CD8 TEM Cells Research Articles

Identification of therapeutic targets and medicines for comorbid Crohn's disease and rheumatoid arthritis: A comprehensive analysis

ObjectiveThe incidence of Crohn's disease (CD) and rheumatoid arthritis (RA) co-morbidity, as well as the number of individuals affected, is on the rise due to their shared molecular and cellular factors. This study aimed to identify potential therapeutic targets and medicines for comorbid CD and RA. MethodsWe integrated single-cell RNA sequencing, Mendelian randomization, and colocalization analysis results from public databases to analyse immune cell subgroups in CD and RA patients and identify candidate therapeutic targets. We further screened potential medicines for the identified candidate targets using the Comparative Toxicogenomics Database (CTD) and molecular docking and molecular dynamics simulations. ResultsThe proportion of CD8 effector memory T cells (Tem) was consistently elevated in the peripheral blood mononuclear cells (PBMCs) of both CD and RA patients. MYBL1 had a causal effect on the onset of both CD (OR = 1.23; 95 % CI, 1.05–1.45; P = 0.046) and RA (OR = 1.45; 95 % CI, 1.14–1.85; P = 0.04). Four potential therapeutic molecules were retrieved from the CTD database, among which tretinoin (docking score: −6.3 kcal/mol) showed the best potential. ConclusionOur comprehensive analysis suggests that CD8 Tem cells are a key cell group in comorbid RA and CD and that MYBL1 has a causal effect. Tretinoin was identified as a potential targeted therapeutic drug, which is of great clinical research value.

Abstract 1167: Prospective analysis of pretreatment circulating CD4 memory T cell features associated with severe immunotherapy toxicity

Abstract Introduction: Immune checkpoint inhibitors (ICIs) have revolutionized melanoma treatment, significantly improving outcomes. Nevertheless, too many melanoma patients undergoing ICI treatment experience severe and often debilitating immune-related adverse events (irAEs). In a retrospective melanoma cohort treated with ICIs, we previously found that elevated baseline levels of circulating CD4 effector memory T (TEM) cells were associated with the development of severe irAEs, irrespective of the affected organ system (Lozano et al., Nature Medicine, 2022). Here, we present data from an extended prospective validation cohort from 47 patients across two academic medical centers. Methods: Of 47 patients, 26 received combination anti-PD1/anti-CTLA4 ICIs, 3 combination anti-PD1/anti-LAG3 ICIs, 17 anti-PD1 ICI, and 1 anti-CTLA4 ICI. Peripheral blood mononuclear cells (PBMCs) were prepared from pretreatment (Cycle 1 Day 1) blood samples obtained from all patients. We performed mass cytometry (CyTOF) on PBMCs using a 38-marker panel to deeply profile immune subsets. We further leveraged bulk RNA-seq from 24 patients to (i) deconvolve CD4 memory T cell abundance using CIBERSORTx and (ii) determine T cell receptor (TCR) diversity using Shannon entropy following MiXCR clonotype assembly. irAEs were graded on a 5-point scale using the Common Terminology Criteria for Adverse Events v5. Results: After treatment initiation, 14 patients experienced severe irAEs (grade 3+) at a median of 12.1 weeks (range 4-54), including 4 with life-threatening (grade 4) irAEs. CyTOF revealed elevated circulating CD4 TEM cells in patients that developed severe irAEs (P<0.0001; AUC=0.87), including those treated with combination ICIs (P=0.0001; AUC=0.89). We further observed a continuous relationship between increasing CD4 TEM levels and irAE grade (P=3e-6, Jonckheere-Terpstra test). We then combined CD4 memory T cell abundance and TCR diversity derived from bulk RNA-seq on 24 of these patients using logistic regression (trained on data from Lozano et al.) to yield a composite model score. The pretreatment composite model score was strongly associated with severe irAE development (P=0.0009, AUC=0.88), and in a median split, identified patients with shorter time to severe irAE development (3 months vs. not reached; P=0.006; HR=10.5). Notably, composite model features and the overall score had no significant association with immunotherapy benefit. Conclusion: In this prospective study, both circulating CD4 TEM levels determined by CyTOF and a composite model score measured by RNA-seq were strongly associated with severe irAE development, independent of response status. These findings hold promise for future pretreatment risk stratification of ICI therapy. Citation Format: Abul Usmani, Noah Earland, Wubing Zhang, Peter K. Harris, Antonietta Bacchiocchi, Chloe M. Sachs, Aishwarya Nene, David Y. Chen, Mario Sznol, Ruth Halaban, Aaron M. Newman, Aadel A. Chaudhuri. Prospective analysis of pretreatment circulating CD4 memory T cell features associated with severe immunotherapy toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1167.

Single-cell RNA landscape of osteoimmune microenvironment in osteoporotic vertebral compression fracture and Kümmell's disease.

