Distribution Of T-cell Subsets Research Articles

Reverse aging to treat lupus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with multifaceted pathogenetic processes, including abnormalities of T-cell subset distribution and function. Accumulation of senescent CD4+ T cells has been found to contribute to the development of the disease. In this issue, Jiang etal. provide compelling evidence that links an expanded pool of CD4+CD57+ senescent T cells in patients with SLE to disease activity favored by interleukin-15. Importantly, treatment of lupus-prone mice with a senolytic drug resulted in decreased autoimmune pathology. The findings of this study suggest possible novel therapeutics to treat patients with SLE.

Diagnostic and prognostic value of double-negative T cells in colorectal cancer

ObjectiveTo evaluate the T-lymphocyte subset distribution and the diagnostic and prognosis value of double-negative T (DNT) cells in colorectal cancer (CRC). MethodsThis retrospective study compared the T-lymphocyte subsets and DNT of 114 patients with CRC with those of 107 healthy controls (HC). The diagnostic potential of DNT and T-lymphocyte subsets was assessed using the receiver operating characteristic (ROC) curve, and prognostic values were evaluated using the Kaplan–Meier curve and the Cox regression model. ResultsThe percentages of CD8+ T cells and DNT cells, and value of carcinoembryonic antigen (CEA), were remarkably higher in patients with CRC than in those with HC, but the ratio of CD4+/CD8+ was decreased. Using ROC curve analysis, DNT cell percentage, CEA, and CD4+/CD8+ ratio all had good diagnostic efficacy, with areas under the curve (AUCs) of 0.865, 0.786 and 0.624, respectively. The combination of DNT cell percentage and CEA had an AUC of 0.905, which was significantly higher than that of any single biomarker (p < 0.05). In univariate analysis, the Tumor Node Metastasis (TNM) clinical stage, CD4+/CD8+ ratio, and DNT cell percentage were significantly associated with overall survival (OS) (p < 0.05). In multivariate analysis, TNM clinical staging (HR = 2.37, 95 % CI: 1.15–4.90), a decreased CD4+/CD8+ ratio (HR = 0.33, 95 % CI: 0.15–0.74), and an increased DNT cell percentage (HR = 2.29, 95 % CI: 1.11–4.73) were independent prognostic factors for CRC. ConclusionThe percentage of DNT cells may be useful as an evaluation index for CRC diagnosis and prognosis, which was even better when combined with serum CEA.

Higher TIGIT+ γδ TCM cells may predict poor prognosis in younger adult patients with non-acute promyelocytic AML.

γδ T cells recognize and exert cytotoxicity against tumor cells. They are also considered potential immune cells for immunotherapy. Our previous study revealed that the altered expression of immune checkpoint T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) on γδ T cells may result in immunosuppression and is possibly associated with a poor overall survival in acute myeloid leukemia (AML). However, whether γδ T-cell memory subsets are predominantly involved and whether they have a relationship with clinical outcomes in patients with AML under the age of 65 remain unclear. In this study, we developed a multicolor flow cytometry-based assay to monitor the frequency and distribution of γδ T-cell subsets, including central memory γδ T cells (TCM γδ), effector memory γδ T cells (TEM γδ), and TEM expressing CD45RA (TEMRA γδ), in peripheral blood from 30 young (≤65 years old) patients with newly diagnosed non-acute promyelocytic leukemia (also known as M3) AML (AMLy-DN), 14 young patients with AML in complete remission (AMLy-CR), and 30 healthy individuals (HIs). Compared with HIs, patients with AMLy-DN exhibited a significantly higher differentiation of γδ T cells, which was characterized by decreased TCM γδ cells and increased TEMRA γδ cells. A generally higher TIGIT expression was observed in γδ T cells and relative subsets in patients with AMLy-DN, which was partially recovered in patients with AMLy-CR. Furthermore, 17 paired bone marrow from patients with AMLy-DN contained higher percentages of γδ and TIGIT+ γδ T cells and a lower percentage of TCM γδ T cells. Multivariate logistic regression analyses revealed the association of high percentage of TIGIT+ TCM γδ T cells with an increased risk of poor induction chemotherapy response. In this study, we investigated the distribution of γδ T cells and their memory subsets in patients with non-M3 AML and suggested TIGIT+ TCM γδ T cells as potential predictive markers of induction chemotherapy response.

