Lack Of CD4 Research Articles

CD4+ T cell help during early acute hepacivirus infection is critical for viral clearance and the generation of a liver-homing CD103+CD49a+ effector CD8+ T cell subset.

In hepatitis C virus (HCV) infection, CD4+ and CD8+ T cells are crucial for viral control. However, a detailed understanding of the kinetic of CD4+ T cell help and its role in the generation of different CD8+ T cell subsets during acute infection is lacking. The absence of a small HCV animal model has impeded mechanistic studies of hepatic antiviral T cell immunity and HCV vaccine development. In this study, we used a recently developed HCV-related rodent hepacivirus infection mouse model to investigate the impact of CD4+ T cell help on the hepatic CD8+ T cell response and viral clearance during hepacivirus infection in vivo. Our results revealed a specific kinetic of CD4+ T cell dependency during acute infection. Early CD4+ T cell help was essential for CD8+ T cell priming and viral clearance, while CD4+ T cells became dispensable during later stages of acute infection. Effector CD8+ T cells directly mediated timely hepacivirus clearance. An analysis of hepatic CD8+ T cells specific for two different viral epitopes revealed the induction of subsets of liver-homing CD103+CD49a+ and CD103-CD49a+ effector CD8+ T cells with elevated IFN-γ and TNF-α production. CD103+CD49a+ T cells further persisted as tissue-resident memory subsets. A lack of CD4+ T cell help and CD40L-CD40 interactions resulted in reduced effector functions and phenotypical changes in effector CD8+ T cells and a specific loss of the CD103+CD49a+ subset. In summary, our study shows that early CD4+ T cell help through CD40L signaling is essential for priming functional effector CD8+ T cell subsets, including unique liver-homing subsets, and hepacivirus clearance.

Galectin 7 leads to a relative reduction in CD4+ T cells, mediated by PD-1

The role of glycan-binding proteins as an activator of immune regulatory receptors has gained attention recently. We report that galectin 7 reduced CD4+ T cell percentage in both in vitro culture and mouse tumor models. Immunohistochemical staining of esophageal cancer patient samples showed a lower percentage of CD4+ cells in the galectin 7 high area. The lack of CD4+ T cell depletion by galectin 7 in PD-1 knockout mice supports the role of PD-1 in mediating the effects of galectin 7. The binding assays demonstrate that galectin 7 binds to the N-glycosylation of PD-1 on N74 and N116 sites and leads to the recruitment of SHP-2. NFAT suppressive activity of galectin 7 was abrogated upon overexpression of the dominant negative SHP-2 mutant or inhibition of PD-1 by siRNA. Glycosylation of PD-1 has been reported to play a critical role in surface expression, stability, and interaction with its ligand PD-L1. This report further expands the significance of PD-1 glycosylation and suggests that galectin 7, a glycan-binding protein, interacts with the immune regulatory receptor PD-1 through glycosylation recognition.

Pumilio1 regulates NPM3/NPM1 axis to promote PD-L1-mediated immune escape in gastric cancer

Abnormal regulation of RNA binding proteins (RBPs) plays an essential role in tumorigenesis and progression, but their functions and mechanisms remain largely elusive. Previously, we reported that Pumilio 1 (PUM1), a RBP, could regulate glycolysis metabolism and promote the progression of gastric cancer (GC). However, the role of PUM1 in tumor immune regulation remains largely elusive. In this study, we report that PUM1 induces immune escape through posttranscriptional regulation of PD-L1 in GC. We used multiplexed immunohistochemistry to analyze the correlation between PUM1 expression and immune microenvironment in GC. The effect of PUM1 deficiency on tumor killing of T cells was examined in vitro and in vivo. The molecular mechanism of PUM1 was evaluated via RNA immunoprecipitation, chromatin immunoprecipitation, Western blot, co-immunoprecipitation, and RNA stability assays. Clinically, elevated PUM1 expression is associated with high-expression of PD-L1, lack of CD8+ T cell infiltration and poor prognosis in GC patients. PUM1 positively regulates PD-L1 expression and PUM1 reduction enhances T cell killing of tumors. Mechanistically, PUM1 directly binds to nucleophosmin/nucleoplasmin 3 (NPM3) mRNA and stabilizes NPM3. NPM3 interacts with NPM1 to promote NPM1 translocation into the nucleus and increase the transcription of PD-L1. PUM1 inhibits the anti-tumor activity of T cells through the PUM1/NPM3/PD-L1 axis. In summary, this study reveals the critical post-transcriptional effect of PUM1 in the modulation of PD-L1-dependent GC immune escape, thus provides a novel indicator and potential therapeutic target for cancer immunotherapy.