Background: Single-cell RNA sequencing (scRNA-seq) enables specific analysis of cell populations at single-cell resolution; however, there is still a lack of single-cell-level studies to characterize the dynamic and complex interactions between osteoporotic vertebral compression fractures (OVCFs) and Kümmell's disease (KD) in the osteoimmune microenvironment. In this study, we used scRNA-seq analysis to investigate the osteoimmune microenvironment and cellular composition in OVCFs and KD. Methods: ScRNA-seq was used to perform analysis of fractured vertebral bone tissues from one OVCF and one KD patients, and a total of 8,741 single cells were captured for single-cell transcriptomic analysis. The cellularity of human vertebral bone tissue was further analyzed using uniform manifold approximation and projection. Pseudo-time analysis and gene enrichment analysis revealed the biological function of cell fate and its counterparts. CellphoneDB was used to identify the interactions between bone cells and immune cells in the osteoimmune microenvironment of human vertebral bone tissue and their potential functions. Results: A cellular profile of the osteoimmune microenvironment of human vertebral bone tissue was established, including mesenchymal stem cells (MSCs), pericytes, myofibroblasts, fibroblasts, chondrocytes, endothelial cells (ECs), granulocytes, monocytes, T cells, B cells, plasma cells, mast cells, and early erythrocytes. MSCs play an immunoregulatory function and mediate osteogenic differentiation and cell proliferation. The differentiation trajectory of osteoclasts in human vertebral bone tissue was also revealed. In addition, ECs actively participate in inflammatory infiltration and coupling with bone cells. T and B cells actively participate in regulating bone homeostasis. Finally, by identifying the interaction of ligand-receptor pairs, we found that immune cells and osteoclasts have bidirectional regulatory characteristics, have the effects of regulating bone resorption by osteoclasts and promoting bone formation, and are essential for bone homeostasis. It is also highlighted that CD8-TEM cells and osteoclasts might crosstalk via CD160-TNFRSF14 ligand-receptor interaction. Conclusion: Our analysis reveals a differential landscape of molecular pathways, population composition, and cell-cell interactions during OVCF development into KD. OVCFs exhibit a higher osteogenic differentiation capacity, owing to abundant immune cells. Conversely, KD results in greater bone resorption than bone formation due to depletion of MSCs and a relatively suppressed immune system, and this immune imbalance eventually leads to vertebral avascular necrosis. The site of action between immune cells and osteoclasts is expected to be a new therapeutic target, and these results may accelerate mechanistic and functional studies of osteoimmune cell types and specific gene action in vertebral avascular necrosis and pathological bone loss diseases, paving the way for drug discovery.

Effector T cell chemokine IP-10 predicts cardiac recovery and clinical outcomes post-myocardial infarction.

Preclinical data suggest that activation of the adaptive immune system is critical for myocardial repair processes in acute myocardial infarction. The aim of the present study was to determine the clinical value of baseline effector T cell chemokine IP-10 blood levels in the acute phase of ST-segment elevation myocardial infarction (STEMI) for the prediction of the left ventricular function changes and cardiovascular outcomes after STEMI. Serum IP-10 levels were retrospectively quantified in two independent cohorts of STEMI patients undergoing primary percutaneous coronary intervention. We report a biphasic response of the effector T cell trafficking chemokine IP-10 characterized by an initial increase of its serum levels in the acute phase of STEMI followed by a rapid reduction at 90min post reperfusion. Patients at the highest IP-10 tertile presented also with more CD4 effector memory T cells (CD4 TEM cells), but not other T cell subtypes, in blood. In the Newcastle cohort (n=47), patients in the highest IP-10 tertile or CD4 TEM cells at admission exhibited an improved cardiac systolic function 12 weeks after STEMI compared to patients in the lowest IP-10 tertile. In the Heidelberg cohort (n=331), STEMI patients were followed for a median of 540 days for major adverse cardiovascular events (MACE). Patients presenting with higher serum IP-10 levels at admission had a lower risk for MACE after adjustment for traditional risk factors, CRP and high-sensitivity troponin-T levels (highest vs. rest quarters: HR [95% CI]=0.420 [0.218-0.808]). Increased serum levels of IP-10 in the acute phase of STEMI predict a better recovery in cardiac systolic function and less adverse events in patients after STEMI.

31-OR: Sex-Specific Differences in Subcutaneous Adipose Tissue in Persons with HIV May Contribute to Diabetes Risk

Background: Men with HIV have greater visceral adipose tissue (VAT) accumulation compared to women, which contributes to insulin resistance and derives, in part, from a failure to sequester lipids in subcutaneous adipose tissue (SAT). We assessed whether sex-specific differences in SAT composition are associated with measures of VAT volume and insulin resistance. Methods: Adults with HIV and consistent viral suppression on antiretroviral therapy had SAT and VAT measured by CT imaging, HOMA2-IR using fasting plasma glucose and insulin, and a subset had abdominal SAT lipoaspirates for single-cell RNA-sequencing to define proportions of CD4 and CD8 T effector memory cells (TEM), perivascular (PVM) and intermediate (IM) macrophages, and fibroblasts. Adjusted ordinal regression models and partial Spearman’s assessed the interaction between sex and cell proportions. Results: We enrolled 111 participants (24% female) with a median age of 48 years, BMI of 31.5 kg/m2, and HOMA2-IR of 3.0. VAT volume was significantly associated with HOMA2-IR. Women had lower VAT volumes and higher proportions of SAT PVMs and lower IMs, as well as fewer cytotoxic CD4 and CD8 T cells compared with men (p ≤ 0.05 for all). The proportions of SAT CD4 TEM, CD8 TEM, myofibroblasts, MYOC fibroblasts, and IMs were associated with VAT volume (p ≤ 0.05 for all), but an interaction by sex was not observed. The proportions of CD4 and CD8 TEM cells were significantly associated with HOMA2-IR and there was evidence of an interaction by sex status (interaction term p = 0.09 & p = 0.06, respectively). Conclusions: Women with HIV have a greater adjusted proportion of PVMs and lower cytotoxic CD4 and CD8 T cells in SAT. While the relationship between immune cell proportions and HOMA-IR only appeared to differ for CD4 and CD8 TEM cells, lower ectopic fat deposition in the context of reduced SAT inflammatory cells in women suggests the sex difference in diabetes risk among persons with HIV may relate to adipose tissue cellular composition. Disclosure S.Bailin: None. M.Mashayekhi: None. C.L.Gabriel: None. L.Hannah: None. S.Kalams: None. S.A.Mallal: None. J.A.Kropski: Advisory Panel; APIE Therapeutics, Research Support; Boehringer Ingelheim Inc., Bristol-Myers Squibb Company, Genentech, Inc. C.N.Wanjalla: None. J.Koethe: Advisory Panel; Merck Sharp & Dohme Corp., Janssen Pharmaceuticals, Inc., Gilead Sciences, Inc., Theratechnologies, Research Support; Merck Sharp & Dohme Corp. Funding National Institutes of Health (R01DK112262, UL1RR024975, T32AI007474, P30AI110527)

Spondyloarthritis mass cytometry immuno-monitoring: a proof of concept study in the tight-control and treat-to target TiCoSpA trial.

