CD4 T-cell Subsets Research Articles

The Effect of Intensive Phototherapy Treatment on CD4 and CD8 T-Lymphocyte subsets in Neonatal Jaundice

Introduction: Jaundice is the most common condition that requires medical attention and hospital readmission in neonates. Serum bilirubin levels may rise excessively, which can be cause lifelong neurologic sequelae (kernicterus). Objectives: To investigate the effect of intensive phototherapy on the expression of CD4 and CD8 lymphocyte subsets in neonates with indirect hyperbilirubinemia. Patients and Methods: This is a prospective study included 30 full term neonates admitted to neonatal intensive care unit, Minia university hospital and 20 full term healthy neonates during the period from October 2016 to October 2017. Results: There were no significant differences regarding CD4%, CD8% and CD4/CD8 ratios in neonates with indirect hyperbilirubinemia either before or after phototherapy. After phototherapy there were no significant differences between cases and controls as regarding CD4% and CD4/CD8 ratio while there was a significant decrease in CD% in cases compared to controls (p value 0.03). A significant positive correlation between CD4% and age (r- value 0.39, p-value 0.02) was present in cases. Conclusions: The results demonstrated that in addition to the well-known positive effect of phototherapy on neonatal serum bilirubin level, this treatment have no effect on the function of the immune system in neonates as regards CD4 and CD8 subsets as well as CD4/CD8 ratio .

Re-defining T-Cell Exhaustion: Subset, Function, and Regulation.

Acute viral infection or vaccination generates highly functional memory CD8 T cells following the Ag resolution. In contrast, persistent antigenic stimulation in chronic viral infection and cancer leads to a state of T-cell dysfunction termed T-cell exhaustion. We and other have recently identified a novel subset of exhausted CD8 T cells that act as stem cells for maintaining virus-specific CD8 T cells in a mouse model of chronic lymphocytic choriomeningitis virus infection. This stem cell-like CD8 T-cell subset has been also observed in both mouse and human tumor models. Most importantly, in both chronic viral infection and tumor models, the proliferative burst of Ag-specific CD8 T cells driven by PD-1-directed immunotherapy comes exclusively from this stem cell-like CD8 T-cell subset. Therefore, a better understanding of the mechanisms how CD8 T-cell subsets are regulated during chronic viral infection and cancer is required to improve the current immunotherapies that restore the function of exhausted CD8 T cells. In this review, we discuss the differentiation of virus-specific CD8 T cells during chronic viral infection, the characteristics and function of CD8 T-cell subsets, and the therapeutic intervention of PD-1-directed immunotherapy in cancer.

Despite early antiretroviral therapy effector memory and follicular helper CD4 T cells are major reservoirs in visceral lymphoid tissues of SIV-infected macaques

Whereas antiretroviral therapy (ART) suppresses viral replication, ART discontinuation results in viral rebound, indicating the presence of viral reservoirs (VRs) established within lymphoid tissues. Herein, by sorting CD4 T-cell subsets from the spleen, mesenteric and peripheral lymph nodes (LNs) of SIVmac251-infected rhesus macaques (RMs), we demonstrate that effector memory (TEM) and follicular helper (TFH) CD4+ T cells harbor the highest frequency of viral DNA and RNA, as well of early R-U5 transcripts in ART-naïve RMs. Furthermore, our results highlight that these two CD4 T cells subsets harbor viral DNA and early R-U5 transcripts in the spleen and mesenteric LNs (but not in peripheral LN) of RMs treated with ART at day 4 post infection suggesting that these two anatomical sites are important for viral persistence. Finally, after ART interruption, we demonstrate the rapid and, compared to peripheral LNs, earlier seeding of SIV in spleen and mesenteric LNs, thereby emphasizing the importance of these two anatomical sites for viral replication dynamics. Altogether our results advance understanding of early viral seeding in which visceral lymphoid tissues are crucial in maintaining TEM and TFH VRs.