Ineffective polarization characterizes CD4 T cell responses to persistent Borrelia burgdorferiinfection

Abstract Borrelia burgdorferi (Bb), the spirochetal agent of Lyme disease, evades immune clearance in infected mice and humans, suggesting ongoing immune suppression. Here we explored the functionality of Bb-induced CD4 T cell responses in infected mice, which are considered Th1 polarized. Surprisingly, in vitro restimulation of CD4 T cells from mice infected for 12 days failed to induce secretion of the hallmark Th1 cytokine IFNg. Consistent with that data, flow cytometry and RNAseq of CD4 T cells from lymph nodes (LNs) of Bb-infected mice at 6, 10, 14, and 21 days post infection (dpi) showed a transient Th1 and Th17 polarization that peaked at 10 dpi before declining, confirmed also by RT-PCR. While Th1 and Th17 gene expression profiles returned to baseline by 14 dpi, increased frequencies of CXCR4+ CXCR5+ PD-1 hiICOS+ BCL6+ T follicular helper (Tfh) cells emerged at that time, concomitant with germinal center induction. The lack of CD4 T cell effector polarization was noted also in the skin, an effector site after Bb infection, as well as in antigen-specific T cells, which were identified using a new MHCII tetramer to the Bb surface antigen arthritis related protein (Arp). Together, our results indicate a failure to sustain effector CD4 T cell polarization after Bb infection, identifying CD4 T cells as a potential target of Bb-mediated immune suppression. This work was supported by grants from the Global Lyme Alliance, the Stephen & Alexander Cohen Foundation, NIH R01AI157007 (NB) and T32 AI060555 (EMH). This work was supported by grants from the Global Lyme Alliance, the Stephen & Alexander Cohen Foundation, NIH R01AI157007 (NB) and T32 AI060555 (EMH).

Mutations in SARS-CoV-2 spike protein impair epitope-specific CD4+ T cell recognition.

CD4+ T cells are essential for protection against viruses, including SARS-CoV-2. The sensitivity of CD4+ T cells to mutations in SARS-CoV-2 variants of concern (VOCs) is poorly understood. Here, we isolated 159 SARS-CoV-2-specific CD4+ T cell clones from healthcare workers previously infected with wild-type SARS-CoV-2 (D614G) and defined 21 epitopes in spike, membrane and nucleoprotein. Lack of CD4+ T cell cross-reactivity between SARS-CoV-2 and endemic beta-coronaviruses suggested these responses arose from naïve rather than pre-existing cross-reactive coronavirus-specific T cells. Of the 17 epitopes located in the spike protein, 10 were mutated in VOCs and CD4+ T cell clone recognition of 7 of them was impaired, including 3 of the 4 epitopes mutated in omicron. Our results indicated that broad targeting of epitopes by CD4+ T cells likely limits evasion by current VOCs. However, continued genomic surveillance is vital to identify new mutations able to evade CD4+ T cell immunity.

EQTL analysis of laying hens divergently selected for feather pecking identifies KLF14 as a potential key regulator for this behavioral disorder.

