TEMRA CD8 Research Articles

ITK Inhibitor Induces Dose-Dependent Th1 Skewing in Normal T Cells and Is Active in Refractory T Cell Lymphomas

Introduction Interleukin 2 inducible T cell kinase (ITK) is a TEC family tyrosine kinase expressed in T cells with an important role both in T cell receptor (TCR) signaling and T helper cell differentiation. Resting lymphocyte kinase (RLK), a closely related kinase, is redundant to ITK and is involved in the differentiation of naïve T cells into Th1 T cells. ITK-/- mice exhibit defects in Th2 differentiation while retaining the ability to differentiate into Th1 cells that secrete IFNγ; whereas, double ITK/RLK knockout mice exhibit profound defects in T cell function. Th1 cells are needed for various cytotoxic T cell functions such as destruction of tumor cells. CPI-818 is a covalent inhibitor of ITK (KD 2.5nM) with > 100 fold selectivity over RLK and other TEC family kinases. CPI-818 is being evaluated in an ongoing phase 1 trial in patients (pts) with refractory T cell lymphomas (TCL). We now report that selective ITK blockade with CPI-818 induces Th1 skewing of normal T cells and demonstrates anti-tumor activity. Methods Normal human peripheral blood mononuclear cells (PBMC) were stimulated in vitro for 6 days with anti-CD3/CD28 in the presence of CPI-818. Th1 cells were measured by flow cytometry based on ratio of IFNγ:IL4 staining cells. Sezary cells were similarly stimulated in the presence of CPI-818. Peripheral blood and tumor samples were stained with antibodies to CD3, 4, 8, 183, 196, 197 and 45RA to enumerate Th1 and other T cell subsets using multicolor flow cytometry. Successive cohorts of TCL pts were treated in a phase 1 trial until disease progression with continuous oral dosing of CPI-818 100mg, 200mg, 400mg or 600mg given BID. Results T cell receptor stimulated PBMC from 12 healthy donors incubated in vitro with CPI-818 demonstrated Th1 skewing at concentrations of 0.1 to < 10uM (Figure 1). Maximal skewing was seen at 1uM; cell viability was unaffected but, proliferation was inhibited at 10uM. Similar studies using Th2 Sezary cells showed inhibition of proliferation with CPI-818 (0.5-2.5uM). Thirty-three pts with TCL with a median age of 62 yrs and median of 4 prior therapies were enrolled. Histologies included: PTCL-NOS (17), AITL (4), and CTCL (12). The most common (≥ 10%) AEs were nausea, chills, vomiting, fatigue, pyrexia, decreased appetite, pruritus, and rash. Treatment-related Grade 3+ AEs were: anemia (1), lymphocytosis (1), WBC decreased (1) and neutropenia (1). The swimmer plot (Figure 2) shows a dose-dependent relationship with an optimum dose of 200mg (N=10) producing 1 CR 25 mo; 1 PR 2+ mo; 1 nodal CR 16 mo (CTCL); 2 SD; 1 PD; 4 not yet evaluated; 6 pts remain on treatment. Pharmacokinetic and target occupancy studies for the 200mg dose showed mean plasma Cmax of 2.3uM (SD 1.9uM) and ITK target occupancy of >75%. Analysis of blood in 4 of 4 pts treated in this cohort showed increases in Th1 cells (CD3+/CD4+/CD8-/CD183+/CD196-) compared to baseline and increases in terminally differentiated T effector memory cells (TEMRA CD3+/CD4-/CD8+/CD197-/CD45RA+). Tumor biopsy from 1 patient taken during response demonstrated an increase in Th1 (0 to 40%) and TEMRA CD8+ T cells (2.83% to 18.6%). CPI-818 reduced blood eosinophils (Eos) in 3 of 3 pts in this cohort with elevated Eos count, consistent with inhibition of Th2 function. Conclusions This study demonstrates that selective inhibition of ITK, while sparing RLK, induces Th1 skewing. As expected, the effects on T cell differentiation and proliferation are dose dependent: lower doses induce Th1 skewing and higher concentrations in vitro and in vivo lead to inhibition of TCR signaling and proliferation. Tumor responses were observed in several patients receiving 200mg BID, which achieves drug plasma levels consistent with Th1 skewing observed in vitro. The increases in Th1 cells and TEMRA CD8+ cells suggest that anti-tumor activity is due to induction of a host anti-tumor response. This raises the possibility that immunomodulation with ITK blockade could be used for immunotherapy of other cancers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD8+ TEMRA Clones Cause Platelet Lysis in Immune Thrombocytopenia