Reduced mitochondrial respiration in peripheral T cells after paediatric heamatopoietic stem cell transplantation.

Recovery and functional differentiation of T-cell subsets are central for the development of immune function and complications after allogeneic hematopoietic stem cell transplantation (HSCT), but little is known about the cellular respiration and factors influencing T-cell metabolic fitness during immune maturation after HSCT. We included 20 HSCT patients and analysed mitochondrial oxidative phosphorylation and mitochondrial fitness in peripheral blood mononuclear cell samples collected at days +90 and +180 after HSCT. Phenotypic analysis revealed lower overall T-cell counts, lower CD4+/CD8+ ratio and a skewed distribution of early T-cell subsets at day +90, gradually recovering by day +180. Although ATP turnover in HSCT patients was similar to healthy controls, the spare respiratory capacity (SRC) of T cells, reflecting the available energy reserve, was significantly reduced at day +90 and +180 compared to healthy controls. This reduction in SRC was not correlated with the occurrence of acute graft-versus-host disease (aGVHD), the intensity of conditioning regimens and markers of T-cell exhaustion. We found significantly depressed SRC until six months post-HSCT, but we were not able to identify transplant-related risk factors or associations with the clinical outcome.

Comparison of Recovery Effect for Sufentanil vs. Remifentanil Anesthesia in Elderly Patients Undergoing Curative Resection for Hepatocellular Carcinoma.

The aim of this work is to evaluate the clinical efficacy and safety of sufentanil vs. remifentanil anesthesia in elderly patients undergoing curative resection for hepatocellular carcinoma (HCC). Medical records of elderly patients aged ≥ 65years who received curative resection for HCC between January 2017 and December 2020 were retrospectively reviewed. The patients were divided into either the sufentanil group or the remifentanil group according to the method of analgesia used. Vital signs including mean arterial pressure (MAP), heart rate (HR), and arterial oxygen saturation (SpO2), distribution of T-cell subsets (CD3, CD4, and CD8 lymphocytes), distribution of the stress response index [cortisol (COR), interleukin (IL)-6, C-reactive protein (CRP), and glucose (GLU)] were recorded prior to anesthesia (T0), after induction of anesthesia (T1), at the end of surgery (T2), 24h after surgery (T3), and 72h after surgery (T4). Postoperative adverse events were collected. Repeated measure analysis of variance (ANOVA) showed that after controlling for baseline patient demographic and treatment characteristics as covariates, both between- and within-group effects were significant (all P < 0.01), and the interaction between time and treatments was also significant (all P < 0.01) in the vital signs (MAP, HR, and SpO2), distribution of T-cell subsets (CD3, CD4, and CD8 lymphocytes), and distribution of the stress response index (COR, IL-6, CRP, and GLU), indicating that sufentanil led to stable hemodynamic and respiratory functions, lower reduction of T-lymphocyte subsets, and stable stress response indices compared to remifentanil. There is no significant difference in adverse reactions between the two groups (P = 0.72). Sufentanil was associated with improved hemodynamic and respiratory function, less stress response, less inhibition of cellular immunity, and similar adverse reactions compared with remifentanil.

T-Cells Subsets in Castleman Disease: Analysis of 28 Cases Including Unicentric, Multicentric and HHV8-Related Clinical Forms.

Castleman disease (CD) is a rare lymphoproliferative disorder that includes various clinico-pathological subtypes. According to clinical course, CD is divided into unicentric CD (UCD) and multicentric CD (MCD). MCD is further distinguished based on the etiological driver in herpes virus-8-related MCD (that can occur in the setting of HIV); in MCD associated with POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin changes); and idiopathic MCD (iMCD). The latter can also be divided in iMCD-TAFRO (thrombocytopenia, anasarca, fever, myelofibrosis, organomegaly) and iMCD not otherwise specified. To date, CD pathogenesis is still uncertain, but CD may represent the histological and clinical result of heterogeneous pathomechanisms. Transcriptome investigations in CD lymph nodes have documented the expression and up-regulation of different cytokines; furthermore, few recent studies have shown alterations of different T-cell subsets in CD patients, suggesting a possible role of the nodal microenvironment in CD development. On this basis, our study aimed to investigate the distribution of T-cell subsets in the clinico-pathological spectrum of CD. We evaluated the CD4/CD8 ratio and the number of T-regulatory (T-reg) FOXP3+ cells in 28 CD cases. In total, 32% of cases showed a decreased CD4/CD8 ratio due to increased CD8+ T-cells, including both UCD, iMCD, and HHV8+ MCD cases. The T-reg subset analysis revealed a statistically significant (p < 0.0001) lower mean number of FOXP3+ T-reg cells in CD cases when compared with non-specific reactive lymph nodes. We did not find statistically significant differences in T-reg numbers between the different CD subtypes. These findings may suggest that alterations in T-cell subpopulations that can lead to disruption of immune system control may contribute to the numerous changes in different cellular compartments that characterize CD.