Single-Antibody Detection of T-Cell Receptor Beta Chain Monotypia Resolves Uncertainties in the Identification and Quantitation of Sézary Cells By Routine Flow Cytometry: Towards Accurate and Unequivocal Blood Staging of Cutaneous T-Cell Lymphomas

BACKGROUND: Sézary syndrome and mycosis fungoides are two clinically distinct neoplasias of CD4-positive skin-resident T-cells that share remarkable morphologic and immunophenotypic similarities, commonly referred together as cutaneous T-cell lymphoma (CTCL). Prognostic clinical staging in CTCL requires flow cytometric quantitation of peripheral blood tumor cells (Sézary cells), based on aberrant loss of CD7 and/or CD26 within the CD4-positive T-cell compartment. Unfortunately, similar T-cell populations are frequently encountered in reactive conditions, creating uncertainty in the diagnostic interpretation. We recently reported a flow cytometric strategy to assess T-cell clonality using a single antibody (JOVI-1) against one of 2 mutually exclusive T-cell receptor beta constant (TRBC) regions randomly selected during T-cell receptor gene rearrangement. We hereby applied this strategy to a diagnostic Sézary cell flow cytometry panel, resulting in rapid, accurate and unequivocal identification and quantitation of clonal CD4-positive T-cells. METHODS: We studied 33 peripheral blood specimens from 24 patients with CTCL, and 28 specimens from patients with no clinical or laboratory evidence of T-cell malignancy. A routine Sézary cell flow cytometry panel (CD2/CD3/CD4/CD5/CD7/CD8/CD26/CD45) was modified to include a fluorescent labeled antibody against TRBC1. Gated CD4 T-cells were studied to identify discrete subsets lacking CD7, CD26 or both, in addition to other immunophenotypic abnormalities. Within each subset, clonality was defined as a dominant TRBC1-positive or TRBC1-negative population greater than 85%, or a dominant abnormal TRBC1-dim population, as previously described. RESULTS: Peripheral blood specimens from patients with CTCL showed subsets of CD4-positive T-cells lacking CD7 (median=35%; range 1.6% to 96%) or CD26 expression (median=54%; range 6.6% to 98%). Fifteen (45%) of these specimens had other identifiable phenotypic abnormalities, including decreased CD2, CD3, CD4 or CD5. Patients without a T-cell malignancy also had CD4-positive T-cells lacking CD7 (median=13%; range 0.5% to 38%) or CD26 expression (median=9.4%; range 0.7% to 30%), albeit to a lesser extent compared to CTCL patients (p<0.01 and p<0.0001 for CD7 and CD26, respectively). One febrile infant showed loss of CD2 on a small T-cell subset. Discrete CD4-positive T-cell subsets with monotypic TRBC expression were identified in 24 specimens (73%) from patients with CTCL (Figure A), ranging from 30 to12,000 cells/µL (median=632). Importantly, no TRBC-restricted CD4-positive T-cells were detected in any of the 28 patients without a T-cell malignancy. Nine specimens from CTCL patients without a TRBC-restricted subset were reviewed based on morphologic, immunophenotypic and clinical features, yielding no definitive evidence of Sézary cells. Absolute numbers of TRBC-restricted T-cells correlated with numbers of Sézary cells estimated based on lack of CD7 or CD26 expression (R=0.99), or absence of both antigens (R=0.7) (Figure B). However, all cases with no detectable clonal TRBC-restricted T-cells showed significant absolute numbers of CD4-positive T-cells lacking CD7 or CD26 (mean=208 cells/µL, range=22 to 1,078 cells/µL), resulting in common diagnostic uncertainties based on quantitation of these subsets. A narrower gating strategy on these same patients based on the combined loss of CD7 and CD26 yielded lower numbers of abnormal cells (mean=51 cells/µL, range=2 to 556 cells/µL), but grossly underestimated the number of TRBC-restricted CD4-positive T-cells by more than 500 cells/µL in 6 patients with CTCL (Figure B). CONCLUSIONS: A single anti-TRBC1 antibody added to a routine Sézary cell flow cytometry assay provides a rapid, simple and low-cost approach to query for clonality within immunophenotypically distinct CD4 T-cell subsets, eliminating the need for a separate T-cell clonality assay. Diagnostic uncertainties and quantitative biases resulting from immunophenotypic overlaps between Sézary cells and reactive T-cells are virtually resolved. Direct quantitation of clonal CD4 T-cells based on TRBC1 restriction is a biologically sound strategy for blood staging in CTCL, effectively overcoming the limitations and uncertainties of staging by immunophenotypic analysis based on current guidelines. Figure Disclosures Horna: MorphoSys AG: Research Funding.

PD-1 Expression and Function of T-Cell Subsets in TILs From Human Lung Cancer.