Mass cytometry (MC) immunoprofiling allows high-parameter phenotyping of immune cells. We set to investigate the potential of MC immuno-monitoring of axial spondyloarthritis (axSpA) patients enrolled in the Tight Control SpondyloArthritis (TiCoSpA) trial. Fresh, longitudinal PBMCs samples (baseline, 24, and 48 weeks) from 9 early, untreated axSpA patients and 7 HLA-B27+ controls were analyzed using a 35-marker panel. Data were subjected to HSNE dimension reduction and Gaussian mean shift clustering (Cytosplore), followed by Cytofast analysis. Linear discriminant analyzer (LDA), based on initial HSNE clustering, was applied onto week 24 and 48 samples. Unsupervised analysis yielded a clear separation of baseline patients and controls including a significant difference in 9 T cell, B cell, and monocyte clusters (cl), indicating disrupted immune homeostasis. Decrease in disease activity (ASDAS score; median 1.7, range 0.6-3.2) from baseline to week 48 matched significant changes over time in five clusters: cl10 CD4 Tnai cells median 4.7 to 0.02%, cl37 CD4 Tem cells median 0.13 to 8.28%, cl8 CD4 Tcm cells median 3.2 to 0.02%, cl39 B cells median 0.12 to 2.56%, and cl5 CD38+ B cells median 2.52 to 0.64% (all p<0.05). Our results showed that a decrease in disease activity in axSpA coincided with normalization of peripheral T- and B-cell frequency abnormalities. This proof of concept study shows the value of MC immuno-monitoring in clinical trials and longitudinal studies in axSpA. MC immunophenotyping on a larger, multi-center scale is likely to provide crucial new insights in the effect of anti-inflammatory treatment and thereby the pathogenesis of inflammatory rheumatic diseases. Key Points • Longitudinal immuno-monitoring of axSpA patients through mass cytometry indicates that normalization of immune cell compartments coincides with decrease in disease activity. • Our proof of concept study confirms the value of immune-monitoring utilizing mass cytometry.

Abstract 6670: Association between circulating CD4 memory T cell levels and severe immune-related adverse events in melanoma patients treated with immune checkpoint blockade

Abstract Introduction: Severe immune-related adverse events (irAEs) occur in up to 60% of melanoma patients treated with immune checkpoint inhibitors (ICIs), causing substantial treatment-related morbidity and in the most severe cases, death. There is no clinical assay to predict who will develop severe ICI-induced irAEs and who will not. Using single-cell profiling assays applied to a retrospective melanoma cohort treated with ICIs, we recently showed that higher baseline levels of circulating CD4 effector memory T (TEM) cells were associated with severe irAE development, independent of the affected organ system (Lozano et al. Nature Medicine, 2022). As part of a prospective validation study of 100 melanoma patients, here we report our initial findings on the first 24 patients accrued to date. Methods: We prospectively collected pre-ICI blood from 24 metastatic melanoma patients from two academic medical centers from February 2021 onward. Peripheral blood was collected pre-treatment on the day of immunotherapy (cycle 1 day 1). We then isolated peripheral blood mononuclear cells (PBMCs) and applied mass cytometry by time of flight (CyTOF) to profile 38 leukocyte markers including markers specific to CD4 TEM cells. Cellular subpopulations were quantified using Cytobank v9. Patients underwent routine medical oncology follow-up during and after ICI treatment including grading of irAEs using the Common Terminology Criteria for Adverse Events v5. Results: Median follow-up time after pre-ICI blood collection was 9.1 months (range 2.6-18.5). Fifteen (63%) patients received combination (anti-PD1/anti-CTLA4) ICIs while the remainder received anti-PD1 monotherapy. Eight patients developed severe (grade 3+) irAEs at a median of 8.5 weeks (range 4-21) after treatment initiation, including 4 who developed life-threatening (grade 4) irAEs. Severe and life-threatening irAE development spanned 9 separate organ systems, most commonly gastrointestinal, dermatological, and hepatic. Using CyTOF, we found that circulating CD4 TEM cells were more abundant in patients who developed severe irAEs compared to those who did not (P = 0.01; AUC = 0.81), irrespective of the involved organ systems. Moreover, median-splitting the patient cohort into two groups based on pretreatment CD4 TEM levels revealed that patients with low CD4 TEMs had significantly longer freedom from severe irAE than those with high CD4 TEMs (not reached vs. 4.6 months; P = 0.013; HR = 6.7). There was no significant difference in pretreatment CD4 TEM levels between patients with and without durable clinical benefit to ICIs (P = 0.75). Conclusion: Circulating CD4 TEM levels measured by CyTOF were associated with severe irAE development, independent of response status, in patients with advanced melanoma. These findings could form the basis for pretreatment ICI risk stratification in the future. Citation Format: Abul Usmani, Noah Earland, Wubing Zhang, Peter K. Harris, Antonietta Bacchiocchi, Aishwarya Nene, David Y. Chen, Mario Sznol, Ruth Halaban, Aaron M. Newman, Aadel A. Chaudhuri. Association between circulating CD4 memory T cell levels and severe immune-related adverse events in melanoma patients treated with immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6670.

Single-cell transcriptome profiling reveals enriched memory T-cell subpopulations in hypertension.