Feather pecking in chickens is a damaging behavior, seriously impacting animal welfare and leading to economic losses. Feather pecking is a complex trait, which is partly under genetic control. Different hypotheses have been proposed to explain the etiology of feather pecking and notably, several studies have identified similarities between feather pecking and human mental disorders such as obsessive-compulsive disorder and schizophrenia. This study uses transcriptomic and phenotypic data from 167 chickens to map expression quantitative trait loci and to identify regulatory genes with a significant effect on this behavioral disorder using an association weight matrix approach. From 70 of the analyzed differentially expressed genes, 11,790 genome wide significantly associated variants were detected, of which 23 showed multiple associations (≥15). These were located in proximity to a number of genes, which are transcription regulators involved in chromatin binding, nucleic acid metabolism, protein translation and putative regulatory RNAs. The association weight matrix identified 36 genes and the two transcription factors: SP6 (synonym: KLF14) and ENSGALG00000042129 (synonym: CHTOP) as the most significant, with an enrichment of KLF14 binding sites being detectable in 40 differentially expressed genes. This indicates that differential expression between animals showing high and low levels of feather pecking was significantly associated with a genetic variant in proximity to KLF14. This multiallelic variant was located 652 bp downstream of KLF14 and is a deletion of 1-3 bp. We propose that a deletion downstream of the transcription factor KLF14 has a negative impact on the level of T cells in the developing brain of high feather pecking chickens, which leads to developmental and behavioral abnormalities. The lack of CD4 T cells and gamma-Aminobutyric acid (GABA) receptors are important factors for the increased propensity of laying hens to perform feather pecking. As such, KLF14 is a clear candidate regulator for the expression of genes involved in the pathogenic development. By further elucidating the regulatory pathways involved in feather pecking we hope to take significant steps forward in explaining and understanding other mental disorders, not just in chickens.

Cancer Vaccine in Cold Tumors: Clinical Landscape, Challenges, and Opportunities.

The idea of cancer immunotherapy is to stimulate the immune system to fight tumors without destroying normal cells. One of the anticancer therapy methods, among many, is based on the use of cancer vaccines that contain tumor antigens in order to induce immune responses against tumors. However, clinical trials have shown that the use of such vaccines as monotherapy is ineffective in many cases since they do not cause a strong immune response. Particular tumors are resistant to immunotherapy due to the absence or insufficient infiltration of tumors with CD8+ T cells, and hence, they are called cold or non-inflamed tumors. Cold tumors are characterized by a lack of CD8+ T cell infiltration, the presence of anti-inflammatory myeloid cells, tumor-associated M2 macrophages, and regulatory T cells. It is very important to determine the stage of the antitumor response that does not work properly in order to use the right strategy. Applying other therapeutic methods alongside cancer vaccines can be more rational for cold tumors, which do not provoke the immune system strongly. Herein, we indicate some combinational therapies that have been used or are in progress for cold tumor treatment alongside vaccines.

Abstract 5208: Circulating T cell: B cell: NK cell axis associated with response to pembrolizumab plus doxorubicin in patients with metastatic triple negative breast cancer