On the basis of the autologous tumor-infiltrating lymphocytes (TILs) or genetically modified TILs for adoptive cell therapy have received more attention. Programmed cell death protein 1 (PD-1) expression on the T cells exert complex response during the tumor immune response. But the composition and function of PD-1T-cell subsets in TILs from human lung cancer still limited. In blood and TILs from human lung cancer patients, we confirmed that PD-1 is expressed in higher levels in CD4T-cell subsets than in CD8T-cell subsets. To further analyze the function of PD-1T cells in TILs, we observed the cytokine production in different T-cell subsets. We found that higher interferon-γ and granzyme B production in CD4/CD8PD-1T-cell subsets in TILs than in peripheral blood mononuclear cells (PBMCs); except for PD-1Tscm, higher tumor necrosis factor-α production was observed in PD-1T-cell subsets in TILs than in PBMCs; the expression level of interleukin-17 were lower in PD-1T cells in TILs than in PBMCs; and perforin expression was significantly reduced in CD4PD-1T cells subsets in TILs compared with peripheral blood. Clarify elucidating the composition and function of PD-1T-cell subsets in TILs will have great value in clinical application for evaluating the sensitivity to PD-1 blockade and selecting the promising candidate T-cell subsets in TILs for combination immunotherapy in human lung cancer.

Recombinant Human Interleukin-15 and Anti-PD-L1 Combination Therapy Expands a CXCR3+PD1-/low CD8 T-Cell Subset in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

The PD1/PD-L1 pathway contributes to the pathogenesis of human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection, and blockade of this pathway may have potential to restore immune function and promote viral control or elimination. In this study, we combined a checkpoint inhibitor anti-PD-L1 (Avelumab) and recombinant human interleukin-15 (rhIL-15) in SIV-infected rhesus macaques (RM). The rhIL-15 was administered as continuous infusion in 2 cycles of 10 days in the context of weekly administration of anti-PD-L1 (Avelumab) in SIV-infected RM receiving combination antiretroviral therapy (cART). Safety, immunological parameters, and viral loads were monitored during the study. Administration of rhIL-15/anti-PD-L1 was safe and well tolerated. Treatment resulted in transient increases in proliferating (Ki67+) natural killer and CD8 T cells. In addition, treatment expanded a CXCR3+PD1-/low CD8 T-cell subset with the ability to secrete cytokines. Despite these effects, no changes in plasma viremia were observed after cART interruption. Expansion of the CXCR3+PD1-/low CD8 T-cell subset with functional capacity and potential to traffic to sites of viral reservoirs in SIV-infected rhesus macaques had no demonstrable effect on plasma viremia after cART interruption.

Antibody-suppressor CD8+ T Cells Require CXCR5.

We previously reported the novel activity of alloprimed CD8 T cells that suppress posttransplant alloantibody production. The purpose of the study is to investigate the expression and role of CXCR5 on antibody-suppressor CD8 T-cell function. C57BL/6 mice were transplanted with FVB/N hepatocytes. Alloprimed CD8 T cells were retrieved on day 7 from hepatocyte transplant recipients. Unsorted or flow-sorted (CXCR5CXCR3 and CXCR3CXCR5) alloprimed CD8 T-cell subsets were analyzed for in vitro cytotoxicity and capacity to inhibit in vivo alloantibody production following adoptive transfer into C57BL/6 or high alloantibody-producing CD8 knock out (KO) hepatocyte transplant recipients. Alloantibody titer was assessed in CD8 KO mice reconstituted with naive CD8 T cells retrieved from C57BL/6, CXCR5 KO, or CXCR3 KO mice. Antibody suppression by ovalbumin (OVA)-primed monoclonal OVA-specific t-cell receptor transgenic CD8+ T cells (OT-I) CXCR5 or CXCR3 CD8 T-cell subsets was also investigated. Alloprimed CXCR5CXCR3CD8 T cells mediated in vitro cytotoxicity of alloprimed "self" B cells, while CXCR3CXCR5CD8 T cells did not. Only flow-sorted alloprimed CXCR5CXCR3CD8 T cells (not flow-sorted alloprimed CXCR3CXCR5CD8 T cells) suppressed alloantibody production and enhanced graft survival when transferred into transplant recipients. Unlike CD8 T cells from wild-type or CXCR3 KO mice, CD8 T cells from CXCR5 KO mice do not develop alloantibody-suppressor function. Similarly, only flow-sorted CXCR5CXCR3 (and not CXCR3CXCR5) OVA-primed OT-I CD8 T cells mediated in vivo suppression of anti-OVA antibody production. These data support the conclusion that expression of CXCR5 by antigen-primed CD8 T cells is critical for the function of antibody-suppressor CD8 T cells.