Background Immune thrombocytopenia (ITP) is traditionally thought of as an antibody-mediated disease. However, there are a number of features to suggest alternative mechanisms of platelet destruction. In this study we use a multi-dimensional approach to explore the role of terminally differentiated effector memory CD8+ T cells (TEMRA). Methods We characterised 83 adults with chronic ITP and 30 age-matched controls using immunophenotyping, next generation sequencing of T cell receptor (TCR), single cell (sc) RNA sequencing and functional assays. Next generation DNA sequencing of TCRβ was carried out using the Illumina MiSeq platform. Analysis of the raw TCR sequences was performed using MiXCR. Single cell immune profiling (Gene expression and TCR) was done using Chromium Single Cell V(D)J and 5' Library kits from 10x Genomics. The constructed library was sequenced on the HiSeq platform (Illumina). Seurat v.2.3.4 was used for gene expression analysis. Interactions between CD8+ T cells and platelets were measured by co-culturing CD8+ T cells with autologous platelets overnight and by perfusing them on the autologous platelet covered VenaFluoro8+ microchip channel. Results We found an expansion of CD8+ CD45RA+ CD62L- TEMRA cells expressing IFN gamma, TNF alpha and granzyme B, with no evidence of physiological exhaustion (failure to upregulate TIM-3 or PD-1 expression) in ITP patients which correlated with disease activity. DNA TCR sequencing revealed that expansion of TEMRA cells in ITP was associated with reduced T cell repertoire diversity (p ≤ 0.0001) and lower Simpson's diversity index (p ≤ 0.05). Patients with ITP had a higher number of T cell clones occupying more than 5% of the T cell repertoire when compared to age matched controls (p ≤ 0.05). Expanded clones were not shared across ITP patients and displayed no known antigen specificities when compared to available databases of TCR sequences, providing evidence for the lack of virally driven clonal expansion. Clonally expanded CD8+ T cells persisted over many years in ITP patients and characterised disease severity. Longitudinal samples from 9 patients showed expanded clones and reduced T cell repertoire diversity during active disease (platelet count of less than 30 x 109/L) compared to stable disease (platelet count greater or equal to 30 x 109/L) (p ≤ 0.01; r = 0.56 - Figure 1 shows an example patient). To better characterise CD8+ T cells, we performed paired scRNA and scTCR sequencing on isolated whole CD8+ T cells from patients with active and stable disease. Paired sequencing enables transcriptomic characterisation of T cell clones. ScRNA sequencing revealed four distinct clusters in CD8+ T cells: naïve, TEMRA, central memory (CM) and natural killer T cells (NKT). By mapping scTCR CD8+ sequencing to single cell gene expression, we found that the largest expanded clones showed an aggregative distribution, indicating transcriptional homogeneity and were almost exclusively comprised of TEMRA cells (Figure 2). Consistent with immunophenotyping and DNA TCR sequencing, expanded TEMRA cell clones were expanded in active disease compared to stable disease. To assess the impact of these CD8+ T cells on platelets in ITP, PBMCs from patients with ITP and HC were passed across a platelet layer at flow rates consistent with venous blood flow. CD8+ T cells in ITP patients interacted with platelets 4x more frequently than HCs. Isolated CD8+ T cells were cultured over night with autologous platelets to further explore these interactions: CD8+ T cell:platelet aggregates were identified more frequently in the co-cultures from patients with ITP (p ≤ 0.05) and were inhibited by blocking MHC Class I (p ≤ 0.01). CD8+ T cells within the aggregates had increased CD107a expression compared to non-aggregated CD8+ T cells (p ≤ 0.01), indicating cytolytic activity and release of Granzyme and Perforin. Correspondingly, platelets in the aggregates showed increased CD62P expression, a marker of activation and impending death (p ≤ 0.001). Conclusions Here we demonstrate clonal expansion of disease-associated TEMRA CD8+ T cells in ITP. These cells bind to platelets and cause TCR-mediated platelet activation and death providing an antibody-independent mechanism of platelet destruction. This could serve as biomarker to direct treatment in patients with refractory ITP and targeting specific T-cell clones could be a novel therapeutic option in refractory ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Eltrombopag Causes Cell Cycle Arrest of T Cells through Iron Chelation and Could Modulate the Immune Response in ITP

Background Thrombopoietin receptor agonists (TPO-RAs) are the recommended second line treatment in most guidelines for immune thrombocytopenia (ITP) allowing patients to avoid immunosuppressive treatments (Provan et al and Neunert et al 2019). Three different TPO-RAs are licensed for use in ITP. Real world evidence has shown that if patients do not respond to one agent, they may respond to another. This is thought to relate to different signalling pathways, and possibly one TPO-RA creating a stronger signal than another (Olnes et al, 2012; D'Arena et al, 2013). Nonetheless, it is difficult to fully explain responses on switching agents, especially a response to Eltrombopag (ELT) when Romiplostim (ROMI) has failed. In addition to being a TPO-RA, ELT is a small molecule peptide, which chelates cations, in particular iron and calcium. We have previously shown its ability to shuttle iron out of cells, and the impact this has on liver, cardiac and megakaryocyte cells in vitro (Roth et al, 2012; Vlachodimitropoulou et al, 2016, Liu et al 2022). We previously reported that ELT has an anti-proliferative effect on stimulated Jurkat and primary T cells, and demonstrated that patients on ELT have a reduced frequency of terminally differentiated effector (TEMRA) CD8 T cells compared to patients on ROMI (Sayed et al, ASH, 2019). We propose that this is related to an intracellular iron chelation effect, which could either explain or be additive to its megakaryocyte stimulating effect in ITP. In this study, we further explored the immunomodulatory effect of ELT on CD8 T cells in ITP. Methods Primary T cells were stimulated with Dynabeads Human T-Activator CD3/CD28 in the presence of ELT, ROMI, Deferoxamine (DFX) or human recombinant TPO (rhTPO) at increasing doses based on their calculated therapeutic serum levels. DFX and rhTPO act as positive control for iron chelation and TPO-R, respectively. Cell cycle was measured using Propidium Iodide (PI) and analysed using flow cytometry. To explore the in vivo effect of ELT, we measured CD8 TEMRA cells before and after treatment in a cohort of patients with ITP treated with ELT. Results Primary T cells treated with ELT resulted in cell cycle arrest to G1 phase. This effect was more pronounced with increasing doses of ELT. DFX exhibited similar effect on primary T cells, however, the induction of G1 phase cell cycle arrest was less potent than ELT. ROMI and rhTPO did not affect cell cycle progression of primary T cells (Figure 1). To confirm whether this cell cycle arrest was due to the iron chelating properties of ELT, we treated the arrested primary T cells with ammonium iron (III) for 24 hours, and found that cell cycle was able to be rescued. We further investigated the immunomodulatory effect of ELT in patients with ITP by assessing CD8 TEMRA cells (CD45RA+CD62L-) before and after ELT treatment. We found that patients who responded to ELT had a reduction in the frequency of TEMRA cells correlating to their platelet count. An example shown in Figure 2 of a patient who had previously failed ROMI, Mycophenolate Mofetil (MMF) and Rituximab, and subsequently responded to ELT. Prior to ELT, at low platelet count, patient presented with high frequency of CD8 TEMRA cells. Response to ELT resulted in an increased platelet count and a corresponding fall in CD8 TEMRA cells. Discussion Here we show that ELT suppresses the proliferation of T cells, through cell cycle arrest via its iron chelating properties. We propose that this could have a dual impact in patients with ITP, increasing platelet production as well as dampening down the immune response, hence explaining why switching to ELT could be effective if other TPO-RAs have failed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Heterogenous CD8+ T Cell Maturation and 'Polarization' in Acute and Convalescent COVID-19 Patients.

