .001). The combination of elevated DNTs and an abnormal in vitro apoptosis functional test was the most useful in identifying all types of ALPS patients; the combination of an abnormal in vitro apoptosis functional test and elevated sFASLs was a predictive marker for ALPS-FAS group identification. Lymphoproliferation, apoptosis functional test, and DNTs are the most sensitive markers; elevated IL-10 and IL-18 are additional indicators for ALPS. The combination of elevated sFASLs and abnormal apoptosis function was the most valuable prognosticator for patients with FAS mutations.

Differential Host Pro-Inflammatory Response to Mycobacterial Cell Wall Lipids Regulated by the Mce1 Operon

The cell wall of wild-type (WT) Mycobacterium tuberculosis (Mtb), an etiologic agent of tuberculosis (TB) and a Mtb strain disrupted in a 13-gene operon mce1 (Δmce1) varies by more than 400 lipid species. Here, we examined Mtb lipid-induced response in murine macrophage, as well as in human T-cell subpopulations in order to gain an insight into how changes in cell wall lipid composition may modulate host immune response. Relative to WT Mtb cell wall lipids, the non-polar lipid extracts from Δmce1 enhanced the mRNA expression of lipid-sense nuclear receptors TR4 and PPAR-γ and dampened the macrophage expression of genes encoding TNF-α, IL-6, and IL-1β. Relative to untreated control, WT lipid-pre-stimulated macrophages from healthy individuals induced a higher level of CD4−CD8− double negative T-cells (DN T-cells) producing TNF-α. Conversely, compared to WT, stimulation with Δmce1 lipids induced higher mean fluorescence intensity (MFI) in IL-10-producing DN T cells. Mononuclear cells from TB patients stimulated with WT Mtb lipids induced an increased production of TNF-α by CD8+ lymphocytes. Taken together, these observations suggest that changes in mce1 operon expression during a course of infection may serve as a strategy by Mtb to evade the host pro-inflammatory responses.

MON-LB75 Stratifying the Risk of Malignancy in Indeterminate Thyroid Nodules

Background Thyroid cancer is the most common endocrine cancer globally, and accounts for 3.4% of all new cancer cases in the US. Although several clinical guidelines to detect/manage thyroid nodules are available, a great deal of controversy still exists around the optimal approach for diagnosis. Approximately 20-30% of cytology results from thyroid fine-needle aspiration (FNA) fall into one of three indeterminate diagnostic categories. In recent years, a number of molecular and gene mutation diagnostic tests have been developed to diagnose the indeterminate thyroid nodules in FNA specimens. However, nearly half of patients recommended for surgery based on these tests were found to have a benign nodule. Therefore, there is a need for a more accurate predictive and prognostic test for thyroid cancer. In case of Euthyroid Hashimoto Thyroiditis condition (EHT), where patient required a partial thyroxin replacement dose, having about 48% risk of thyroid cancer. However, there is still not a very accurate predictive marker for early detection of thyroid cancer in EHT. Invention DescriptionResearchers at the University of Toledo have found that thyroid nodule microenvironment cell profiling can be used as a predictive and prognostic marker for thyroid cancer. This approach uniquely focuses on the phenotype, rather than the genotype, of the microenvironment. Recently described Double Negative (DN) T-cells were significantly more abundant in lymphocytic infiltrates of thyroid cancer. They were shown to downregulate proliferation and cytokine production of activated effector T cells present in the tumor microenvironment and contribute to tumor tolerance and active avoidance of tumor immunity. If the quantify of DN T cells exceeds the defined threshold, it indicates a likelihood of cancer presence. Aside from immune cell profiling, our approach further establishes an integration of the information derived from transcriptome/meta-analysis of the genome and cytokine/chemokine signal analysis all from thyroid FNA. Applications[[Unsupported Character - Symbol Font •]] Diagnosis of thyroid cancer from FNA samples[[Unsupported Character - Symbol Font •]] Predictive tool of severity of disease Advantages[[Unsupported Character - Symbol Font •]] Microenvironment profiling can provide a unique way to diagnose and assess disease progression[[Unsupported Character - Symbol Font •]] Sheds light on cellular cross-talk; more accurate diagnose; can prevent unnecessary surgery IP Status:​Patent Pending