Peripheral differentiation patterns of human T cells.

Long‐term T‐cell memory is dependent on the maintenance of memory T cells in the lymphoid tissues, and at the surface interfaces that provide entry routes for pathogens. However, much of the current information on human T‐cell memory is based on analyzing circulating T cells. Here, we have studied the distribution and age‐related changes of memory T‐cell subsets in samples from blood, mesenteric LNs, spleen, and ileum, obtained from donors ranging in age from 5 days to 67 years of age. Our data show that the main reservoir of polyclonal naive cells is found in the LNs, and the resting memory subsets capable of self‐renewal are also prominent there. In contrast, nondividing but functionally active memory subsets dominate the spleen, and especially the ileum. In general, the replacement of naive cells with memory subsets continues throughout our period of observation, with no apparent plateau. In conclusion, the analysis of lymphoid and nonlymphoid tissues reveals a dynamic pattern of changes distinct to each tissue, and with substantial differences between CD4+ and CD8+ compartments.

Altered T-cell subset distribution in the viral reservoir in HIV-1-infected individuals with extremely low proviral DNA (LoViReTs).

BackgroundHIV cure strategies aim to eliminate viral reservoirs that persist despite successful antiretroviral therapy (ART). We have previously described that 9% of HIV‐infected individuals who receive ART harbor low levels of provirus (LoViReTs).MethodsWe selected 22 LoViReTs matched with 22 controls ART suppressed for more than 3 years with fewer than 100 and more than 100 HIV‐DNA copies/106 CD4+ T cells, respectively. We measured HIV reservoirs in blood and host genetic factors. Fourteen LoViReTs underwent leukapheresis to analyze replication‐competent virus, and HIV‐DNA in CD4+ T‐cell subpopulations. Additionally, we measured HIV‐DNA in rectum and/or lymph node biopsies from nine of them.ResultsWe found that LoViReTs harbored not only lower levels of total HIV‐DNA, but also significantly lower intact HIV‐DNA, cell‐associated HIV‐RNA, and ultrasensitive viral load than controls. The proportion of intact versus total proviruses was similar in both groups. We found no differences in the percentage of host factors. In peripheral blood, 71% of LoViReTs had undetectable replication‐competent virus. Minimum levels of total HIV‐DNA were found in rectal and lymph node biopsies compared with HIV‐infected individuals receiving ART. The main contributors to the reservoir were short‐lived transitional memory and effector memory T cells (47% and 29%, respectively), indicating an altered distribution of the HIV reservoir in the peripheral T‐cell subpopulations of LoViReTs.ConclusionIn conclusion, LoViReTs are characterized by low levels of viral reservoir in peripheral blood and secondary lymphoid tissues, which might be explained by an altered distribution of the proviral HIV‐DNA towards more short‐lived memory T cells. LoViReTs can be considered exceptional candidates for future interventions aimed at curing HIV.

Increased cytotoxic T-cells in the airways of adults with former bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) in preterm-born infants is a risk factor for chronic airway obstruction in adulthood. Cytotoxic T-cells are implicated in COPD, but their involvement in BPD is not known. To characterise the distribution of airway T-cell subsets in adults with a history of BPD. Young adults with former BPD (n=22; median age 19.6 years), age-matched adults born preterm (n=22), patients with allergic asthma born at term (n=22) and healthy control subjects born at term (n=24) underwent bronchoalveolar lavage (BAL). T-cell subsets in BAL were analysed using flow cytometry. The total number of cells and the differential cell counts in BAL were similar among the study groups. The percentage of CD3+CD8+ T-cells was higher (p=0.005) and the proportion of CD3+CD4+ T-cells was reduced (p=0.01) in the BPD group, resulting in a lower CD4/CD8 ratio (p=0.007) compared to the healthy controls (median 2.2 versus 5.3). In BPD and preterm-born study subjects, both CD3+CD4+ T-cells (rs=0.38, p=0.03) and CD4/CD8 ratio (rs=0.44, p=0.01) correlated positively with forced expiratory volume in 1 s (FEV1). Furthermore, CD3+CD8+ T-cells were negatively correlated with both FEV1 and FEV1/forced vital capacity (rs= -0.44, p=0.09 and rs= -0.41, p=0.01, respectively). Young adults with former BPD have a T-cell subset pattern in the airways resembling features of COPD. Our findings are compatible with the hypothesis that CD3+CD8+ T-cells are involved in mechanisms behind chronic airway obstruction in these patients.