Lymph Node Innate Lymphoid Cells (ILCs) in HIV Infected Children are Highly Activated Despite Sustained Depletion in Blood

Innate lymphoid cells (ILCs) are important for response to infection and immune development in early life. HIV infection in adults has a profound effect on ILCs, but the impact from birth is unknown. Here we studied vertically HIV infected children from birth, and found severe and persistent depletion of all circulating ILCs. In contrast to CD4 T-cells, circulating ILCs are not restored by long-term antiretroviral therapy, but could be rescued by treatment from birth. Remaining helper ILCs in blood were transcriptionally silent, whilst NK cells display limited regulation of genes associated with functional impairment, and CD4 T-cells show strong transcriptional activity. In stark contrast to blood, lymphoid (tonsil) tissue ILCs from infected children had a robust and coordinated transcriptional response to infection, while CD4 T-cell subsets were transcriptionally silent. These data suggest an important and on-going role for ILCs in lymphoid tissue of HIV infected children that requires further investigation.

Tuberculosis Antigen-Specific T-Cell Responses During the First 6 Months of Antiretroviral Treatment.

The reconstitution of Mycobacterium tuberculosis antigen-specific CD4 T cells in a cohort of HIV-infected persons starting antiretroviral treatment (ART) in a high tuberculosis endemic area is described. Restoration of the antigen-specific CD4 T-cell subsets mirrored the overall CD4 T-cell compartment. Activation (assessed by HLA-DR expression) decreased during ART but remained elevated compared to HIV-uninfected persons. Despite known M. tuberculosis sensitization determined by interferon-γ release assay, 12/23 participants had no M. tuberculosis-specific CD4 T cells detectable by flow cytometry, combined with overall elevated T-cell activation and memory differentiation, suggesting heightened turnover. Our data suggest early ART initiation to maintain polyfunctional immune memory responses.

Effect of Intensive Phototherapy Treatment of Neonatal Jaundice on CD4 and CD8 T-Lymphocyte subsets

Effect of Intensive Phototherapy Treatment of Neonatal Jaundice on CD4 and CD8 T-Lymphocyte subsets

Effect of homeostatic T-cell proliferation in the vaccine responsiveness against influenza in elderly people

BackgroundSeasonal influenza virus infection is a significant cause of morbimortality in the elderly. However, there is poor vaccine efficacy in this population due to immunosenescence. We aimed to explore several homeostatic parameters in the elderly that could impact influenza vaccine responsiveness.MethodsSubjects (> 60 years old) who were vaccinated against influenza virus were included, and the vaccine response was measured by a haemagglutination inhibition (HAI) test. At baseline, peripheral CD4 and CD8 T-cells were phenotypically characterized. Thymic function and the levels of different inflammation-related biomarkers, including Lipopolysaccharide Binding Protein (LBP) and anti-cytomegalovirus (CMV) IgG antibodies, were also measured.ResultsInfluenza vaccine non-responders showed a tendency of higher frequency of regulatory T-cells (Tregs) before vaccination than responders (1.49 [1.08–1.85] vs. 1.12 [0.94–1.63], respectively, p = 0.061), as well as higher expression of the proliferation marker Ki67 in Tregs and different CD4 and CD8 T-cell maturational subsets. The levels of inflammation-related biomarkers correlated with the frequencies of different proliferating T-cell subsets and with thymic function (e.g., thymic function with D-dimers, r = − 0.442, p = 0.001).ConclusionsAge-related homeostatic dysregulation involving the proliferation of CD4 and CD8 T-cell subsets, including Tregs, was related to a limited responsiveness to influenza vaccination and a higher inflammatory status in a cohort of elderly people.