Abstract B61: High-dimensional analysis of tumor-resident CD4 and CD8 double-negative T-cell subset in multiple tumor types

Abstract Background: Checkpoint blockade therapies like PD-1 antibodies elicit durable long-lasting immunity in a subset of patients of certain tumor types. However, many patients across a range of tumor types are resistant to checkpoint blockade. Mechanisms that confer local response or resistance to checkpoint blockade are not well understood. Mounting evidence points to local suppression of T cell function as one of the most substantial barriers to effective antitumor immunity. Methods: The study involved multiparametric flow cytometry and genomic characterization of a subset of T cells in the tumors and lymphoid organs of syngeneic tumors, genetically engineered mouse model of cancers and human tumors. Results: A significant fraction (10-40%) of α/β TCR+ CD3+ T cells do not express CD4, CD8 or NKp46 in the tumors. The presence of double-negative T-cell subset (DN T cells) is restricted to tumor microenvironment and is relatively scarce in tumor draining lymph nodes and in spleen. To identify if DN T cells are antigen-specific, we performed AH-1 tetramer staining. While a substantial fraction of CD8 T cells are AH-1 tetramer positive, majority of DN T cells are AH-1 tetramer negative in CT26 tumors. Recently, it has been shown that higher frequency of CD39-negative bystander T cells correlated to lack of response to PD-1 therapy in non-small cell lung cancer patients. Mounting evidence also reveals presence of a specific subset of CD8 T cells with features of stem cell memory in tumors as defined by CXCR5 and TCF1 expression. Efforts are under way to understand if DN T cells exhibit bystander cell and/or memory stem-like phenotype. Majority of the DN T cells are proliferating (Ki67+) and express very low levels of PD-1 (in contrast to CD8 T cells) and Granzyme B. Treatment of mice bearing syngeneic tumors with PD-1 checkpoint blockade therapy does not impact the frequency of DN T-cell subset consistent with lower levels of PD-1 expression. Moreover, a substantial frequency (30-40%) of DN T cells in both syngeneic and KPC pancreatic GEMM tumors express CD103, a tissue resident marker. Furthermore, DN T cells are CD44+ CD127+ KLRG1- and may represent a memory precursor cell population. Upon ex vivo stimulation, these DN T cell subsets proliferate and express modest levels of PD-1. Preliminary analysis of single-cell RNA sequencing data from FACS sorted murine DN T cells reveals lack of CD4, CD8 expression and retention of CD2, CD44, CD69, CCR7 and CXCR5 expression. Flow cytometry analysis of human CD2+ TILs reveals presence of DN T cells in human tumors. Single-cell transcriptome analysis of CD2+ T cells isolated from human colorectal tumors revealed presence of DN T cells. Current efforts are focused on further characterization of this subset in both mouse and human tumors. Conclusions: In this study, we describe a tumor-resident CD4 and CD8 double-negative T-cell subset that may represent a bystander cell population with features of memory precursors and potential implications in modulation of response to checkpoint blockade therapy in cancer. Citation Format: Murali Gururajan, Ravi Kandasamy, Jessica Wong, Kalpit Shah, Shuoguo Wang, Adam Bata, Amy Truong, Sunil Kuppasani, Ching-Ping Ho, Robert Graziano, Michael Quigley, Anwar Murtaza, Jinqi Liu. High-dimensional analysis of tumor-resident CD4 and CD8 double-negative T-cell subset in multiple tumor types [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B61.

Use of gastrin vaccine to increase gamma-delta and NKT cells and alter pancreas tumor microenvironment to improve survival.