Background. The adaptive antiviral immune response requires interaction between CD8+ T cells, dendritic cells, and Th1 cells for controlling SARS-CoV-2 infection, but the data regarding the role of CD8+ T cells in the acute phase of COVID-19 and post-COVID-19 syndrome are still limited. Methods.. Peripheral blood samples collected from patients with acute COVID-19 (n = 71), convalescent subjects bearing serum SARS-CoV-2 N-protein-specific IgG antibodies (n = 51), and healthy volunteers with no detectable antibodies to any SARS-CoV-2 proteins (HC, n = 46) were analyzed using 10-color flow cytometry. Results. Patients with acute COVID-19 vs. HC and COVID-19 convalescents showed decreased absolute numbers of CD8+ T cells, whereas the frequency of CM and TEMRA CD8+ T cells in acute COVID-19 vs. HC was elevated. COVID-19 convalescents vs. HC had increased naïve and CM cells, whereas TEMRA cells were decreased compared to HC. Cell-surface CD57 was highly expressed by the majority of CD8+ T cells subsets during acute COVID-19, but convalescents had increased CD57 on ‘naïve’, CM, EM4, and pE1 2–3 months post-symptom onset. CXCR5 expression was altered in acute and convalescent COVID-19 subjects, whereas the frequencies of CXCR3+ and CCR4+ cells were decreased in both patient groups vs. HC. COVID-19 convalescents had increased CCR6-expressing CD8+ T cells. Moreover, CXCR3+CCR6- Tc1 cells were decreased in patients with acute COVID-19 and COVID-19 convalescents, whereas Tc2 and Tc17 levels were increased compared to HC. Finally, IL-27 negatively correlated with the CCR6+ cells in acute COVID-19 patients. Conclusions. We described an abnormal CD8+ T cell profile in COVID-19 convalescents, which resulted in lower frequencies of effector subsets (TEMRA and Tc1), higher senescent state (upregulated CD57 on ‘naïve’ and memory cells), and higher frequencies of CD8+ T cell subsets expressing lung tissue and mucosal tissue homing molecules (Tc2, Tc17, and Tc17.1). Thus, our data indicate that COVID-19 can impact the long-term CD8+ T cell immune response.

SARS-CoV-2 Infection in Pediatric Solid Organ Transplant Recipients: A Single Center Observation.

Objectives:This is a descriptive study to characterize rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in pediatric solid organ transplant (SOT) recipients during the early days of the pandemic. We hypothesized that asymptomatic infection may represent a large proportion of SARS-CoV-2 infection in pediatric SOT recipients.Methods:We queried Organ Transplant Tracking Record (OTTR) for all pediatric SOT recipients followed at our center and reviewed medical records to identify patients tested for SARS-CoV-2 between March 15, 2020 and June 30, 2021. Patients were tested by polymerase chain reaction (PCR): prior to planned procedures or because of symptoms; OR: tested by measurement of IgG to spike protein with their routine labs q 2-monthly. A positive PCR was called acute infection. A positive IgG with negative PCR was called convalescence. For immunologic studies, blood was obtained when the PCR or IgG was positive. Statistical comparisons were made between (1) acute infection versus convalescence; (2) acute infection versus SOT recipients without infection (called healthy controls); (3) liver transplant (LT) versus small bowel (SB)/multivisceral transplant (MVT); (4) positive versus negative test result.Results:Of 257 LT recipients, 99 were tested: 6 were PCR positive, 13 were antibody positive. Of 150 SB/MVT recipients, 55 were tested: 4 were PCR positive, 6 were antibody positive. Of 8 simultaneous liver, kidney transplant recipients, 3 were tested: 1 was PCR positive. Symptoms when present were mostly mild. Patients with a positive test result were younger (6.3 vs 10.0 years; P = 0.017). We observed a rapid decline in viral load within 96 hours without a change in immunosuppression. Antibody lasted >8 months beyond the time it was monitored. Acute infection was associated with increased CD4 and CD8 TEM cell frequency (P = 0.04, P = 0.03, respectively), decreased interferon (IFN)-γ production from T-cells (2.8% vs 11.3%; P = 0.006), and decreased CD8 TEMRA frequency (4.56% vs 11.70%; P = 0.006).Conclusions:Early in the pandemic, COVID-19 disease was mostly mild in pediatric SOT recipients with no rejection, patient death, or graft loss observed.

Identification of Pathogenic Immune Cell Subsets Associated With Checkpoint Inhibitor-Induced Myocarditis.