Evolving Exhaustion of T Cells during the Course of the Disease in AML Can be Abrogated By CD33 BiTE ® Construct Mediated Cytotoxicity

Evolving Exhaustion of T Cells during the Course of the Disease in AML Can be Abrogated By CD33 BiTE ® Construct Mediated Cytotoxicity

P-064: Heterogeneity of bone marrow biopsy and bone marrow aspirate (BMA) in patients with heavily pretreated relapsed/refractory Multiple Myeloma (RRMM)

<h3>Background</h3> Despite new regimens approved for RRMM, patients still relapse, and new therapies are needed. T-cell directed therapies that target bone marrow plasma cells (BMPCs), including CD3 bispecific antibodies, have shown promise for the treatment of RRMM. REGN5458, a B-cell maturation antigen (BCMA) xCD3 bispecific antibody, demonstrated an acceptable safety and tolerability profile with early, deep, and durable efficacy in heavily pretreated and triple refractory patients with RRMM [1]. <h3>Aim</h3> To elucidate disease aspects amenable to T-cell-directed therapies targeting plasma cells, such as REGN5458, we characterized the plasma and immune composition of baseline BMA and core biopsies from patients with RRMM, and evaluated relationships between bone marrow profile, secretory measures of disease, and response. <h3>Methods</h3> Patients with progressive RRMM (triple refractory or intolerant to prior lines of systemic therapy, including a proteasome inhibitor, immunomodulatory agent, and anti-CD38 antibody), were treated with REGN5458 (NCT03761108). Matched BMA and biopsy samples obtained at screening were analyzed by multiplex immunohistochemistry (IHC, N=48) and multiparameter flow cytometry (N=81). Cell numbers and phenotypes were correlated to secretory measures of disease, including serum M-protein, immunoglobulin-free light chain (FLC) assays and R-ISS staging. <h3>Results</h3> Biopsy analysis revealed a variable BMPC burden in RRMM patients. Median percentage of BMPCs by CD138 IHC was 22.5% (range 1–95%). There was no correlation between BMPC burden and serum M-protein levels, but a modest correlation between BMPC burden and serum FLC (rho=0.66; p=0.01). The majority of CD138 biopsy BMPCs in RRMM patients expressed BCMA by IHC; levels varied between patients (median BCMA+ H scores: 45 and 200 for membrane and cytoplasmic staining). BCMA IHC expression was not associated with IMWG response to REGN5458,¹ and did not correlate with soluble BCMA levels at study initiation. BMA analysis by flow cytometry revealed variable levels of abnormal BMPCs in patients with RRMM (median: 2.2% [range 0–68.7%]). BCMA expression was variable in all BMPCs; 53.1% of patients had higher BCMA staining intensity relative to CD38: ratios ranged from 1.2–6.6. Frequency of CD3+ T-cells in CD45+ BMA from RRMM patients ranged from 1.0–63.5%. Preliminary analysis suggests a higher frequency of T cells in R-ISS stage I compared to stage II. T-cell subset distribution in BMA from patients with RRMM reflected low CD8 T_CM (5.0%) and predominantly T_EM (37.0%) and T_EMRA (41.0%). <h3>Conclusions</h3> Analyses of matched BMA and biopsy samples from heavily pretreated RRMM patients, reveal that most BMPC express BCMA, though soluble vs membrane-bound BCMA levels are heterogenous. Diversity in immune and plasma cell markers and subsets is also observed. Ongoing analyses include correlations of plasma and immune cell profile with response to T-cell-targeting therapies such as REGN5458.