Introduction: The adaptive immune response mediated by T cells plays a vital role in the initiation and maintenance of blood pressure (BP) elevation. Memory T cells, which are antigen-specific T cells, can respond specifically to repeated hypertensive stimuli. Although the roles of memory T cells in animal models are well studied, their maintenance and functions in hypertensive patients are poorly understood. Method: Here, we focused on the circulating memory T cells of hypertensive patients. By using single-cell RNA sequencing technology, subsets of memory T cells were identified. Differentially expressed genes (DEGs) and functional pathways were explored for related biological functions in each population of memory T cells. Result and Discussion: Our study identified four subsets of memory T cells in the blood of hypertensive patients, with CD8 effector memory T (TEM) cells accounting for more cells and demonstrating more biological functions than CD4 TEM cells. CD8 TEM cells were further analyzed using single-cell RNA sequencing technology, and subpopulation 1 was demonstrated to contribute to BP elevation. The key marker genes CKS2, PLIN2, and CNBP were identified and validated by mass-spectrum flow cytometry. Our data suggest that CD8 TEM cells as well as the marker genes could be preventive targets for patients with hypertensive cardiovascular disease.

CTIM-14. COMPREHENSIVE BIOMARKER ANALYSIS OF RESPONDERS AND NON-RESPONDERS IN A PHASE IIA TRIAL OF A CMV VACCINE IMMUNOTHERAPEUTIC CANDIDATE (VBI-1901)

Abstract Cytomegalovirus (CMV) antigens have been reported in over 90% of GBMs. CD4+ and CD8+ T cells are most frequently directed against the gB and pp65 antigens, respectively, which are immunogenic targets in a CMV-based GBM immunotherapeutic. First-recurrent GBM patients were enrolled, with Karnofsky Performance Status of at least 70, to receive VBI-1901 (a gB/pp65 enveloped virus-like particle [eVLP]) adjuvanted with GM-CSF and given intradermally (NCT03382977). Patients were vaccinated with VBI-1901 every 4 weeks, with serologic immune-monitoring 2 weeks after each vaccination and surveillance brain MRI scans every 6 weeks. Sixteen patients (8 women, 8 men) with a median age of 55 (33-67 yrs) were enrolled into the GM-CSF arm. Seven tumor responses, including two durable partial responses, were observed which led to an 18-month OS rate of 25% and mOS of 56 weeks. Detailed immunological testing was performed to identify potential correlations at baseline and after treatment between biomarkers and tumor responses. Analyses included class I and II HLA typing, modulation of plasma cytokine and chemokine responses, boosting of CMV-specific antibody and IFN-g ELISPOT responses, and modulation of CMV-specific CD4 Tem cells, of which only differential trafficking of the CD4 Tem cells appears to distinguish patients with tumor responses from those with tumor progression. Single cell, RNAseq is underway in an attempt to understand mechanistic differences in these immune cell populations in Tumor Responders vs. Non-Responders. The U.S. FDA granted Fast Track Designation to VBI-1901 adjuvanted with GM-CSF in first-recurrent GBM patients, and an expansion of the ongoing trial with this formulation in this patient population, with the addition of randomization with a contemporaneous control arm, is anticipated to begin in Q3 2022.

HL-370 Fragmentóme Profiling Reveals Distinct Molecular Subgroups of Classic Hodgkin Lymphoma

Classic Hodgkin lymphoma (cHL) is a phenotypically heterogeneous disease with enigmatic biology and pathogenesis. By leveraging ctDNA analysis, here we identify molecular groups of cHL with phenotype- and outcome-associated signatures. Identification and biological characterization of novel cHL subgroups. We sequenced 202 cHL patients targeting ~344 kb of genomic space. The length of ctDNA mapped reads was extracted, and unsupervised clustering was performed. PET/CT scan and 71 plasma cytokines were obtained for clinical correlation. Single-cell RNA sequencing was performed for 8 patients. To identify molecular subgroups within cHL, we focused on the fragmentation profile of cfDNA, a non-genetic way of detecting tumor-derived DNA in the cfDNA samples. We identified two distinct clusters, one with a fragmentation profile close to healthy (N=135), named mono-nucleosomal, and a submono-nucleosomal cluster characterized by a global shift toward shorter fragments, whose length was less than the typical wrap around the nucleosome (N=67). We explored whether cHL subtypes defined by the cfDNA fragmentation profile are clinically and biologically validated. Patients with the submono-nucleosomal fragmentome showed more frequent advanced-stage B symptoms and elevated ESR at the clinical level, higher tumor volume (TMTV and TLG) at the radiomic level, and lower PFS in terms of outcome. Tumor mutations and immune microenvironment are pathophysiologic features of cHL. The submono-nucleosomal cluster carried a higher tumor mutation burden because of heavier aberrant somatic hypermutation (ASHM). The submono-nucleosomal cluster had an immune-suppressive microenvironment enriched of T-regs and higher plasmatic levels of T-regs chemoattractants and inducers of PD1 expression (IL6, CCL2, CCL4). Conversely, mono-nucleosomal cHL had a microenvironment enriched of TFH cells and cytotoxic CD8 TEM cells and associated with cytokines and chemokines that are known for being attractants and activators of effector T cells (IL-1a, CCL21, CXCL12, IL-17A). Our observations indicate that cHL subgroups belonging to the submono-nucleosomal cluster have a more aggressive disease, a higher mutation load and more neoantigens likely due to denser ASHM, and an immune-suppressive microenvironment. Instead, cHL subgroups belonging to the mono-nucleosomal cluster have a less aggressive disease, a lower mutation load and lower neoantigens likely due to lighter ASHM, and a tumor-suppressive microenvironment.

Evaluation of tumor responses and overall survival in patients with recurrent glioblastoma (GBM) from a phase IIa trial of a CMV vaccine immunotherapeutic candidate (VBI-1901).