Immune checkpoint inhibitors (ICIs) are monoclonal antibodies used to activate the immune system against tumor cells. Despite therapeutic benefits, ICIs have the potential to cause immune-related adverse events such as myocarditis, a rare but serious side effect with up to 50% mortality in affected patients. Histologically, patients with ICI myocarditis have lymphocytic infiltrates in the heart, implicating T cell-mediated mechanisms. However, the precise pathological immune subsets and molecular changes in ICI myocarditis are unknown. To identify immune subset(s) associated with ICI myocarditis, we performed time-of-flight mass cytometry on peripheral blood mononuclear cells from 52 individuals: 29 patients with autoimmune adverse events (immune-related adverse events) on ICI, including 8 patients with ICI myocarditis, and 23 healthy control subjects. We also used multiomics single-cell technology to immunophenotype 30 patients/control subjects using single-cell RNA sequencing, single-cell T-cell receptor sequencing, and cellular indexing of transcriptomes and epitopes by sequencing with feature barcoding for surface marker expression confirmation. To correlate between the blood and the heart, we performed single-cell RNA sequencing/T-cell receptor sequencing/cellular indexing of transcriptomes and epitopes by sequencing on MRL/Pdcd1-/- (Murphy Roths large/programmed death-1-deficient) mice with spontaneous myocarditis. Using these complementary approaches, we found an expansion of cytotoxic CD8+ T effector cells re-expressing CD45RA (Temra CD8+ cells) in patients with ICI myocarditis compared with control subjects. T-cell receptor sequencing demonstrated that these CD8+ Temra cells were clonally expanded in patients with myocarditis compared with control subjects. Transcriptomic analysis of these Temra CD8+ clones confirmed a highly activated and cytotoxic phenotype. Longitudinal study demonstrated progression of these Temra CD8+ cells into an exhausted phenotype 2 months after treatment with glucocorticoids. Differential expression analysis demonstrated elevated expression levels of proinflammatory chemokines (CCL5/CCL4/CCL4L2) in the clonally expanded Temra CD8+ cells, and ligand receptor analysis demonstrated their interactions with innate immune cells, including monocytes/macrophages, dendritic cells, and neutrophils, as well as the absence of key anti-inflammatory signals. To complement the human study, we performed single-cell RNA sequencing/T-cell receptor sequencing/cellular indexing of transcriptomes and epitopes by sequencing in Pdcd1-/- mice with spontaneous myocarditis and found analogous expansions of cytotoxic clonal effector CD8+ cells in both blood and hearts of such mice compared with controls. Clonal cytotoxic Temra CD8+ cells are significantly increased in the blood of patients with ICI myocarditis, corresponding to an analogous increase in effector cytotoxic CD8+ cells in the blood/hearts of Pdcd1-/- mice with myocarditis. These expanded effector CD8+ cells have unique transcriptional changes, including upregulation of chemokines CCL5/CCL4/CCL4L2, which may serve as attractive diagnostic/therapeutic targets for reducing life-threatening cardiac immune-related adverse events in ICI-treated patients with cancer.

Clonally expanded CD8 T cells characterize amyotrophic lateral sclerosis-4.

Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state.

Engineered, fully human nanobody-based CAR T cells have enhanced antitumor activity against hepatocellular carcinoma in preclinical models.

e14512 Background: Glypican 3 (GPC3), a cell surface protein associated with early development, is an established biomarker of hepatocellular carcinoma (HCC). In a vast compendium of work, HN3, a human GPC3 nanobody (VH) isolated from a phage library, was shown to abrogate HCC tumors and block the distally located Wnt binding domain of GPC3. To date, it remains challenging how to precisely engineer CAR T cells for improved efficacy in solid tumors. In the context of hepatocellular carcinoma, we determined how hinges and transmembrane portions of varying structures and sizes affect CAR T cell function. Here, we show how rationally designed, engineered Chimeric Antigen Receptor (CAR) T cells containing HN3, demonstrate enhanced CAR T activity in HCC. Methods: We generated and compared multiple permutations of GPC3 targeted CAR T cells containing HN3, CD8, CD28, IgG4 and Fc domains. Incorporation of HN3, into multiple CAR formats yielded nine constructs, which were screened for antigen-specific and antigen-independent signaling using a luciferase reporter system in Hep3B, Huh7 and HepG2 cell lines. For in vivo assessment, immunodeficient NSG mice were intraperitonially injected with Hep3B or Huh7 cells and treated with a single dose (5 or 15 million) of engineered HN3 CAR T cells. Blood was collected at 2, 4 and 5 weeks to assess proliferation, T cell subtypes and exhaustion. Results: In vitro, the HN3-IgG4H-CD28TM CAR T cells induced high specificity and cytotoxic activity. HN3-IgG4H-CD28TM CAR T cells markedly improved HN3 cell killing activity by 30-40% in low (1.6:1) and high (25:1) effector to target ratios. Similarly, in vivo, in a high antigen environment, two determinative changes in the hinge region and transmembrane domains led to complete tumor eradication in immunodeficient mice bearing HCC tumors within 7-10 days. Moreover, HN3-IgG4H-CD28TM CAR T cells maintained specific tumor killing and averted exhaustion by producing a clear T cell response signature of enriched cytotoxic-memory (Temra) CD8+ along with a subset of naïve T cells. Conclusions: Engineered nanobody based CAR T cells containing the appropriate hinge and transmembrane domains can lead to determinative T cell signaling capable of inducing swift and durable eradication of HCC tumors. Altogether, we show that engineered HN3 CAR T cell therapy demonstrates strong potential for treatment of aggressive HCCs enabled by Wnt dysregulation.

Low Memory T Cells Blood Counts and High Naïve Regulatory T Cells Percentage at Relapsing Remitting Multiple Sclerosis Diagnosis.

ObjectiveThe aim of this study is to assess the peripheral immune system of newly diagnosed patients with relapsing remitting multiple sclerosis (RRMS) and compare it to healthy controls (HC).MethodsThis cross-sectional study involves 30 treatment-naïve newly diagnosed patients with RRMS and 33 sex- and age-matched HC. Peripheral blood mononuclear cells were analyzed regarding: i) thymic function surrogates [T cell receptor excision circles (TRECs) and recent thymic emigrants (RTEs)]; ii) naïve and memory CD4+ and CD8+ T cells subsets; iii) T helper (Th) phenotype and chemokine receptors expression on CD8+ T cells subsets; iv) regulatory T cell (Tregs) phenotype; and exclude expression of activating/inhibitory receptors by natural killer (NK) and NKT cells. Analyses were controlled for age, sex, and human cytomegalovirus (HCMV) IgG seroprevalence.ResultsNewly diagnosed patients with RRMS and HC have equivalent thymic function as determined by similar numbers of RTEs and levels of sjTRECs, DJβTRECs, and sj/DJβTREC ratio. In the CD8+ T cells compartment, patients with RRMS have a higher naive to memory ratio and lower memory cell counts in blood, specifically of effector memory and TemRA CD8+ T cells. Interestingly, higher numbers and percentages of central memory CD8+ T cells are associated with increasing time from the relapse. Among CD4+ T cells, lower blood counts of effector memory cells are found in patients upon controlling for sex, age, and anti-HCMV IgG seroprevalence. Higher numbers of CD4+ T cells (both naïve and memory) and of Th2 cells are associated with increasing time from the relapse; lower numbers of Th17 cells are associated with higher MS severity scores (MSSS). Patients with RRMS have a higher percentage of naïve Tregs compared with HC, and lower percentages of these cells are associated with higher MSSS. Percentages of immature CD56bright NK cells expressing the inhibitory receptor KLRG1 and of mature CD56dimCD57+ NK cells expressing NKp30 are higher in patients. No major alterations are observed on NKT cells.ConclusionCharacterization of the peripheral immune system of treatment-naïve newly diagnosed patients with RRMS unveiled immune features present at clinical onset including lower memory T cells blood counts, particularly among CD8+ T cells, higher percentage of naïve Tregs and altered percentages of NK cells subsets expressing inhibitory or activating receptors. These findings might set the basis to better understand disease pathogenesis.