The transcription factor NFIL3 controls regulatory T-cell function and stability

Regulatory T (Treg) cells are a CD4 T-cell subset with an important role in immune tolerance; however, the mechanisms underlying Treg cell differentiation and function are incompletely understood. Here, we show that NFIL3/E4BP4, a transcription factor, plays a key role in Treg cell differentiation and function. Microarray analysis showed that Treg cells had lower Nfil3 expression than all other CD4 T-cell subsets. Overexpression of Nfil3 in Treg cells led to diminished expression of Foxp3 and other signature Treg genes, including Il2ra, Icos, Tnfrsf18, and Ctla4. Furthermore, Nfil3-overexpressing Treg cells exhibited impaired immunosuppressive activity in vitro and in vivo. We discovered that NFIL3 directly binds to and negatively regulates the expression of Foxp3. In addition, bisulfite sequencing revealed that NFIL3 induces methylation at Foxp3 locus regulatory CpG sites, which contributes to the control of Treg cell stability. Together, these data indicate that NFIL3 impairs Treg cell function through the downregulation of Foxp3 expression.

Limited HIV-2 reservoirs in central-memory CD4 T-cells associated to CXCR6 co-receptor expression in attenuated HIV-2 infection.

The low pathogenicity and replicative potential of HIV-2 are still poorly understood. We investigated whether HIV-2 reservoirs might follow the peculiar distribution reported in models of attenuated HIV-1/SIV infections, i.e. limited infection of central-memory CD4 T lymphocytes (TCM). Antiretroviral-naive HIV-2 infected individuals from the ANRS-CO5 (12 non-progressors, 2 progressors) were prospectively included. Peripheral blood mononuclear cells (PBMCs) were sorted into monocytes and resting CD4 T-cell subsets (naive [TN], central- [TCM], transitional- [TTM] and effector-memory [TEM]). Reactivation of HIV-2 was tested in 30-day cultures of CD8-depleted PBMCs. HIV-2 DNA was quantified by real-time PCR. Cell surface markers, co-receptors and restriction factors were analyzed by flow-cytometry and multiplex transcriptomic study.HIV-2 DNA was undetectable in monocytes from all individuals and was quantifiable in TTM from 4 individuals (median: 2.25 log10 copies/106 cells [IQR: 1.99–2.94]) but in TCM from only 1 individual (1.75 log10 copies/106 cells). HIV-2 DNA levels in PBMCs (median: 1.94 log10 copies/106 PBMC [IQR = 1.53–2.13]) positively correlated with those in TTM (r = 0.66, p = 0.01) but not TCM. HIV-2 reactivation was observed in the cells from only 3 individuals. The CCR5 co-receptor was distributed similarly in cell populations from individuals and donors. TCM had a lower expression of CXCR6 transcripts (p = 0.002) than TTM confirmed by FACS analysis, and a higher expression of TRIM5 transcripts (p = 0.004). Thus the low HIV-2 reservoirs differ from HIV-1 reservoirs by the lack of monocytic infection and a limited infection of TCM associated to a lower expression of a potential alternative HIV-2 co-receptor, CXCR6 and a higher expression of a restriction factor, TRIM5. These findings shed new light on the low pathogenicity of HIV-2 infection suggesting mechanisms close to those reported in other models of attenuated HIV/SIV infection models.

Inflammatory Cytokines and T-Lymphocyte Subsets in Serum and Sputum in Patients with Bronchial Asthma and Chronic Obstructive Pulmonary Disease.

BackgroundIt can be difficult to distinguish between bronchial asthma and chronic obstructive pulmonary disease (COPD) clinically, although these conditions are associated with different profiles of inflammatory cytokines and immune cells. This study aimed to compare T-lymphocyte subsets and inflammatory cytokines in the serum and sputum of patients with bronchial asthma and COPD who had respiratory function testing.Material/MethodsThe study included 42 patients with bronchial asthma, 48 patients with COPD, and 45 patients with bronchial asthma complicated with COPD. The percentage predicted values of the forced expiratory volume in one second (FEV1), the forced vital capacity (FVC), and the peak expiratory flow (PEF) rate were measured. Serum and sputum levels of interleukin (IL)-4, IL-5, IL-9, IL-13, IL-1β, IL-6 and tumor necrosis factor-α (TNF-α) were measured using an enzyme-linked immunosorbent assay (ELISA). Flow cytometry measured the CD4 and CD8 T-lymphocyte subsets, and the CD4: CD8 ratio was calculated.ResultsThe FEV1, FVC, and PEF were significantly lower in patients with COPD compared with the other two patient groups. Serum and sputum levels of IL-4, IL-5, IL-9 and IL-13 were significantly increased in the COPD patient group, and levels of TNF-α, IL-1β and IL-6 were significantly increased in the bronchial asthma patient group. The CD4: CD8 ratio in sputum was lowest in bronchial asthma patient group and highest in COPD patient group.ConclusionsThe detection of serum and sputum inflammatory cytokines and T-lymphocyte subsets may distinguish between bronchial asthma and COPD.