2014 Background: Cytomegalovirus (CMV) antigens have been reported in over 90% of GBMs. CD4+ and CD8+ T cells are most frequently directed against the gB and pp65 antigens, respectively, which are immunogenic targets in a CMV-based GBM immunotherapeutic. Methods: A total of 20 first-recurrent GBM patients were enrolled, with Karnofsky Performance Status of at least 70, across 2 arms of the Phase IIa extension phase to receive VBI-1901 (a gB/pp65 enveloped virus-like particle [eVLP]) adjuvanted with either GM-CSF (given intradermally) or AS01B (given intramuscularly) (NCT03382977). Patients were vaccinated with VBI-1901 every 4 weeks, with serologic immune-monitoring 2 weeks after each vaccination and surveillance brain MRI scans every 6 weeks. Results: 10 patients (6 women, 4 men) with a median age of 58 (33-67 yrs) were enrolled into the GM-CSF arm and 10 patients (3 women, 7 men) with a median age of 65 (40-67) enrolled into the AS01B arm. The 12-month OS rates for the GM-CSF and AS01B arms were 60% and 70%, respectively; the 18-month OS rate for the GM-CSF arm was 30%, and for the AS01B arm is expected to be 30%-40% (data has not yet matured). Two durable partial responses (locally determined by RANO) have been observed in the GM-CSF arm, with one patient progression-free and on protocol after 2 years with a tumor size reduction of 93% relative to start of treatment. Immunological analyses demonstrate that prolonged, monthly dosing with VBI-1901 does not lead to immunological tolerance. Dynamic boosting and loss in the peripheral blood of CMV-specific CD4 Tem cells after treatment with VBI-1901 formulated with GM-CSF may correlate with tumor responses. Conclusions: The U.S. FDA granted Fast Track Designation to VBI-1901 adjuvanted with GM-CSF in first-recurrent GBM patients, and an expansion of the ongoing trial with this formulation in this patient population, with the addition of randomization with a contemporaneous control arm, is anticipated to begin in H1 2022. Acknowledgement: GlaxoSmithKline Biologicals SA provided the AS01B adjuvant used in this study. Clinical trial information: NCT03382977.

Conserved T-cell epitopes of respiratory syncytial virus (RSV) delivered by recombinant live attenuated influenza vaccine viruses efficiently induce RSV-specific lung-localized memory T cells and augment influenza-specific resident memory T-cell responses

Respiratory syncytial virus (RSV) can cause recurrent infection in people because it does not stimulate a long-lived immunological memory. There is an urgent need to develop a safe and efficacious vaccine against RSV that would induce immunological memory without causing immunopathology following natural RSV infection. We have previously generated two recombinant live attenuated influenza vaccine (LAIV) viruses that encode immunodominant T-cell epitopes of RSV M2 protein in the neuraminidase or NS1 genes. These chimeric vaccines afforded protection against influenza and RSV infection in mice, without causing pulmonary eosinophilia or inflammatory RSV disease. The current study assessed the formation of influenza-specific and RSV-specific CD4 and CD8 T-cell responses in the lungs of mice, with special attention to the lung tissue-resident memory T cell subsets (TRM). The RSV epitopes did not affect influenza-specific CD4 effector memory T cell (Tem) levels in the lungs. The majority of these cells formed by LAIV or LAIV-RSV viruses had CD69+CD103- phenotype. Both LAIV+NA/RSV and LAIV+NS/RSV recombinant viruses induced significant levels of RSV M282 epitope-specific lung-localized CD8 Tem cells expressing both CD69 and CD103 TRM markers. Surprisingly, the CD69+CD103+ influenza-specific CD8 Tem responses were augmented by the addition of RSV epitopes, possibly as a result of the local microenvironment formed by the RSV-specific memory T cells differentiating to TRM in the lungs of mice immunized with LAIV-RSV chimeric viruses. This study provides evidence that LAIV vector-based vaccination can induce robust lung-localized T-cell immunity to the inserted T-cell epitope of a foreign pathogen, without altering the immunogenicity of the viral vector itself.

Integrated Immune Signature Analyses Identifies Evolution of Distinct Immunoregulatory Cell Populations Which Control Alloreactivity after Allogeneic HSCT

Background. Treatment failure after allogeneic haematopoietic stem-cell transplantation (AHST) using reduced-intensity conditioning (RIC) results from too much alloreactivity and harmful acute Graft-versus-Host Disease (aGVHD). Studies have identified many reconstituting immune cell subsets associated with development of clinical alloreactivity but the functionally dominant parameters at different time-points remain unknown. We therefore used mass cytometry (MS) to simultaneously assess multiple alloreactive and immunoregulatory cell populations to identify dominant immune reconstitution signatures associated with subsequent development of aGvHD after AHST. Methods. Phenotypic markers identifying more than 30 immune cell subsets known to influence alloreactivity were combined in a single MS panel. Peripheral blood from 58 patients with haematological cancers was analysed after T-replete HLA-matched RIC-AHST using uniform conditioning. Normalization of individual test samples spiked with CD45-barcoded healthy control cells was used to reduce batch effects. Complementary high-dimensional analytic tools were used to generate cellular profiles across the whole cohort and identify differences between patients grouped by subsequent development of aGVHD. Results. Unsupervised clustering analysis identified 40 phenotypically distinct T, B and NK cell clusters post-transplant. Significant batch effects were effectively reduced with a novel R-based algorithm normalising data to control cells. Cluster diversity analysis early post-transplant demonstrated lower cluster diversity in patients who subsequently developed aGvHD consistent with perturbation of phenotypic clusters in these patients. Two specific clusters were significantly different in abundance at D+30 in patients who went on to develop aGvHD and those who remained aGVHD-free. A cluster with a CD56brightCD16negCD27+/- regulatory NK cell (NKreg) phenotype was reduced in patients going on to develop aGvHD using both Phenograph and FlowSOM algorithms (p=0.001). These findings were validated by forward analysis using the CITRUS algorithm, revealing a similar differentiating cell population. CD56bright NKreg reconstitution was independent of CMV reactivation and did not impede reconstitution of WT1 and PR1 tumor-associated antigen-specific T cells. The reduction in NKreg in patients who subsequently developed aGvHD was accompanied by a significant increase in alloreactive CCR5+CD45RA-CCR7- CD4 effector memory T cells (Tem). We next used correlation analysis of cluster abundance across the whole cohort to identify all clusters contributing to the immune 'regulome' (those inversely correlated with alloreactive CD4 EM and/or CD8 EM T cell clusters). Notably, at D+30 the regulome consisted of 4 phenotypically distinct CD56bright NKreg clusters and a CD4+CD8+ double positive (DP) cluster, but not FOXP3+ CD4 regulatory T cells (Treg), Figure 1A Both the identity of differentiating clusters between patients subsequently developing aGVHD and those who remained aGvHD-free, and the dominant constituents of the regulome changed over time. By D+60 a CD56bright NKreg cluster (with a distinct phenotype to the differentiating cluster identified at D+30) and a DP T cell cluster were significantly reduced in patients subsequently developing aGvHD. The D+60 regulome consisted of multiple distinct CD56bright clusters, a DP T cell cluster and CD4 Treg, Figure 1B. Importantly by D+90 the immune regulome consisted of a reduced number of CD56bright NKreg clusters and increasing dominance of CD4Treg, Figure 1C. Conclusion. We show proof-of-concept that a novel acquisition and analysis pipeline can be applied to MS data to identify multiple immunoregulatory cells after AHST that contribute to the control of reconstituting alloreactive T cells. This approach identified a loss of NK cell-mediated control of alloreactive CD4 Tem cells as the dominant immune process preceding the development of aGvHD early post-transplant. Importantly, we show that specific immunoregulatory subsets are dominant at different time-points, with increasing influence of DP T cells and CD4 Treg at later time points. Our data provide mechanistic insight into the dynamic pattern of control of alloreactivity over time and show that strategies to expand or potentiate immunoregulatory cells to prevent aGvHD should be time-dependent. Disclosures Gribben: Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding.