Assessment of changes in immune status linked to COVID-19 convalescent and its clinical severity in patients and uninfected exposed relatives

IntroductionThe immune response during and after SARS-CoV-2 infection can be complex and heterogeneous, and it can be affected by the severity of the disease. It can also contribute to an unfavorable evolution and bring about short and long term effects. The aim of this study was to characterize the lymphocyte composition according to the severity of COVID-19, as well as its degree of relationship to the specific humoral response to SARS-CoV-2 in convalescents up to 106 days after the infection and in their exposed relatives. MethodsAn applied research was carried out with a cross-section analytical design, from March 11 to June 11, 2020 in Cuba. The sample consisted of 251 convalescents from COVID-19 over 18 years of age and 88 exposed controls who did not become ill. The B and T cell subpopulations, including memory T cells, as well as the relationship with the humoral immune response against SARS-CoV-2, were identified by flow cytometry and enzyme immunoassay. ResultsConvalescent patients, who evolved with severe forms, showed a decrease in frequency and a greater proportion of individuals with values ​​lower than the minimum normal range of B cells, CD3 + CD4 + cells and the CD4 + / CD8 + ratio, as well as a higher frequency and a greater proportion of individuals with values ​​above the normal maximum range of CD3 + CD8 + and NK cells. Convalescent patients with severe forms of COVID-19 that exhibited IgG / RBD titers ≥ 1/200 had a lower frequency of TEMRA CD8 + cells (p = 0.0128) and TEMRA CD4 + (p = 0.0068). IgG / RBD titers were positively correlated with the relative frequency of CD4 + CM T memory cells (r = 0.4352, p = 0.0018). ConclusionsThe identified alterations of B and T lymphocytes suggest that convalescent patients with the severe disease could be vulnerable to infectious, autoimmune or autotinflammatory processes; therefore, these individuals need medical follow-up after recovering from the acute disease. Furthermore, the role of T cells CD4 + CM in the production of antibodies against SARS-CoV-2 is confirmed, and it is noted that the defect of memory T cells CD8 + TEMRA could contribute to the development of severe forms of COVID-19.

Immunophenotypic and functional analysis of lymphocyte subsets in common variable immunodeficiency patients without monogenic defects.

Common variable immunodeficiency (CVID) is accompanied by various lymphocyte abnormalities believed to be mostly responsible for disease features in patients with no diagnosed monogenic defects. Here, we evaluated the association of B and T lymphocyte abnormalities with the incidence of CVID. Twenty-six genetically unsolved CVID patients were examined for B and T lymphocyte subsets by flow cytometry and CD4+ T-cell proliferation by carboxyfluorescein succinimidyl ester (CFSE) test. We detected a reduction in total, naive, memory B cells and plasmablasts, and also total, naive, central memory and regulatory CD4+ T cells, besides naive CD8+ T cells. There was an increase in CD21low and transitional B cells, effector memory (EM) and terminally differentiated effector memory (TEMRA ) CD4+ T-cell subsets as well as total, EM, TEMRA , activated and cytotoxic CD8+ T cells among non-monogenic CVID patients. CD4+ T-cell proliferation response was reduced regarding both division index and percent divided. In conclusion, regarding the similarity of lymphocyte abnormalities between patients without genetic defects and those with monogenic defects, genetic mutations are not responsible for these specific lymphocyte changes. However, the novel correlations observed between lymphocyte alterations among genetically unsolved CVID patients may serve as a guide to predict the potential of future CVID development for hypogammaglobulinemia children.

Abstract 12876: Identification of Pathogenic Immune Cell Subsets in Immunotherapy-Induced Myocarditis

Introduction: Immune checkpoint inhibitors (ICIs) are monoclonal antibodies used to activate the immune system against tumor cells. Despite therapeutic benefits, ICIs have the potential to cause immune related adverse events (irAE) such as myocarditis, a rare but serious side effect with up to 50% mortality in affected patients. Histologically, patients with ICI myocarditis have lymphocytic infiltrates in the heart, implicating T-cell-mediated mechanisms. However, the precise pathologic immune subsets and molecular changes in ICI myocarditis are unknown. Hypothesis: We hypothesize that ICI myocarditis is associated with significant quantitative and molecular changes in pathogenic immune cell subsets. Methods: To identify immune subset(s) associated with ICI myocarditis, we performed time-of-flight mass cytometry (CyTOF) on peripheral blood mononuclear cells from 40 patients and controls with autoimmune adverse events (irAE) on ICI including four patients with ICI myocarditis. We also profiled 15 patients/controls using single cell T-cell receptor (TCR) sequencing and RNA sequencing (scRNA-seq) with feature barcoding for surface marker expression confirmation. Results: With both methods, we found expansion of cytotoxic CD8+ T effector cells re-expressing CD45RA (Temra CD8+ cells) in ICI myocarditis patients compared to controls. Using TCR sequencing, these CD8+ Temra cells were clonally expanded in myocarditis patients. Transcriptomic analysis of these Temra CD8+ clones confirmed a highly activated and cytotoxic phenotype with expression of granzyme/perforin. Longitudinal study demonstrated progression of these Temra CD8+ cells into an exhausted phenotype two months after treatment with glucocorticoids. Differential expression of proinflammatory chemokines (CCL4/CCL4L2/CCL5) was uncovered in clonally expanded Temra CD8+ cells, and ligand-receptor analysis implicated their regulation of other inflammatory cells such as monocytes and NK cells. Conclusions: Clonal cytotoxic Temra CD8+ cells are significantly increased in patients with ICI myocarditis, and have unique transcriptional changes, including upregulation of chemokines CCL4/CCL4L2/CCL5, which may serve as attractive diagnostic/therapeutic targets.