Abstract Introduction: The use of immune checkpoint inhibitors (ICI) in combination with chemotherapy is now standard of care for patients with programmed death ligand 1-positive (PD-L1+) metastatic triple negative breast cancer (mTNBC). However, the mechanisms of response to ICIs are still poorly understood in patients with mTNBC. Here we investigate immune correlates of response in patients with mTNBC treated with programmed cell death protein 1 (PD-1) receptor-targeting pembrolizumab and doxorubicin (n=9). Methods: Patients received pembrolizumab and doxorubicin every 3 weeks for a total of 6 cycles followed by pembrolizumab maintenance until disease progression. Objective response rates (ORR per RECIST 1.1) were: N=1 complete response (CR), N=4 partial responses (PR), N=3 stable disease (SD), and N=1 progression of disease (PD). Baseline tumor biopsies were collected for PD-L1 (22C3 antibody) and TILs analysis, and peripheral blood was collected for immune correlatives. Circulating peripheral blood mononuclear cells (PBMCs) were assessed by high parameter flow cytometry with samples collected at timepoints cycle 1 day 1 (C1D1), cycle 2 day 1 (C2D1), and cycle 3 day 1 (C3D1) Results: No association between response and tumor infiltrating lymphocyte (TIL) scoring or PD-L1 expression were observed. Circulating exhausted CD8+ T cells (PD-1 high CD39+) were identified at baseline in all patients, with the patient with PD having a low frequency of exhausted CD8+ T cells (0.08% in PD vs. 0.3% mean in CR/PR/SD) at baseline. In the patient with PD, the following results were observed relative to other patients: high levels of antibody-secreting B cells (ASC, 15.2% in PD vs. 1.5% mean in CR/PR/SD), CD4+ follicular helper T cells (Tfh, 8.47% PD vs. 2.6% mean in CR/PR/SD), and terminally differentiated NK cells (11.0% PD vs. 6.3% mean CR/PR/SD) at baseline. From baseline to C2D1, the patient who achieved CR demonstrated robust expansion of exhausted CD8+ T cells (4.4-fold change in CR vs. 1.4-fold change in PR/SD/PD). Conclusion: Our data suggests that both baseline immune profile ‘setpoints’ and dynamic remodeling of immune features, including expansion of exhausted CD8+ T cells, are associated with response to ICIs in patients with mTNBC. Lack of response to ICIs is defined not only by a lack of CD8+ T cell expansion, but also by high levels of circulating ASCs, Tfh, and terminal NK cells at baseline. Additional studies to further explore and validate features of this T cell: B cell: NK cell axis and response to ICIs in patients with mTNBC are ongoing. Citation Format: Colt A. Egelston, Weihua Guo, Peter P. Lee, Susan E. Yost, James R. Waisman, Yuan Yuan. Circulating T cell: B cell: NK cell axis associated with response to pembrolizumab plus doxorubicin in patients with metastatic triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5208.

Staphylococcus aureus specific lung resident memory CD4+ Th1 cells attenuate the severity of influenza virus induced secondary bacterial pneumonia

Staphylococcus aureus is a major cause of severe pulmonary infections. The evolution of multi-drug resistant strains limits antibiotic treatment options. To date, all candidate vaccines tested have failed, highlighting the need for an increased understanding of the immunological requirements for effective S. aureus immunity. Using an S. aureus strain engineered to express a trackable CD4+ T cell epitope and a murine model of S. aureus pneumonia, we show strategies that lodge Th1 polarised bacterium specific CD4+ tissue resident memory T cells (Trm) in the lung can significantly attenuate the severity of S. aureus pneumonia. This contrasts natural infection of mice that fails to lodge CD4+ Trm cells along the respiratory tract or provide protection against re-infection, despite initially generating Th17 bacterium specific CD4+ T cell responses. Interestingly, lack of CD4+ Trm formation after natural infection in mice appears to be reflected in humans, where the frequency of S. aureus reactive CD4+ Trm cells in lung tissue is also low. Our findings reveal the protective capacity of S. aureus specific respiratory tract CD4+ Th1 polarised Trm cells and highlight the potential for targeting these cells in vaccines that aim to prevent the development of S. aureus pneumonia.

Progranulin mediates immune evasion of pancreatic ductal adenocarcinoma through regulation of MHCI expression

Immune evasion is indispensable for cancer initiation and progression, although its underlying mechanisms in pancreatic ductal adenocarcinoma (PDAC) are not fully known. Here, we characterize the function of tumor-derived PGRN in promoting immune evasion in primary PDAC. Tumor- but not macrophage-derived PGRN is associated with poor overall survival in PDAC. Multiplex immunohistochemistry shows low MHC class I (MHCI) expression and lack of CD8+ T cell infiltration in PGRN-high tumors. Inhibition of PGRN abrogates autophagy-dependent MHCI degradation and restores MHCI expression on PDAC cells. Antibody-based blockade of PGRN in a PDAC mouse model remarkably decelerates tumor initiation and progression. Notably, tumors expressing LCMV-gp33 as a model antigen are sensitized to gp33-TCR transgenic T cell-mediated cytotoxicity upon PGRN blockade. Overall, our study shows a crucial function of tumor-derived PGRN in regulating immunogenicity of primary PDAC.