Sustained treatment-free remission in chronic myeloid leukaemia is associated with an increased frequency of innate CD8(+) T-cells.

Immunological mechanisms of treatment-free remission (TFR) in chronic myeloid leukaemia (CML) are poorly defined and, to date, no correlation between successful TFR and CD8(+) T-cell subsets has been found. We analysed a new identified human subset of CD8(+) T-cells, namely innate CD8(+) T-cells, in CML patients with TFR≥2years. We demonstrated a dramatic increase of functionally active innate CD8(+) T-cells in these patients as compared to control subjects and patients in remission under tyrosine kinase inhibitors. Moreover, we found a positive correlation between frequencies of innate CD8(+) T-cells and natural killer cells, possibly representing a new innate biomarker profile of successful TFR.

Abstract P3-07-08: Multi-omic predictor of rapid and late relapse in primary triple negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease. Clinically, we observe three distinct TNBC outcomes: 1) rapid relapse (rrTNBC) characterized by aggressive drug resistant disease; 2) late relapse (lrTNBC) characterized by indolent or treatment responsive disease; and 3) no relapse (NoRTNBC). We hypothesized that distinct clinical and genomic features of primary tumors define rapid versus late relapse in TNBC. Approach: Using three publicly-available datasets (METABRIC, TCGA, and a prior gene expression meta-analysis), we identified 455 patients diagnosed with primary TNBC with adequate follow-up to be characterized as rrTNBC (relapse or death within 2 years of diagnosis), lrTNBC (relapse or death more than 2 years after diagnosis), or NoRTNBC (no relapse/death with at least 5 years follow-up). We compiled basic clinical (n=455 patients) and primary tumor multi-omic data, including whole transcriptome (n=455), whole genome copy number (n=317), and mutation data for 171 cancer-related genes (n=317). We evaluated intrinsic subtypes (PAM50, TNBCtype), 125 gene expression signatures, CIBERSORT immune subsets, copy number, and mutation frequency. Results: We first evaluated patients with relapse (rrTNBC+lrTNBC) vs. NoRTNBC. There was no significant difference in age, grade, stage at diagnosis, or PAM50 or TNBC subtype proportion between relapse and NoRTNBC. Among 125 expression signatures, five immune signatures were significantly higher in NoRTNBCs (FDR p<0.05) suggesting increased immune activity in patients who do not relapse. Using CIBERSORT inferred immune subsets, anti-tumor CD8 T-cell, M1 macrophage, and gamma-delta T-cell subsets were all highly correlated to these immune signatures (all Pearson's r >= 0.3, all p<1.2e-8). Among genomic features, patients who relapsed were significantly more likely to harbor a mutation in PIK3CA (Fisher exact FDR p=0.02) but there was no significant difference in tumor mutation burden or percent genome altered (Student's t-test p=0.83 and p=0.99, respectively). We then evaluated primary TNBC genomic data in patients who ultimately developed rapid vs. late relapse. Patients with rrTNBC were more likely to be higher stage (p<0.0001) while lrTNBC were more likely to be non-basal PAM50 subtype (p=0.03). Among 11 significantly altered gene expression signatures (FDR p<0.05), 6 estrogen/luminal signatures were significantly higher in lrTNBC. Mutations in DNAH11 and PIK3CA were more common in lrTNBC (Fisher exact FDR p=0.04 and p=0.05, respectively) but there were no significant differences in tumor mutation burden or copy number burden (Student's t-test p=0.13 and p=0.45, respectively). Using 317 cases with full genomic data divided into training and validation datasets, we will report a comparison of machine learning models for predicting relapse versus no relapse and rapid versus late relapse. Conclusions: Primary TNBC tumors destined for rapid, late, or no relapse reflect distinct genomic features. Anti-tumor immune signatures and subsets are enriched in patients who do not relapse yet no difference in mutational or copy number burden. Relative to rapid relapse TNBCs, late relapse TNBCs are enriched for non-basal tumors, estrogen/luminal expression signatures, and mutations in DNAH11 and PIK3CA. Citation Format: Zhang Y, Nock W, Asad S, Adams E, Singh J, Damicis A, Lustberg MB, Noonan A, Reinbolt R, Sardesai S, VanDeusen J, Wesolowski R, Williams N, Ramaswamy B, Stover DG. Multi-omic predictor of rapid and late relapse in primary 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 P3-07-08.