Expansion of Phenotypically Senescent CD57+ CD8 T Cells Is Associated with Impaired Immunocompetence after Allogeneic Hematopoietic Stem Cell Transplantation

Background: T cell senescence is a physiological process typically associated with aging. In addition, lymphopenia and chronic immune activation can result in T cell senescence. CD57, a member of the N-CAM family initially described as a natural killer cell marker, has been reported as a marker of human senescent CD8 T cells. Percentages of CD57+ CD8 T cells increase during aging as well as during chronic viral infections. Early studies have reported increased proportions of CD57-expressing CD8 T cells after autologous and allogeneic hematopoietic stem cell transplantation (HSCT). Whether these cells can be considered a counterpart of the senescent population found during aging is at present unknown. More importantly, whether the expansion of CD57+ CD8 T cells is associated with impaired immunocompetence after allogeneic HSCT remains to be investigated.Materials and Methods: With written informed consent, peripheral blood mononuclear cells were collected from healthy controls (HC, n=21) and patients undergoing allogeneic HSCT (n=115) at Geneva University Hospitals. Proportions of CD57+ CD8 T cells as well as phenotypic and functional characteristics of CD57+ CD8 T cells were assessed by flow cytometry. Virus-specific CD8 T cells were identified by flow cytometry based on IFNg and/or TNFa intra-cytoplasmic expression after 6h in vitro stimulation with peptides derived from CMV, EBV, HHV6, BKV and Adenovirus. Torque Teno Virus (TTV) replication was quantified by quantitative PCR as previously described (Pradier et al., Front Immunol 2020; doi: 10.3389/fimmu.2020.00998).Results: CD8 T cells recovered from allogeneic HSCT displayed significantly higher proportions of CD57+ cells compared with healthy controls (HC, median 23% [range 6%-67%]; HSCT 58% [11%-97%]; p= 8.1e−06; Figure 1A-B). Such a difference was detected at early time points after transplantation and further increased at later time points (Figure 1B). After taking into account T cell subsets heterogeneity, we observed higher proportions of CD57+ cells in CD45RA- CCR7+ CD27+ central memory (CM; p= 0.00057), CD45RA- CCR7- CD27+ transitional memory (TM; p=1.1e-05) and CD45RA- CCR7- CD27- effector memory (EM; p=2.6e−06) CD8 T cells from allogeneic HSCT recipients compared with healthy controls. We did not observe any significant differences in CD57 expression in naïve nor in TEMRA CD8 T cells. Phenotypically, CD57+ CD8 T cells from allogeneic HSCT recipients displayed a senescent immunophenotype characterized by the low surface expression of CD127 that was further downregulated in CD57+ CD8 T cells from allogeneic HSCT recipients compared with healthy controls (p=0.00067). Functionally, CD57+ CD8 T cells from allogeneic HSCT recipients displayed a similar capacity to produce IFN-g, TNF-a, granzyme B and perforin when compared to CD57+ CD8 T cells from healthy controls. Virus-specific CD8 T cells identified upon stimulation with CMV, EBV, HHV6, BKV and Adenovirus peptides mainly displayed a CD27- effector memory phenotype in HSCT recipients (Figure 1C) and expressed higher levels of CD57 in HSCT recipients compared with healthy controls for CMV (p=0.017(; EBV p=0.018; Figure 1C-D). with a trend not reaching significance was for adenovirus, HHV6 and BK-virus-specific CD8 T cells. Using the replication of the non-pathogenic anellovirus TTV as a measure of impaired immunocompetence, we observed that the proportion of CD57-expressing cells among effector memory CD8 T cells positively correlated with TTV titers in allogeneic HSCT recipients (R=0.32, p=0.019; Figure 1E).Conclusion: These results show that the proportion of phenotypically senescent CD57+ CD8 T cells increases after allogeneic HSCT as a result of an increased expression at the surface of memory CD8 T cells, including virus-specific cells. Moreover, CD57 expression at the surface of EM CD8 T cells, a highly enriched population in allogeneic HSCT recipients, correlated with higher replication of TTV, reflecting a status of impaired immunocompetence after allogeneic HSCT. Studies are ongoing to determine the utility of CD57 expression on T cells as a biomarker to predict infectious complications after allogeneic HSCT. [Display omitted] DisclosuresChalandon: Incyte, BMS, Pfizer, Abbie, MSD, Roche, Novartis, Gilead, Amgen, Jazz, Astra Zenec: Other: Travel EXpenses, Accomodation; Incyte: Speakers Bureau; Incyte, BMS, Pfizer, Abbie, MSD, Roche, Novartis, Amgen: Other: Advisory Board. Simonetta: BMS Cellgene: Other: Ad-Hoc Advisory Board.

Horticultural Therapy Reduces Biomarkers of Immunosenescence and Inflammaging in Community-Dwelling Older Adults: A Feasibility Pilot Randomized Controlled Trial.