Single-cell transcriptome analysis revealed a suppressive tumor immune microenvironment in EGFR mutant lung adenocarcinoma

BackgroundsImmunotherapy is less effective in patients with epidermal growth factor receptor (EGFR) mutant non-small-cell lung cancer (NSCLC). Lower programmed cell death-ligand 1 (PD-L1) expression and tumor mutation burden (TMB) are...

Lack of CD8+ T cell effector differentiation during priming mediates checkpoint blockade resistance in non-small cell lung cancer.

In non–small cell lung cancer (NSCLC), response to immune checkpoint blockade (ICB) is associated with programmed cell death ligand 1 expression that is induced by interferon-γ–producing, tumor-infiltrating CD8+ T cells. However, not all tumors with a CD8+ T cell infiltrate respond to ICB, and little is known about the mechanisms governing ICB resistance in T cell–infiltrated NSCLC. We used an orthotopic NSCLC mouse model to study ICB-refractory CD8+ T cell responses. Single-cell RNA sequencing of the NSCLC mouse tumors revealed that lung cancer–specific tumor-infiltrating CD8+ T cells exhibited clonal expansion but lacked expression of genes associated with effector and exhausted T cell responses, indicating that they underwent a differentiation program distinct from conventional T cell exhaustion. This lung cancer–specific T cell dysfunction program was established early during priming in the mediastinal lymph node and was characterized by robust proliferation but a failed up-regulation of effector and exhausted T cell characteristics. Intriguingly, CD8+ T cells from patients with NSCLC expressed an analogous gene expression program, which appeared distinct from conventional T cell exhaustion. Administration of recombinant interleukin-2 (IL-2) and IL-12 was sufficient to restore effector T cell differentiation and induce control of KP lung tumors. These findings imply that a CD8+ T cell differentiation trajectory, activated during T cell priming in the mediastinal lymph node, limits the response of CD8+ T cells to ICB and thereby may contribute to failure of ICB in a subset T cell–infiltrated NSCLC.

A novel mouse model for liver metastasis of prostate cancer reveals dynamic tumour‐immune cell communication

ObjectivesIn contrast to extensive studies on bone metastasis in advanced prostate cancer (PCa), liver metastasis has been under‐researched so far. In order to decipher molecular and cellular mechanisms underpinning liver metastasis of advanced PCa, we develop a rapid and immune sufficient mouse model for liver metastasis of PCa via orthotopic injection of organoids from PbCre+; rb1f/f;p53f/f mice.Materials and MethodsPbCre+;rb1f/f;p53f/f and PbCre+;ptenf/f;p53f/f mice were used to generate PCa organoid cultures in vitro. Immune sufficient liver metastasis models were established via orthotopic transplantation of organoids into the prostate of C57BL/6 mice. Immunofluorescent and immunohistochemical staining were performed to characterize the lineage profile in primary tumour and organoid‐derived tumour (ODT). The growth of niche‐labelling reporter infected ODT can be visualized by bioluminescent imaging system. Immune cells that communicated with tumour cells in the liver metastatic niche were determined by flow cytometry.ResultsA PCa liver metastasis model with full penetrance is established in immune‐intact mouse. This model reconstitutes the histological and lineage features of original tumours and reveals dynamic tumour‐immune cell communication in liver metastatic foci. Our results suggest that a lack of CD8+ T cell and an enrichment of CD163+ M2‐like macrophage as well as PD1+CD4+ T cell contribute to an immuno‐suppressive microenvironment of PCa liver metastasis.ConclusionsOur model can be served as a reliable tool for analysis of the molecular pathogenesis and tumour‐immune cell crosstalk in liver metastasis of PCa, and might be used as a valuable in vivo model for therapy development.

Helpless Priming Sends CD8+ T Cells on the Road to Exhaustion.