Abstract A186: A new strategy to identify and expand tumor-reactive CD8 TILs in human solid tumors

Abstract The immune system can recognize and destroy tumor cells through T-cell mediated mechanisms. Hence, identifying tumor antigen-specific T-cells from cancer patients and expanding them in large numbers in vitro has important implications for immunotherapy diagnostics and therapeutics. Here we show that tumor-reactive T-cells are enriched in a subset of tumor-infiltrating CD8 T-cells (CD8 TILs) identified by co-expression of CD103 and CD39 both in primary and metastatic tumors. The CD103+CD39+ CD8 TILs are present at high frequencies in melanoma and mismatch repair-deficient colon cancer but at low frequencies in microsatellite stable (MSS) colon cancer and colorectal liver metastasis. This cell population displays a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. Importantly, we show in a MSS colon cancer patient that a very low number of CD103+CD39+ CD8 TILs can be expanded in vitro and that those cells recognize tumor-specific neoantigens. Finally, patients with head and neck cancer whose CD8 TILs contained a higher frequency of CD103+CD39+ cells experienced a greater overall survival. Our work suggests that CD103+CD39+ CD8 TILs are key players in the patient’s antitumor response and describe an approach for detecting and expanding those cells, which will help improve adoptive TIL therapy for cancer patients. Citation Format: Thomas Duhen, Rebekka Duhen, Ryan Montler, Tarsem Moudgil, Jitske van den Bulk, Bernard A. Fox, Shu-Ching Chang, Gary Grunkemeier, Els M.E. Verdegaal, Noel F. de Miranda, Rom Leidner, Richard B. Bell, Andrew D. Weinberg. A new strategy to identify and expand tumor-reactive CD8 TILs in human solid tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A186.

Abstract B200: Single-cell RNA-sequencing (ScRNA-seq) reveals broad heterogeneity among CD8 T-cells during chronic viral infection and identifies a critical role for CD4 help in promoting the differentiation of a potent cytotoxic CD8 T-cell subset

Abstract During chronic viral infection and cancer, CD8 T-cells undergo a differentiation process commonly referred to as T-cell exhaustion. This process is traditionally defined by a stepwise loss of effector functions, eventually leading to cell death. Despite their inability to completely clear the infection, exhausted T-cells are still necessary for limiting viral replication during infection. Thus, it has been proposed that functional adaptation is a more appropriate term for T-cell exhaustion, as CD8 T-cells may be undergoing a multifaceted process of differentiation to better meet the needs of a chronic infection. In line with this hypothesis, it has recently been demonstrated that CD8 T-cells responding to chronic infection are non-homogenous and can be compartmentalized into at least two major subsets, with a TCF-1+ subset serving as a progenitor population that can give rise to a more terminally exhausted TCF-1- subset. However, whether additional heterogeneity exists among CD8 T-cells responding to persistent infection remains unclear. Here, we used ScRNA-seq to fully characterize the heterogeneity of CD8 T-cells during chronic LCMV Cl13 infection. We identified that several transcriptionally distinct subsets of CD8 T-cells develop during chronic LCMV infection, with 3 particular clusters, Slamf6, Pdcd1, and Cx3cr1 cell subsets dominating the antiviral CD8 T-cell response. Importantly both ScRNA-seq and flow cytometric analyses demonstrated that differential expression of cell surface receptors CX3CR1 and Ly108 (encoded by Slamf6) can distinguish these 3 major T-cell subsets. Notably, Ly108 cells shared similar characteristics to the previously described progenitor population and displayed elevated expression of TCF-1. Conversely, CX3CR1 CD8 T-cells displayed increased expression of killer cell lectin-like receptors Klre1 and Klra9, and the TFs T-bet and Zeb2, whereas CX3CR1-Ly108- (DN) cells exhibited elevated expression of multiple co-inhibitory receptors and the TFs Eomes and Nr4a2. Ex vivo functional analyses further indicated that Ly108 CD8 T-cells exhibit an enhanced capacity to co-produce IFN-γ and TNF-α upon GP33 peptide stimulation, whereas CX3CR1 CD8 T-cells display augmented cytotoxicity against peptide-pulsed targeT-cells. Sc trajectory modeling using Monocle analyses predicted that Ly108 CD8 T-cells give rise to both CX3CR1 and DN subsets, with the DN subset branch appearing closer in pseudotime to the Ly108 progenitor subset. To determine the in vivo differentiation trajectory, proliferative potential, and phenotypic stability of these 3 subsets, we performed adoptive transfer experiments using congenically marked CD8 T-cells. Importantly, and consistent with our Monocle predictions, our results demonstrate that Ly108 CD8 T-cells display robust secondary proliferation and give rise to both CX3CR1 and DN subsets. By contrast, CX3CR1 cells retained high CX3CR1 and T-bet expression and did not differentiate into Ly108 or DN CD8 T-cells. Intriguingly, although the DN subset appeared to be the most phenotypically and functionally exhausted subset, more than half of their progeny acquired high CX3CR1 and T-bet expression, indicating that this subset may not be as terminally differentiated as its CX3CR1 counterpart. Notably, our ScRNA-seq analyses also identified that DN cells displayed the highest levels of IL-21R expression, suggesting a potential role for CD4 help in regulating the differentiation of this subset. Strikingly, depletion of CD4 T-cells or deletion of IL-21R signaling in P14 transgenic CD8 T-cells abrogated the development of the CX3CR1 CD8 subset, indicating a critical role for CD4 help in facilitating the differentiation of exhausted CD8 T-cells into a potent cytotoxic CD8 subset. Collectively, our work supports a new model of CD8 T-cell differentiation during chronic viral infection and has important implications for T-cell-based immunotherapies aimed at treating persistent infections and/or cancer. Citation Format: Ryan Zander, David Schauder, Gang Xin, Christine Nguyen, Xiaopeng Wu, Weiguo Cui. Single-cell RNA-sequencing (ScRNA-seq) reveals broad heterogeneity among CD8 T-cells during chronic viral infection and identifies a critical role for CD4 help in promoting the differentiation of a potent cytotoxic CD8 T-cell subset [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B200.