696 Background: Pancreatic cancer (PC) has been called a “cold” tumor as it responds poorly to immune-based therapies. Strategies to render PC susceptible to immunotherapy are under investigation by methods that alter immune cell signatures such as increasing the population of double negative CD4-CD8-T-cells, or changing the polarization of tumor-associated macrophages (TAMs). Our current aim was to determine if a gastrin vaccine, Polyclonal Antibody Stimulator (PAS) influences double negative T-cell phenotype and polarization of TAMs to improve survival of PC. Methods: Two cohorts of C57BL/6 mice were injected either sc or orthotopically with syngeneic mT3 murine pancreatic cancer cells. After 1 week, groups were treated with PBS; PAS (100μg); PD-1 antibody (150μg); or the combination of PAS and PD-1 Ab. PAS was given ip at weeks 0, 1 and 3. Anti-PD-1 was given on days 0, 4, 8, 15 and 21. Spleens were collected from the sc experiment for T-cell surface analysis by flow cytometry. Orthotopic tumors were measured for growth rate, metastases, and stained for M1 (inos) and M2 (arginase) polarized TAMs, and mouse survival was analyzed. Results: PAS therapy increased expression of double negative T-cells. The percentage of gamma-delta T-cells in the (CD3+/CD4−/CD8−/CD44−/CD62L−) TEMRA subpopulation of mice treated with PAS or combinations of PAS with PD-1 Ab were increased approximately 40% compared to PBS-treated controls or PD-1 Ab-treated mice. NKT cells from PAS-vaccinated mice were 2.5-fold higher than controls. M2+ TAMS were significantly decreased in tumors of PAS treated mice compared to PBS treated mice (p = 0.017). PAS monotherapy resulted in a nearly 10-fold increase in M1 TAMs relative to controls (p = 0.002). PAS monotherapy decreased tumor metastases by 69% and prolonged survival. Primary tumor growth decreased with PAS in combination with PD-1 Ab (p=0.0025). Conclusions: PAS decreases PC tumor growth and metastases by increasing CD4-CD8-, gamma-delta, and NKT T-cell populations. Furthermore, PAS monotherapy significantly increases M1 and decreases M2 TAMs. These immune cell changes with PAS vaccination may render tumors more susceptible to other cancer therapies and improve survival.

The Co-inhibitor BTLA Is Functional in ANCA-Associated Vasculitis and Suppresses Th17 Cells.

Objectives: The activation and inhibition of T-cells has been well-studied under physiological conditions. Co-inhibition is an important mechanism to keep effector T-cells in check. Co-inhibitors mediate peripheral self-tolerance and limit the immune response. Dysfunctional co-inhibition is associated with loss of T-cell regulation and induction of autoimmunity. Therefore, we investigated the co-inhibitor B- and T-Lymphocyte attenuator (BTLA) in ANCA-associated vasculitis (AAV).Methods: Fifty-six AAV patients and 32 healthy controls (HC) were recruited. Flow cytometry was performed to investigate the expression of BTLA on T-cells. Double negative T-cells were defined as CD3+CD4−CD8−. To assess the functionality of BTLA, CFSE-labeled T-cells were stimulated in presence or absence of an agonistic anti-BTLA antibody. In addition, impact of BTLA-mediated co-inhibition on Th17 cells was studied.Results: AAV patients in remission had a decreased expression of BTLA on double negative T-cells (CD3+CD4−CD8−). On all other subtypes of T-cells, expression of BTLA was comparable to healthy controls. TCR-independent stimulation of T-cells resulted in down-regulation of BTLA on Th cells in AAV and HC, being significantly lower in HC. Co-inhibition via BTLA led to suppression of T-cell proliferation in AAV as well as in HC. As a result of BTLA mediated co-inhibition, Th17 cells were suppressed to the same extent in AAV and HC.Conclusion: BTLA expression is altered on double negative T-cells but not on other T-cell subsets in quiescent AAV. BTLA-induced co-inhibition has the capacity to suppress Th17 cells and is functional in AAV. Thus, BTLA-mediated co-inhibition might be exploited for future targeted therapies in AAV.

Double-negative T Cells Inhibit Proliferation and Invasion of Human Pancreatic Cancer Cells in Co-culture.