Persistent antigen exposure in chronic infection and cancer has been proposed to lead to cytotoxic T lymphocyte (CTL) “exhaustion”, i.e., loss of effector function and disease control. Recent work identifies a population of poorly differentiated TCF-1+PD-1+ CD8+ T cells as precursors of the terminally exhausted CTL pool. These “predysfunctional” CTLs are suggested to respond to PD-1 targeted therapy by giving rise to a pool of functional CTLs. Supported by gene expression analyses, we present a model in which lack of CD4+ T cell help during CD8+ T cell priming results in the formation of predysfunctional CTLs. Our model implies that predysfunctional CTLs are formed during priming and that the remedy for CTL dysfunction is to provide “help” signals for generation of optimal CTL effectors. We substantiate that this may be achieved by engaging CD4+ T cells in new CD8+ T cell priming, or by combined PD-1 blocking and CD27 agonism with available immunotherapeutic antibodies.

Abstract 3276: Molecular heterogeneity of anti-PD-1/PD-L1 immunotherapy efficacy is correlated with tumor immune microenvironment in non-small cell lung cancer from East-Asian patients

Abstract Background: Anti-PD-1/PD-L1 immunotherapy is widely used to treat patients with advanced non-small-cell lung cancer (NSCLC). Although PD-1/PD-L1 inhibitor efficacy across NSCLC is now better understood, it is largely unknown how the tumor immune microenvironment differs regarding tumor genomics and its impact on prognosis and response to immunotherapy in East-Asian NSCLC patients. Methods: We performed an integrated analysis on publicly available data to explore associations between anti-PD-1/PD-L1 immunotherapy efficacy and classic driver oncogene mutations, including EGFR, KRAS, and ALK fusion mutations, in East-Asian NSCLC patients. Four pooled and clinical cohort analyses were used to correlate driver oncogene mutation status and tumor microenvironment based on PD-L1 and CD8+ tumor-infiltrating lymphocytes (TILs). Immune infiltrating patterns were also established for genomic NSCLC subgroups by CIBERSORT algorithm. Results: Based on East-Asian NSCLC patients, TIDE analysis revealed that for anti-PD-1/PD-L1 immunotherapy, EGFR-mutant and ALK-rearranged tumors may yield inferior responses; however, KRAS-mutant tumors may respond better. Pooled and clinical cohort analyses demonstrated tumor immune microenvironment heterogeneity with oncogenic addiction. Data showed remarkably higher PD-L1- and TIL-positive KRAS-mutant tumors, suggesting KRAS mutations may drive an inflammatory phenotype with adaptive immune resistance. However, the EGFR-mutant or ALK-rearranged groups showed a remarkably higher proportion of PD-L1−/TIL− tumors, suggesting an uninflamed phenotype with immunological ignorance. Notably, similar to triple wild-type NSCLC, EGFR L858R-mutant tumors positively correlated with an inflammatory phenotype, suggesting responsiveness to anti-PD-1/PD-L1 immunotherapy. Furthermore, CIBERSORT algorithm results revealed that apart from enriched CD8+ T cells, KRAS-mutant NSCLC showed a similar intratumoral immune profile to triple wild-type NSCLC. Strikingly, EGFR-mutant and ALK-rearranged tumors were characterized by enriched resting memory CD4+ T cells, as well as a lack of CD8+ and activated memory CD4+ T cells. Conclusion: Our study highlights the complex relationship between immune heterogeneity and immunotherapeutic response in East-Asian NSCLC patients regarding oncogenic addiction. Importantly, our findings provide support for individualized treatments according to NSCLC molecular subtype. Keywords: NSCLC, East-Asian, oncogene mutations, PD-1/PD-L1 inhibitors, immune landscape Citation Format: Chengming Liu, Runsen Jin, Sufei Zheng, Xinfeng Wang, Nan Sun, Jie He. Molecular heterogeneity of anti-PD-1/PD-L1 immunotherapy efficacy is correlated with tumor immune microenvironment in non-small cell lung cancer from East-Asian patients [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 3276.

Impact of immunophenotypic characteristics on genetic subgrouping in childhood acute lymphoblastic leukemia: Tokyo Children's Cancer Study Group (TCCSG) study L04-16.