Abstract P4-06-03: Unravelling triple-negative breast cancer tumor microenvironment heterogeneity using an integrative multiomic analysis

Abstract Introduction: Triple negative breast cancer (TNBC) constitute 10-20% of all breast cancers and is associated with a worse prognosis and limited treatment options. Recent trials evaluating immune checkpoint blockade in TNBC demonstrated encouraging results for a subset of patients. TNBC is highly heterogeneous and its tumour microenvironment (TME) has been recognized as a critical determinant of its behavior and clinical outcome. Genome-wide gene expression profiling analyses have already improved our understanding of the complexity of this disease and have defined 6 different molecular subtypes namely Basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL) and luminal androgen receptor (LAR), exhibiting distinct biological and clinical characteristic. In this study, we aim to dissect the molecular diversity of the TME and more specifically to assess the immune landscape according to TNBC molecular subtypes. Methods: A cohort of 485 TNBC patient with publicly available data (RNA-Seq and Illumina HT-12 v3) from the METABRIC and the TCGA consortia were used in the gene expression analysis. Gene signatures reflecting different features or cellular components (immune, stromal, angiogenesis, lymphangiogenesis, hypoxia, metabolism) of the TME were used to evaluate multiple biological processes known to contribute to tumorogenesis. A compendium of 17 immune specific gene signatures and T cell localisation classification were used to evaluate the immune composition and spatial pattern of immune infiltrates. All parameters were compared using a logistic regression model to evaluate their relative contribution according to each molecular subtype. Results: Our analyses demonstrated that each molecular subtype exhibits different TME profiles, as well as specific immune composition and localisation. IM tumors were associated with the highest expression of immune-related gene signatures, enriched with adaptive immune cells and with a fully inflamed spatial pattern. MSL tumors were mostly associated with the expression of Lymphangiogenesis and Stromal TME signatures. They also exhibited some immune activity through the expression of immune gene signatures capturing innate immune and adaptive immunosuppressive cells. This subtype was mainly associated with margin restricted and to some extent with fully inflamed spatial pattern. BL1 tumors were associated with the expression of Metabolism TME signatures, along with fully inflamed and stroma restricted spatial pattern. To a lesser extent, this subtype was also associated with activated DC and CD4 Tem cells. LAR and M tumors exhibited an immune cold phenotype. They were associated with Stromal and Metabolism TME signatures, enriched in margin restricted spatial pattern and negatively associated with every immune cells. Conclusions: Our results demonstrate for the first time the huge heterogeneity that characterizes the TME of TNBCs. Identification of specific TME profiles could help to design more rationale and appropriate synergistic therapeutic combinations targeting TME elements in this high-risk disease. Citation Format: Bareche Y, Buisseret L, Gruosso T, Girard E, Venet D, Dupont F, Desmedt C, Park M, Rothé F, Stagg J, Sotiriou C. Unravelling triple-negative breast cancer tumor microenvironment heterogeneity using an integrative multiomic analysis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-03.

Associations between inflammation and senescent/exhaustion markers, CD57, KLRG1, and PD-1 differ in elderly and young

Abstract Background Inflammatory indices such as IL-6 and soluble CD14 are elevated in the elderly, and aging is also associated with immune senescence and exhaustion. Soluble indices of inflammation and immune senescence have independently been associated with mortality and morbidity in the elderly. Whether these pathways are mechanistically linked is not clear. Methods This pilot study examined the relationship of plasma levels of IL-6, IP10, and sCD14 to T cell expression of immune checkpoint markers PD-1, CD57, and KLRG1 inelderly (≥70 years old; n=10) and young (≤40 years old;n=10) participants. Results Elderly participants had elevated plasma levels of IL-6 (p= &amp;lt;0.0001), sCD14 (p=0.0009), and IP10 (p= 0.012) compared to young participants. Surface expression of PD-1 (p= 0.002), CD57 (p= 0.0005), and KLRG1 (p= 0.04) was elevated on memory CD4 T cell in elderly participants. Co-expression of immune checkpoint markers PD-1, CD57, and KLRG1 on CD4 T cells inversely correlated with plasma levels of IL-6 in the elderly; whereas coexpression of checkpoint markers on CD8 T cells positively correlated with plasma levels of IP10 in young participants. Plasma levels of sCD14 positively associated with PD-1 expression on CD4 TEM cells in elderly but inversely correlated with PD-1 expression onCD8 CM and TEM cells in young participants. Conclusion The relationship between soluble indices of inflammation and immune check pointmarker expression varies between elderly and young and between CD4 and CD8 T cells. Further studies are warranted to understand the complex interactions of inflammation and immune senescence and how they contribute tomortality and morbidity in the elderly.