PI3K-δ Inhibition Influences T-Cell Populations and Anti-Tumoral Immune Function in Preclinical Models

Chronic lymphocytic leukemia (CLL) is characterized by continuously activated B-cell receptor (BCR) signaling. Phosphoinositide 3-kinase delta (PI3K-δ) is an important mediator of BCR signaling, which controls cell survival and proliferation through activation of AKT. The PI3K-δ inhibitor idelalisib (IDELA) is a highly effective treatment option for relapsed/refractory CLL patients. In the frontline setting, IDELA in treatment-naïve patients was associated with frequent adverse effects (AEs), such as an increased infection rate, colitis, and pneumonitis. Therefore, understanding the impact of PI3K-δ inhibition on the immune cells in the CLL microenvironment is of importance to identify novel treatment strategies to overcome these AEs.Methods and Results: In this study, we specifically focused on the effect of PI3K-δ inhibition on T-cell function in CLL using the Eµ-TCL1 adoptive transfer (TCL1 AT) model. In brief, Eµ-TCL1 tumor cells were transferred intravenously into syngeneic littermates, followed by continuous treatment after reaching a tumor load of 50% in peripheral blood. GS-649443, a tool compound with more favorable murine pharmacokinetic properties than IDELA, was used for PI3K-δ inhibition in vivo . Tumor load, as well as the immune microenvironment, was analyzed after 2 weeks of continuous treatment of the drug.Since PI3K-δ signaling has been previously demonstrated to be important for CD4+ regulatory T-cell (Treg)-mediated suppression of antitumor immunity, we studied the impact of PI3K-δ inhibition on differentiation and activation of Tregs. GS-649443 treatment resulted in a dramatic reduction in numbers of Foxp3+ CD25+ CD4+ Tregs, as well as a decrease in Treg maturation and activation markers (CD103, GITR, KLRG1, and CD69) compared to vehicle treatment.We further assessed the impact of PI3K-δ inhibition on TCL1 AT-induced changes in CD8+ T-cell differentiation and subset distribution. Notably, treatment with GS-649443 resulted in a dose-dependent decrease in effector T-cell numbers, accompanied by a decrease in activation markers such as CD69 and CD137. In line with these findings, effector cell proliferation, as measured by Ki-67, significantly decreased in GS-649443-treated mice. In accordance with that, degranulation capacity, granzyme B, and IFNγ production by CD8+ T cells was substantially reduced by GS-649443 treatment. These results were further confirmed in a treatment study with PI-3065, another PI3K-δ-specific inhibitor.To investigate a direct impact of PI3K-δ inhibition on CD8+ T cells, we performed in vitro studies with GS-649443. For that purpose, we used splenocytes from Nur77-GFP mice, a reporter mouse model for T-cell receptor (TCR) activity, and stimulated these with aCD3 antibody after pre-treatment with GS-649443 or vehicle. PI3K-δ inhibition led to a profound reduction of TCR signaling, which was accompanied by decreased expression of activation markers like CD69, CD44, CD25, and CD137 on CD8+ T-cells. Furthermore, GS-649443 pre-treatment resulted in significant dose-dependent decrease in CD8+ T-cell proliferation rates, as measured by CFSE dilution. These findings were confirmed using aCD3-stimulated human peripheral blood mononuclear cells that were pretreated with IDELA in vitro, which resulted in an impairment of CD8+ T-cell activation measured by CD69 and CD25 expression, as well as proliferation measured by CFSE dilution.Conclusion: This study clearly shows that PI3K-δ inhibition abrogates the immunosuppressive activity of Tregs in an in vivo model of CLL. Additionally, PI3K-δ inhibition of CD8+ T cells leads to decreased activation and proliferation of these cells, which might contribute to the immune-mediated AEs observed with IDELA when used as frontline treatment. DisclosuresStilgenbauer:Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding.

Cytomegalovirus infection is associated with an increase in aortic stiffness in older men which may be mediated in part by CD4 memory T-cells.