Immunophenotyping was performed in 1044 consecutive childhood acute lymphoblastic leukemia (ALL) patients enrolled in the Tokyo Children's Cancer Study Group L04-16 trial, revealing novel findings associated with genetic abnormalities. In addition to TCF3-PBX1 and MEF2D fusions, the CD10(+) subtype of KMT2A-MLLT3-positive ALL frequently exhibited the cytoplasmic-μ(+) pre-B ALL immunophenotype. Although ETV6-RUNX1 was significantly correlated with myeloid antigen expression, more than half of patients expressed neither CD33 nor CD13, while the CD27(+) /CD44(-) immunophenotype was maintained. Expression of CD117 and CD56 in B-cell precursor-ALL was limited to certain subtypes including ETV6-RUNX1 and KMT2A-MLLT3. Besides BCR-ABL1, CRLF2, hyperdiploidy, and hypodiploidy, CD66c was also expressed in Ph-like kinase fusion-, PAX5 fusion-, and DUX4 fusion-positive ALL, but not in MEF2D fusion-positive ALL, indicating constant selectivity of CD66c expression. In T-ALL, SIL-TAL1-positive patients were likely to exhibit a more mature immunophenotype. Expression of CD21 and CD10 was not rare in T-ALL, while lack of CD28 was an additional feature of early T-cell precursor-ALL. Considering the immunophenotype as a prognostic maker, MEF2D fusion-positive ALL with CD5 expression may be associated with a poorer prognosis in comparison with those lacking CD5 expression. In cases with characteristic marker expression, the presence of certain fusion transcripts could be predicted accurately.

Prognostic relevance of CD4+ T-cells in the microenvironment of newly diagnosed follicular lymphoma (FL) patients is independent of the tumor gene expression profile.

8052 Background: A significant proportion of patients with FL experience an early relapse and a subsequent poor outcome. While several prognostic indices have been developed, none were designed to predict early failure. Recently, we established that lack of intrafollicular CD4+ T-cell expression predicted risk of early failure, and integrating this microenvironment biomarker with the Follicular Lymphoma International Prognostic Index, termed BioFLIPI, further improved identification of FL patients at risk of early failure ( Blood 2019;134(suppl1):121). However, the microenvironment may be influenced by the genetic composition of tumor. We investigated whether the CD4 biomarker and BioFLIPI were impacted by genetic features of the tumor as assessed by a 23-gene expression prognostic score ( Lancet Oncol 2018;19:549-61). Methods: Of the 186 cases with FL grade 1-3A treated with immunochemotherapy (IC) in our prior study, 152 had digital expression quantification of 23 selected genes (23-GEP score), which used RNA from formalin-fixed, paraffin-embedded samples. Event-free survival (EFS) was defined as time from diagnosis to progression, relapse, retreatment, or death. Early failure was defined as failing to achieve EFS at 24 months. Risk of early failure was estimated using odds ratios (ORs) and 95% confidence intervals from logistic regression models. We also used Cox regression to assess associations with continuous EFS and overall survival (OS). Results: 28% of patients failed to achieve EFS24. Lack of CD4+ intrafollicular expression (38% of patients, OR = 2.33, p = 0.024) and high risk 23-GEP score (26% of patients, OR = 3.52, p = 0.001) each predicted early failure, and in a multivariable model that included FLIPI, both CD4+ (OR = 2.26, p = 0.046) and 23-GEP score (OR = 2.26, p = 0.0.057) remained predictors. Similarly, BioFLIPI modeled as a continuous score (1-4, OR per one point increase = 2.31, p < 0.001) predicted early failure, and the association remained (OR = 2.14, p < 0.001) when the high risk 23-GEP score (OR = 2.79, p = 0.013) was included in the model. When stratified on 23-GEP score, BioFLIPI was a stronger predictor of early failure in low risk (74%, OR = 2.51, p = 0.002) relative to high risk (26%, OR = 1.55, p = 0.27) patients. Similar patterns were observed for EFS and OS. Conclusions: CD4+ T-cell infiltrate and tumor gene expression appear to be independently predictive of early failure in newly diagnosed FL patients treated with IC. Future studies should integrate and validate these measures.