Double-negative T (DNT) cells are phenotypically CD3+CD4-CD8-T cells. This study aimed to investigate the anti-cancer activity of DNT cells against pancreatic cancer cells. DNT cells were isolated from human peripheral blood. The effect of DNT cells on proliferation and invasion of the human pancreatic cell line Panc-1 was assessed. Expression of Nrf2 and Fas in Panc-1 cells co-cultured with DNT cells was analyzed with RT-PCR. The supernatants of Panc-1 and DNT co-cultures were analyzed with ELISA for IFN-r and FasL levels. The isolated DNT cell phenotype was CD4-CD8-CD56- CD3+TCR (T cell receptor) α/β+ T cells with more than 90% purity. Panc-1 cell proliferation was significantly inhibited by co-culture with DNT cells. Panc-1 cells co-cultured with DNT cells showed significantly reduced cell invasion. Panc-1 cells co-cultured with DNT cells showed increased Nrf2 and Fas mRNA expression. Increased INF-r and FasL levels were detected in the supernatants of co-cultures of DNT and pancreatic cells. DNT cells inhibited proliferation and invasion of human pancreatic cancer cells. The INF-r, Fas/FasL pathway and Nrf2 may be involved in the anti-cancer effect of DNT cells against human pancreatic cancer.

Double negative T cells mediate Lag3-dependent antigen-specific protection in allergic asthma

Allergic asthma is an inflammatory disorder of the airway without satisfactory traditional therapies capable of controlling the underlying pathology. New approaches that can overcome the detrimental effects of immune dysregulation are thus desirable. Here we adoptively transfer ovalbumin (OVA) peptide-primed CD4−CD8− double negative T (DNT) cells intravenously into a mouse model of OVA-induced allergic asthma to find that OVA-induced airway hyperresponsiveness, lung inflammation, mucus production and OVA-specific IgG/IgE production are significantly suppressed. The immunosuppressive function of the OVA-specific DNT cells is dependent on the inhibition of CD11b+ dendritic cell function, T follicular helper cell proliferation, and IL-21 production. Mechanistically, Lag3 contributes to MHC-II antigen recognition and trogocytosis, thereby modulating the antigen-specific immune regulation by DNT cells. The effectiveness of ex vivo-generated allergen-specific DNT cells in alleviating airway inflammation thus supports the potential utilization of DNT cell-based therapy for the treatment of allergic asthma.

Unique T-Cell Populations Define Immune-Inflamed Hepatocellular Carcinoma.

Background & AimsThe characterization of T cells infiltrating hepatocellular carcinoma (HCC) provides information on cancer immunity and also on selection of patients with precise indication of immunotherapy. The aim of the study was to characterize T-cell populations within tumor tissue and compare them with non-neoplastic liver tissue as well as circulating cells of the same patients.MethodsThe presence of unique cell populations was investigated in 36 HCC patients by multidimensional flow cytometry followed by t-distributed stochastic neighbor embedding analysis. Functional activity of tumor-infiltrating T cells was determined after activation by phorbol 12-myristate 13-acetate and ionomycin.ResultsWithin the tumor there were more cells expressing CD137 and ICOS than in non-neoplastic liver tissue, possibly after recent antigenic activation. These cells contained several populations, including the following: (1) functionally impaired, proliferating CD4+ cells co-expressing Inducible T-cell costimulator (ICOS) and T cell immunoreceptor with Ig and ITIM domains (TIGIT); (2) functionally active CD8+ cells co-expressing CD38 and Programmed cell-death protein 1 (PD1); and (3) CD4-CD8 double-negative T-cell receptor αβ and γδ cells (both non–major histocompatibility complex–restricted T cells). When the identified clusters were compared with histologic classification performed on the same samples, an accumulation of activated T cells was observed in immune-inflamed HCC. The same analyses performed in 7 patients receiving nivolumab treatment showed a remarkable reduction in the functionally impaired CD4+ cells, which returned to almost normal activity over time.ConclusionsUnique populations of activated T cells are present in HCC tissue, whose antigen specificity remains to be investigated. Some of these cell populations are functionally impaired and nivolumab treatment restores their responsiveness. The finding of ongoing immune response within the tumor shows which lymphocyte populations are impaired within the HCC and identifies the patients who might take benefit from immunotherapy.