Human Cytomegalovirus (CMV) infection is associated with atherosclerosis, higher cardiovascular disease (CVD) risk, and an increase in memory T-cells (Tmem). T-cells have also been implicated in CVD, independently of CMV infection. To better understand the CMV-associated CVD risk, we examined the association between CMV (IgG) serostatus and central aortic (carotid-to-femoral) pulse wave velocity (cfPWV), an early, independent predictor of CVD. We also investigated if such an association might be reflected by the distribution of Tmem and/or other T-cell subsets.Methods: Healthy older volunteers (60-93 years) underwent routine clinical and laboratory evaluation, including assessment of cfPWV in eligible participants. Flow-cytometry was used to assess proportions of memory T-cells, CD28null T-cells, and CMV-specific T-cells. The following associations were examined; CMV serostatus/cfPWV, CMV serostatus/proportion of Tmem, proportion of Tmem/cfPWV, CD28null T-cells/cfPWV, and CMV-specific T-cells/cfPWV. Linear regression models were used to adjust for age, sex, socioeconomic status, smoking, waist-to-hip ratio, cholesterol, and blood pressure as required.Results: Statistically significant positive associations were found (P-values for the fully adjusted models are given); CMV serostatus/cfPWV in men (P ≤ 0.01) but not in women, CMV serostatus/proportions of CD4 Tmem in men (P ≤ 0.05) but not in women; proportions of CD4 Tmem/cfPWV among CMV seropositive (CMV+) people (P ≤ 0.05) but not CMV seronegative (CMV-) people.Conclusion: CMV infection increases the CVD risk of older men by increasing cfPWV. This may be mediated in part by increased proportions of CD4 Tmem, higher numbers of which are found in CMV+ older people and more so among men than women. Given the high prevalence of CMV worldwide, our findings point to a significant global health issue. Novel strategies to mitigate the increased CVD risk associated with CMV may be required.

Baseline Biomarkers of T-Cell Function Correlate with Clinical Responses to Odronextamab (REGN1979), and Loss of CD20 Target Antigen Expression Identified As a Mechanism of Treatment Resistance

Baseline Biomarkers of T-Cell Function Correlate with Clinical Responses to Odronextamab (REGN1979), and Loss of CD20 Target Antigen Expression Identified As a Mechanism of Treatment Resistance

Altered T-cell subset repertoire affects treatment outcome of patients with myelofibrosis.

Phenotypic characterization of T cells in myelofibrosis is intriguing because of increased inflammation, markedly elevated pro-inflammatory cytokines, and altered distribution of T-cell subsets. Constitutive activation of Janus kinase-2 (JAK2) in the majority of patients with myelofibrosis contributes to the expression of the programmed cell death protein-1 (PD1) and T-cell exhaustion. We wondered whether T-cell activation affects treatment outcome of patients with myelofibrosis and sought to determine whether the JAK1/2 inhibitor ruxolitinib affects the activation of T-cell subsets. T cells from 47 myelofibrosis patients were analyzed and the percentages of either helper (CD4+) or cytotoxic (CD8+) naïve, central memory, effector memory, or effector T cells; and fractions of PD1-expressing cells in each subset were assessed. Higher numbers of T cells co-expressing CD4/PD1 and CD8/PD1 were found in myelofibrosis patients than in healthy controls (n=28), and the T cells were significantly skewed toward an effector phenotype in both CD4+ and CD8+ subsets, consistent with a shift from a quiescent to an activated state. Over the course of ruxolitinib treatment, the distribution of aberrant T-cell subsets significantly reversed towards resting cell phenotypes. CD4+ and CD8+ subsets at baseline correlated with monocyte and platelet counts, and their PD1+ fractions correlated with leukocyte counts and spleen size. Low numbers of PD1+/CD4+ and PD1+/CD8+ cells were associated with complete resolution of palpable splenomegaly and improved survival rate, suggesting that low levels of exhausted T cells confer a favorable response to ruxolitinib treatment.

Increased systemic inflammation and altered distribution of T-cell subsets in postmenopausal women.