Lack of CD8+ T-cell co-localization with Kaposi's sarcoma-associated herpesvirus infected cells in Kaposi's sarcoma tumors.

Despite the close association between Kaposi’s sarcoma (KS) and immune dysfunction, it remains unclear whether tumor infiltrating immune cells (TIIC), by their absence, presence, or dysfunction, are mechanistically correlated with KS pathogenesis. Therefore, their potential capacity to serve as prognostic biomarkers of KS disease progression or control is unclear. Because epidemic-KS (EpKS) occurs with HIV-1 co-infection, it is particularly important to compare TIIC between EpKS and HIV-negative African endemic-KS (EnKS) to dissect the roles of HIV-1 and Kaposi Sarcoma-associated herpesvirus (KSHV) in KS pathogenesis. This cross-sectional study of 13 advanced KS (4 EnKS, 9 EpKS) patients and 3 healthy controls utilized single-color immunohistochemistry and dual-color immunofluorescence assays to characterize and quantify KSHV infected cells in relation to various TIIC in KS biopsies. Analysis of variance (ANOVA) and Mann-Whitney tests were used to assess differences between groups where P-values < 0.05 were considered significant. The abundance of KSHV infected cells was heterogeneous in KS biopsies. Despite the presence of T-cell chemoattractant chemokine CxCL-9 in biopsies, CD8+ T-cells were sparsely distributed in regions with evident KSHV infected cells but were readily detectable in regions devoid of KSHV infected cells (P < 0.0001). CD68+ (M1) macrophages were evenly and diffusely distributed in KS biopsies, whereas, the majority of CD163+ (M2) macrophages were localized in regions devoid of KSHV infected cells (P < 0.0001). Overall, the poor immune cell infiltration or co-localization in KS biopsies independent of HIV-1 co-infection suggests a fundamental tumor immune evasion mechanism that warrants further investigation.

CD4+CD28null T cells are expanded in moderately active systemic lupus erythematosus and secrete pro-inflammatory interferon gamma, depending on the Disease Activity Index.

Pathogenic CD4+CD28null cells are characterized by inflammatory cytokine synthesis and tropism to the inflamed tissues. Recent studies showed the involvement of CD28null T cells in a severe clinical outcome of lupus. However, their role in moderately active disease is still unresolved. We examined the levels of circulating CD4+CD28null cells and CD8+CD28null suppressor T cells. We also compared the CD4+CD28null and CD4+CD28+ T-cell functional properties, including the expression of interferon gamma (IFN-γ) and Ki67 among systemic lupus erythematosus (SLE) patients (n = 20) and healthy controls (n = 20). All the patients were under immunosuppressive treatment and exhibited moderate SLE activity (median SLE Disease Activity Index (SLEDAI) = 6). In patients, we found elevated CD4+CD28null and unchanged levels of suppressor CD8+CD28null T cells. There was no difference between patients and controls in IFN-γ and Ki67-expressing CD4+, CD4+CD28+, and CD4+CD28null T cells, except for higher IFN-γ levels in CD4+CD28+ T cells in SLE. In each studied group, we observed a higher preponderance of IFN-γ- and Ki67-expressing cells among CD4+CD28null T cells and lower levels of IFN-γ in CD4+CD28null T cells compared to the CD28+ subset. Similarly, Ki67 intensity was decreased in healthy CD4+CD28null cells, whereas in patients, comparably high expression was observed in both subsets. IFN-γ intensity in CD4+CD28null T cells correlated with SLEDAI. SLE with a moderately active clinical course is characterized by peripheral blood expansion of CD4+CD28null T cells and a normal abundance of suppressor CD8+CD28null T cells. The demonstration that these pathogenic CD4+ T cells, despite the lack of CD28, maintain the ability to produce pro-inflammatory IFN-γ positively correlated with disease activity as well as relatively high proliferative capacity may suggest their potentially predictive role in SLE flares.

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.