BackgroundWith the challenges that aging populations pose to health care, interventions that facilitate alleviation of age-related morbidities are imperative. A prominent risk factor for developing age-related morbidities is immunosenescence, characterized by increased chronic low-grade inflammation, resulting in T-cell exhaustion and senescence. Contact with nature and associated physical activities have been shown to boost immunity in older adults and may be promoted in the form of horticultural therapy (HT). We aimed to examine the effects of HT on immunosenescence.MethodWe conducted a randomized controlled trial with 59 older adults assigned to either the HT intervention or waitlist control group. Older adults in the HT intervention group underwent HT intervention program over 6 months. Venous blood was drawn at baseline and at the third and sixth month from the commencement of this study. For participants who attended all 3 blood collection time points (HT: n = 22; waitlist: n = 24), flow cytometry analysis was performed on whole blood samples to evaluate the kinetics of lymphocyte subsets over the intervention period, revealing the composition of CD4+ and CD8+ subsets expressing exhaustion markers—CD57, CTLA4, and KLRG1. Enzyme-linked immunosorbent assays were employed to measure changes in plasma IL-6 levels.ResultsHT is associated with increased numbers of naive CD8+ T cells and fewer CTLA4-expressing terminally differentiated effector CD4+ and CD8+ memory T cells re-expressing CD45RA (TEMRA). Furthermore, IL-6 levels were reduced during HT, and the frequencies of naive and TEMRA CD8+ T cells were found to be associated with IL-6 levels.ConclusionHT is associated with a reduction in the levels of biomarkers that measure the extent of T-cell exhaustion and inflammaging in older adults. The positive effects of HT on T-cell exhaustion were associated with the reduction of IL-6 levels.

Peripheral immunological response to treatment with checkpoint inhibitor in recurrent/metastatic nasopharyngeal carcinoma.

e18519 Background: A better understanding of peripheral cellular phenotypes in association with checkpoint inhibitors (CPI) responsiveness in recurrent/metastatic NPC could profoundly impact our knowledge of NPC immunopathology. Methods: Blood were collected from 11 patients with recurrent/metastatic NPC who received CPI (pembrolizumab) after failing second-line chemotherapy. Response to pembrolizumab was assessed by imaging and EBV DNA. Patients who achieved CR/PR were considered responsive and those with SD/PD were considered non-responsive. Four patients in the responsive group also had baseline blood before pembrolizumab. PBMC were freshly isolated and stored until analysis. For surface staining, cells were rested overnight at 37°C before resuspended in BD Brilliant stain with 2.5ng/µl Fc block for 15mins. Cells were then co-stained: 7-AAD, CD19-BV510, CD3-Alexa Fluo 700, CD4-BV510, CD279(PD-1)-BB515, CD197(CCR7)-BV421, CD45RO-APC, CD45RA-Pe-Cy5, CD8-APC-H7, CD27-PE, CD95-PE-Cy7, incubated on ice for 30mins. Samples were washed and acquired on NovoCyte Quanteon. Immune phenotypes were correlated with clinical response. Results: We found that: 1) %CD3 was upregulated in responsive group; 2) CD4/CD8 ratio did not directly stratify drug responsiveness; 3) frequency of PD1-expressing CD8+ T cells was significantly reduced in responsive group; 4) lower frequency of CCR7+PD1+CD8+ T cells in responsive group, suggesting that these may be highly differentiated and have the ability to move into peripheral sites in response to inflammatory chemokines; 5) frequency of naïve and TEMRA CD8+ T cells, but not Tscm was upregulated in responsive group; 6) finally, an interesting finding of sustained CD19+ subsets was observed in non-responsive group. Conclusions: Our results suggest that peripheral blood analysis may provide valuable insights into NPC patients’ responses to PD-1-targeted therapies. [Table: see text]

Terminally Differentiated Effector Memory CD8+ T Cells Identify Kidney Transplant Recipients at High Risk of Graft Failure.

Identifying biomarkers to predict kidney transplant failure and to define new therapeutic targets requires more comprehensive understanding of the immune response to chronic allogeneic stimulation. We investigated the frequency and function of CD8+ T cell subsets-including effector memory (EM) and terminally differentiated EM (TEMRA) CD8+ T cells-in blood samples from 284 kidney transplant recipients recruited 1 year post-transplant and followed for a median of 8.3 years. We also analyzed CD8+ T cell reactivity to donor-specific PBMCs in 24 patients who had received living-donor kidney transplants. Increased frequency of circulating TEMRA CD8+ T cells at 1 year post-transplant associated with increased risk of graft failure during follow-up. This association remained after adjustment for a previously reported composite of eight clinical variables, the Kidney Transplant Failure Score. In contrast, increased frequency of EM CD8+ T cells associated with reduced risk of graft failure. A distinct TEMRA CD8+ T cell subpopulation was identified that was characterized by expression of FcγRIIIA (CD16) and by high levels of proinflammatory cytokine secretion and cytotoxic activity. Although donor-specific stimulation induced a similar rapid, early response in EM and TEMRA CD8+ T cells, CD16 engagement resulted in selective activation of TEMRA CD8+ T cells, which mediated antibody-dependent cytotoxicity. At 1 year post-transplant, the composition of memory CD8+ T cell subsets in blood improved prediction of 8-year kidney transplant failure compared with a clinical-variables score alone. A subpopulation of TEMRA CD8+ T cells displays a novel dual mechanism of activation mediated by engagement of the T-cell receptor or of CD16. These findings suggest that TEMRA CD8+ T cells play a pivotal role in humoral and cellular rejection and reveal the potential value of memory CD8+ T cell monitoring for predicting risk of kidney transplant failure.

High numbers of differentiated CD28null CD8+ T cells are associated with a lowered risk for late rejection and graft loss after kidney transplantation.