Stabilized β-Catenin Ameliorates ALPS-Like Symptoms of B6/lpr Mice.

Autoimmune lymphoproliferative syndrome (ALPS) is an incurable disease mainly caused by the defect of Fas-mediated apoptosis and characterized by nonmalignant autoimmune lymphoproliferation. Stabilized β-catenin could not only potentiate Fas-mediated T cell apoptosis via upregulating the expression of Fas on activated T cells, but also potentiate T cell apoptosis via intrinsic apoptotic pathway. In the present study, we introduced β-catTg into lpr/lpr mice and aimed to explore the potential role of stabilized β-catenin (β-catTg) in the development of ALPS-like phenotypes of lpr/lpr mice. We found that the total splenocyte cells and some compositions were slightly downregulated in β-catTglpr/lpr mice, especially the CD4 and CD8 TEM cells were significantly reduced. Meanwhile, stabilized β-catenin obviously decreased the numbers of spleen TCRβ+CD4−CD8− T (DNT) cells, and the levels of some serum proinflammatory factors also were lowered in β-catTglpr/lpr mice. Beyond that, stabilized β-catenin slightly lowered the levels of the serum autoantibodies and the scores of kidney histopathology of β-catTglpr/lpr mice compared with lpr/lpr mice. Our study suggested that stabilized β-catenin ameliorated some ALPS-like symptoms of lpr/lpr mice by potentiating Fas-independent signal-mediated T cell apoptosis, which might uncover a potential novel therapeutic direction for ALPS.

Phenotypes and HIV-1-specific T cell responses of KIR3DL1 positive CD8 cells in patients with early HIV-1 infection

Objective To investigate the phenotypes and the HIV-1-specific T cell responses of KIR3DL1 positive CD8 cells in patients with early HIV-1 infection. Methods Fifty-six HIV-1 antibody negative individuals and thirty-two patients with early HIV-1 infection were enrolled in the study. Fluorescence-activated cell sorting (FACS) was performed to detect the phenotypes of KIR3DL1 receptor expressed on the surface of CD8 cells. The levels of IFN-γ were measured by intracellular cytokine staining assay after the PBMCs were stimulated with an HIV-1 Gag peptide pool. Results The percentages of KIR3DL1+ CD8 T cells in HIV-1 negative individuals and patients with early HIV-1 infection were 1.45% (0.12%-8.4%) and 0.82% (0.14%-6.14%), respectively, and there was no significant difference between them. The percentages of KIR3DL1+ CD8 Temra cells in HIV-1 negative individuals and patients with early HIV-1 infection were (4.55±3.84)% and (6.71±8.50)%, respectively, which were significantly higher than the percentages of KIR3DL1+ CD8 Tem cells, which were (0.50±0.59)% and (1.18±1.39)%, respectively (all P<0.01). Moreover, the percentages of KIR3DL1+ CD8 Tem cells in patients with early HIV-1 infection were higher than those in HIV-1 negative individuals (P=0.001 2). The percentage of KIR3DL1+ CD8 Temra cells was positively correlated with the HIV-1 viral load in patients with early HIV-1 infection (rs=0.576, P=0.000 9). The percentages of KIR3DL1+ CD8 Temra cells in HIV-1 patients, whose viral loads were larger than 4.0log, were much higher than those in HIV-1 patients with viral loads less than 4.0 log (P=0.002). Additionally, the levels of IFN-γ secreted by KIR3DL1 positive CD8 cells were much lesser than those secreted by KIR3DL1 negative CD8 cells (P<0.000 1). Conclusion The receptor of KIR3DL1 was mainly expressed on CD8 Temra cells in both HIV-1 negative subjects and patients with early HIV-1 infection. High HIV-1 viremia was associated with the high percentage of KIR3DL1+ CD8 Temra cells. The KIR3DL1 positive CD8 cells induced lower HIV-1-specific T cell responses. Key words: Early HIV-1 infection; KIR3DL1 receptor; CD8 cells

Persistent BCG bacilli perpetuate CD4 T effector memory and optimal protection against tuberculosis

Tuberculosis (TB) remains one of the most important infectious diseases of man and animals, and the only available vaccine (BCG) requires urgent replacement or improvement. To facilitate this, the protective mechanisms induced by BCG require further understanding. As a live attenuated vaccine, persistence of BCG bacilli in the host may be a crucial mechanism.We have investigated the long term persistence of BCG following vaccination and the influence on the induced immune response and protection, using an established murine model. We sought to establish whether previously identified BCG-specific CD4 TEM cells represent genuine long-lived memory cells of a relatively high frequency, or are a consequence of continual priming by chronically persistent BCG vaccine bacilli.By clearing persistent bacilli, we have compared immune responses (spleen and lung CD4: cytokine producing T effector/TEM; TCR-specific) and BCG-induced protection, in the presence and absence of these persisting vaccine bacilli. Viable BCG bacilli persisted for at least 16 months post-vaccination, associated with specific CD4 T effector/TEM and tetramer-specific responses. Clearing these bacilli abrogated all BCG-specific CD4 T cells whilst only reducing protection by 1log10.BCG may induce two additive mechanisms of immunity: (i) dependant on the presence of viable bacilli and TEM; and (ii) independent of these factors.These data have crucial implications on the rational generation of replacement TB vaccines, and the interpretation of BCG induced immunity in animal models.