Long-Term Thymic Function and Reconstitution of the T Cell Compartment after T Cell-Replete Haplo-Identical Allografting

INTRODUCTION Post-transplant cyclophosphamide (PTCY) has expanded the application of haploidentical stem cell transplantation (haplo-HSCT). Thymic function may play a pivotal role in long-term clinical outcomes. OBJECTIVES To evaluate the kinetics of long-term immune thymus-dependent reconstitution after PTCY haplo-HSCT. METHODS Twenty-nine patients (median age 53) underwent T-cell replete haplo-HSCT with PTCY. Blood samples were collected before conditioning and at 1, 3, 6, 12, 18, 24 months after transplantation. Analyses of CD4 and CD8 T-cell subsets by flow-cytometry were correlated by generalized linear models with Real-Time PCR quantification of signal joint T-cell receptor excision DNA circles (sjTRECs), specific marker of naive T-cells thymopoiesis. A) Naive; b) central; c) memory; and d) revertant CD4 and CD8 T-cells were defined as follows: a) CD45RA+CD62L+; b) CD45RO+CD62L+; c) CD45RO+CD27-; and d) CD45RA+/45RO+, respectively. SjTRECs real-time PCR was performed on genomic DNA (100 ng) extracted from sorted CD4 and CD8 T-cells. RESULTS Following PTCY induced T-cell depletion, a constant gradual increase in absolute numbers of all CD4 and CD8 T cell subsets and of sjTRECs copies from the first month up to two years post-transplant was observed (Figure 1). Overall, at two years, CD4 and CD8 T-cell levels and sjTRECs levels were however lower than those observed in healthy donors. sjTRECs kinetics was associated with the increase in naive T-cells (overall, p CONCLUSIONS Active thymic function despite age-dependent involution, substantially contributes to T-cell reconstitution after haplo-HSCT. Chronic GVHD and older age are however significantly correlated with lower thymic activity. Overall, lower production of sjTRECs after haplo-HSCT as compared after HLA identical sibling HSCT may partly be due to a higher degree of “mismatching” of MHC molecules during thymic re-education.

Peeking under the Hood of Naive T Cells

Signaling and transcriptional regulation of metabolic reprogramming upon Tcell activation has been studied intensively. In this issue of Cell Metabolism, Ricciardi etal. (2018) show that translational regulation of key metabolic enzymes GLUT1 and ACC1 plays a novel role in human naive CD4 Tcell activation and subset differentiation.