The hypothesis was tested that parameters of an aged T-cell compartment associate with the risk for late rejection after kidney transplantation. Recipients of a kidney transplant in the period 2007-2013 were (N = 365) were included. T cells were characterized prior to transplantation by flow cytometry as naive (CD45RO-CCR7+), central-memory (CD45RO+CCR7+), effector-memory (CD45RO-CCR7-) or terminally differentiated CD8+ Temra (CD45RO-/CCR7-/CD28-) cells. T cell telomere length and thymic output were assessed prior to transplantation in 202 recipients. Follow-up was until December 2018. The date of the first time of biopsy-proven late rejection (>6 months after transplantation) was used to calculate the rejection-free survival time. Fifty cases of biopsy-proven rejection were recorded. Thymic output and T cell telomere length did not associate with late rejection-free survival. However, the percentage and absolute numbers of CD8+Temra and CD28null CD8+ T cells were significantly lower in patients with late rejection. Specifically, in the highest tertile of percentages of CD28null CD8+ T cells, the cumulative incidence of late rejection at 5 and 10 years was only 5% and 8% compared to 16% and 20% in the middle to lowest tertile (p = 0.002). Multivariate proportional hazard analysis showed that percentage and absolute number of CD28null CD8+ T cells remained significantly associated with late rejection and rejection-related graft loss. High numbers of differentiated CD28null CD8+ T cells decrease the risk for late rejection and rejection-related graft loss after kidney transplantation.

Altered T Cell Subsets and Function in Patients with Acute Lymphoblastic Leukemia

Altered T Cell Subsets and Function in Patients with Acute Lymphoblastic Leukemia

TEMRA CD8 T Cells from Human Kidney Transplant Recipients Exhibit Potent Anti-Donor Reactivity and Induce GVHD in Humanized Mouse Model

Introduction We have shown that an increase of TEMRA CD8 (CD45RA+CCR7-) evidenced 1 year post-transplantation is associated with a higher risk of kidney graft failure. The functionality of TEMRA CD8 and especially their reactivity upon donor-specific stimulation have never been investigated. In this study, we characterize before and one year after transplantation the functional response of TEMRA CD8 from Kidney Transplantation (KT) recipients after donor-specific stimulation and we setup a model of humanized NSG mice to investigate in vivo their pro-inflammatory response. Methods PBMC from 24 living-donor kidney-transplant recipients have been prospectively collected before and 12 month post-transplantation. Extensive phenotype of CD8 T cell subsets have been performed using multi-parameter flow cytometry. Naïve, TEMRA and EM CD8 from KT recipients have been cultured with irradiated donor-specific CD3-depleted PBMC. Expression of activation marker (CD25, CD69), cytotoxic marker (CD107a) and secretion of pro-inflammatory cytokines (Luminex Assay) have been analyzed after 24h and 48h of stimulation. Proliferation (CPD dilution) has been investigated after 5 days of culture. One to 5.106 TEMRA CD8 purified from KT recipients were co-injected i.v. with 107 T- and NK-depleted autologous PBMC in NSG HLA-A*0201 transgeneic mice. Xenogeneic GVHD was monitored by daily evaluation of the body weight and CD8 T cell infiltration was investigated using fluorescent microscopy in different organs. Results Kidney transplantation results in an increase of TEMRA CD8 (25 vs 39%), characterized by a high expression of GZMb and effector-associated transcription factor T-bet, and a concomitant decrease of NAÏVE CD8 whereas frequency of EM CD8 remains unchanged. Donor-specific stimulation results in the early expression of CD25+CD69+CD107a+ by TEMRA CD8 whereas NAÏVE CD8 failed to upregulate CD25 or CD107a. Expression of cytotoxic molecule CD107a by TEMRA was even enhanced after kidney transplantation. Donor specific stimulation results in the proliferation of TEMRA CD8. Finally, the infusion of TEMRA CD8 from KT recipients induces GVHD in 7 to 10 days with an infiltration of TEMRA in colon. Conclusion We demonstrate that TEMRA CD8 exhibit potent pathogenic function both in vitro when challenged with donor cells and in vivo resulting in an acute GVHD in immunodeficient mice. Our study highlights the need to design TEMRA specific therapy to improve kidney graft outcome. IHU Cesti (ANR-10-IBHU-005). FP7 VISICORT (602470). Fondation Centaure. Labex IGO (ANR-11-LABX-0016-01).

Inclusion of CD8 Monitoring Improves the Prognostic Capacities of the Kidney Transplant Failure Score

Introduction Beside the classical monitoring of kidney graft function using serum creatinine or proteinuria levels, the assessment of other non-invasive biomarkers has been proposed to identify patients at-risk of kidney rejection. To reach a true clinical utility, the prognostic capacities of a biomarker have to be higher than other available metrics such as clinical-based scoring system. We have previously shown that an increase in TEMRA CD8 T cells is associated with a 2-fold higher risk of long-term graft dysfunction. In this study, we evaluate if the monitoring of CD8-related biomarkers could improve the prognostic capacities of a clinical-based scoring system (Kidney Transplant Failure Score; KTFS). Methods 286 kidney-transplant recipients have been prospectively enrolled and followed for more than 8 years. At the end of the follow-up time, 51 patients returned to dialysis. Targeted analysis of 22 CD8 T cell subsets have been performed on blood samples retrieved 12 months post-transplantation. Results The frequency of EM and TEMRA CD8 measured at one year post-transplantation is correlated with the risk to return in dialysis during the 8 years follow-up. Moreover, we show that the prognostic capacity of the KTFS can be improved by the inclusion of the CD8 markers as the AUC of the biomarker-updated KTFS is 0.75 as compared to 0.71 a single predictor. Finally, when clinical based KTFS was used as inclusion criteria, we demonstrate that the use of one-year frequency of TEMRA CD8 allows the discrimination of patients that will lose their graft from those that will maintain stable graft function. Conclusion The combination of CD8-related biomarkers with clinical-parameters based KTFS allows to better predict patients at-risk of kidney graft failure and to target those with a more specific immunologic risk. Such score, after further validation on large external cohorts, could be useful as decision tool in the clinical management of kidney transplant recipients. Labex IGO (ANR-11-LABX-0016-01). FP7 VISICORT (602470). ANR-11-JSV1-0008-01. ITMO Santé Publique (A13053NS). Fondation Centaure. IHU CESTI (ANR-10-IBHU-005).