The increased type-1 and type-2 chemokine levels in children with acute RSV infection alter the development of adaptive immune responses.

Severe RSV infections and frequent recurrence could be related to the altered polarization of type-2/type-1 T cells. This increases the importance of determining distinctive chemokines and chemokine receptor profiles on memory T cells. We analyzed systemic adaptive T cell response in the acute (n = 17) and convalescent phase (n = 7) of RSV-infected children, in the acute (n = 11) and convalescent phase (n = 6) of children with other viral respiratory infections (adenovirus and influenza virus), and in healthy children (n = 18). Expression of CCR4 and CXCR3 on effector-memory (TEM) and central-memory (TCM) T cells was compared between tested groups. Serum concentrations of specific chemokines were determined. High CXCL10 levels were detected in acutely infected children regardless of virus pathogen, whereas increased CCL17 production was RSV-specific. Higher percentages of CCR4+ CD4 TEM cells in acute RSV infection were accompanied with higher percentages of CXCR3+ CD8 TEM cells, whereas the development of long-lived memory CXCR3+ CD4 and CD8 TCM cells seems to be compromised, as only children with other viral infections had higher percentages in the convalescent phase. Presence of type-2 and type-1 adaptive antiviral immune response, together with insufficient development of long-lived type-1 T cell memory, could play an important role in RSV pathogenesis and reinfection.

Tracking T Cell Dynamics In The First Month After Haplo-HSCT With Post-Transplant Cyclophosphamide Reveals a Predominant Contribution Of Memory Stem T Cells To The Early Phase Of Immune Reconstitution

Haploidentical hematopoietic stem cell transplantation (HSCT) with T–replete grafts and post-transplant cyclophosphamide (PT-Cy) has gained much interest in the transplantation community for the low rates of GvHD, non-relapse mortality and opportunistic infections. This platform, devoid of anti-thymocyte globulins, allows a thorough analysis of circulating cells in the early phase post-HSCT. Indeed, several biological events that play a critical role for transplant outcome occur within the first month after HSCT; while engraftment and hematological reconstitution are carefully monitored, the shape of T cell dynamics within this timeframe remains largely unknown. We characterized immune reconstitution (IR) during the first month post HSCT in 18 high-risk leukemia patients receiving myeloablative conditioning, T-replete haploidentical peripheral blood stem cell graft (PBSCs), and GvHD prophylaxis consisting of PT-Cy (day 3, 4), followed by mycophenolate mofetil and sirolimus from day 5. Infused PBSCs and blood samples harvested at day 1, 3, 5, 8, 15 and 30 post HSCT were analyzed by multiparametric flow cytometry. T cells were infused in the absence of immunosuppressive agents, and by day 3, prior to PT-Cy, a large fraction of memory lymphocytes, possibly enriched for allo-specificities, proliferated (assessed by Ki-67 staining). Conversely, naïve T cells (TN) were scantly Ki-67+ (P&lt; 0.001). A high CD4:CD8 ratio was observed at this time-point (10). PT-Cy efficiently abated T cell proliferation and appeared to affect CD4 more than CD8 T cells. Nevertheless, T cell numbers progressively increased (mean CD3 counts, day 5: 19 cells/µL; day 8: 27 cells/µL; day 15: 97 cells/µL), suggesting that residual proliferation in extravascular sites was likely to fuel the surge in circulating T cells. Consistently, we observed an expansion of antigen-experienced T cells including central memory (TCM), effector memory (TEM), effectors (TEFF) and the recently described stem memory T cells (TSCM). TSCM are a subset of memory cells hierarchically superior to TCM and TEM, for self-renewal, long-term persistence and functional capacity. Similarly to TN, TSCM coexpress CD45RA and CD62L but differently from TN, TSCM express CD95, a marker of memory cells. As early as day 8 post HSCT, the T cell compartment was predominantly composed by TSCM cells (P &lt; 0.01 compared to all other subsets). Such enrichment in TSCM was not due to a selective resistance to PT-Cy, as suggested by the lack of activity of the ALDH enzyme, which converts Cy to a non-toxic metabolite, in TSCM infused with the graft. Rather, we hypothesized that TSCM expansion came directly from the differentiation of TN infused within the graft, which escaped the purging effect of PT-Cy thanks to a delayed activation kinetics compared to alloreactive memory T cells. We demonstrated the in vivo differentiation of WT1 and PRAME specific TN cells, present in the graft, into memory lymphocytes, comprising TSCM cells, in 4/7 patients suitable for dextramer tracking. Such tumor specific T cells were detected in the peripheral blood and bone marrow of treated patients, suggesting that PT-Cy did not hamper GvL players. Of note in the remaining patients for whom tumor-specific TN were not detectable in the graft, no tumor response could be documented in vivo. From day 15 post HSCT, TSCM were outnumbered by other memory subsets, suggesting their differentiation into more committed TCM TEM and TEFF. The quality of IR correlated with clinical events. The percentage of circulating Ki-67+ CD8 TEMcells at day 8 post HSCT accurately predicted the occurrence of periengraftment syndrome, observed in 4 patients with a median time to onset of 15 days. Acute GvHD (Grade I/II in 6 patients, grade III/IV in 4) was accompanied by a rise in circulating Ki-67+ CD8 cells, and response to therapy resulted in a drop in Ki-67 expression. No immunological parameter correlated with chronic GvHD, observed in 2 patients. In all patients with a CMV-seropositive donor, CMV-specific T cells were tracked in the graft, at early time-points and up to 180 days post HSCT, indicating that virus-specific T cells escaped PT-Cy. These results suggest that PT-Cy acts mainly on alloreactive memory T cells infused within the graft, while sparing infused virus-specific, non cross-reactive, memory cells and TN, which can differentiate predominantly in TSCM, but also in TCM TEM and TEFF, thus promoting a rapid and broad IR. Disclosures: Bonini: MolMed SpA: Consultancy.