AimTo investigate markers of systemic inflammation in pre- and postmenopausal women and identify possible predictors of systemic inflammation with menopause.MethodsCross-sectional study of 69 healthy women between 45- and 60 years. Blood samples were collected to assess leukocyte subsets and plasma cytokines. MRI and DXA scans were performed to assess body composition. Through uni- and multivariate analyses, follicle-stimulating hormone (FSH), visceral fat mass and age were evaluated as predictors of systemic inflammation in relation to menopause.ResultsPostmenopausal women tended to have higher leukocyte counts (5.4 x109 vs. 4.9 x109 cells/l, p = 0.05) reflected in increased total lymphocytes (1.8 x109 vs. 1.6 x109 cells/l, p = 0.01) and monocytes (0.5 x109 vs. 0.4 x109 cells/l, p = 0.02), compared to premenopausal women. Increased visceral fat mass was a strong predictor of high leukocyte subsets. Postmenopausal women had higher plasma TNF-α (2.24 vs. 1.91 pg/ml, p = 0.01) and IL-6 (0.45 vs. 0.33 pg/ml, p = 0.004) compared to premenopausal women and high FSH was a significant predictor of increased plasma TNF-α, IL-1β and IL-6. Menopause was further associated with increased T-cells (1,336 vs. 1,128 cells/μl, p = 0.04) reflected in significantly higher counts of exhausted-, senescent-, and memory CD4+ T-cell subsets.ConclusionsMenopause is associated with increased systemic inflammation as well as exhausted- and senescent T-cells. We suggest, that both increased visceral fat mass and declining sex hormone levels might contribute to postmenopausal systemic inflammation and calls for further large-scale studies to confirm these findings.

Regulatory T-cell subset distribution in children with primary hypertension is associated with hypertension severity and hypertensive target organ damage.

The relationship between circulating regulatory T-cell (Tregs) subset distribution and hypertension severity in children with primary hypertension is not known. We aimed to find out if target organ damage (TOD) in children with primary hypertension is related to defects in Tregs distribution reflected by their phenotype characteristics. The study constituted 33 nontreated hypertensive children and 35 sex-matched and age-matched controls. Using multicolor flow cytometry technique, we assessed a distribution of the total Tregs (CD4CD25CD127) and their subsets (CD45RA-naive Tregs, CD45RA memory/activated Tregs, CD45RACD31 recent thymic emigrants Tregs and mature naive CD45RACD31 Tregs) in the whole blood. Hypertensive children showed decreased percentage of the total Tregs, the CD45RA-naive Tregs, the total CD31 Tregs and the recent thymic emigrants Tregs but elevation of the CD45RA memory/activated Treg and mature naive CD45RACD31 Tregs. Decreased frequency of the total Tregs, naive Tregs and CD31-bearing Treg cell subsets (CD31 total Tregs, CD45RACD31 recent thymic emigrants Tregs) negatively correlated to TOD markers, arterial stiffness and blood pressure elevation. In contrast, increased percentage of memory Tregs and CD31 Tregs subsets positively correlated to organ damage markers, arterial stiffness and blood pressure values. These changes were independent of BMI, age, sex and hsCRP. Both diagnosis of hypertension, TOD and arterial stiffness in hypertensive children were associated with decreased population of total CD4 Tregs, limited output of recent thymic emigrants Tregs, and increased pool of activated/memory Tregs. Hypertension was an independent predictor of the circulating Treg subsets distribution irrespective of hsCRP.

Clonality, Antigen Recognition, and Suppression of CD8+ T Cells Differentially Affect Prognosis of Breast Cancer Subtypes.

In breast cancer, response rates to immune therapies are generally low and differ significantly across molecular subtypes, urging a better understanding of immunogenicity and immune evasion. We interrogated large gene-expression data sets including 867 node-negative, treatment-naïve breast cancer patients (microarray data) and 347 breast cancer patients (whole-genome sequencing and transcriptome data) according to parameters of T cells as well as immune microenvironment in relation to patient survival. We developed a 109-immune gene signature that captures abundance of CD8 tumor-infiltrating lymphocytes (TIL) and is prognostic in basal-like, her2, and luminal B breast cancer, but not in luminal A or normal-like breast cancer. Basal-like and her2 are characterized by highest CD8 TIL abundance, highest T-cell clonality, highest frequencies of memory T cells, and highest antigenicity, yet only the former shows highest expression level of immune and metabolic checkpoints and highest frequency of myeloid suppressor cells. Also, luminal B shows a high antigenicity and T-cell clonality, yet a low abundance of CD8 TILs. In contrast, luminal A and normal-like both show a low antigenicity, and notably, a low and high abundance of CD8 TILs, respectively, which associates with T-cell influx parameters, such as expression of adhesion molecules. Collectively, our data argue that not only CD8 T-cell presence itself, but rather T-cell clonality, T-cell subset distribution, coinhibition, and antigen presentation reflect occurrence of a CD8 T-cell response in breast cancer subtypes, which have been aborted by distinct T-cell-suppressive mechanisms, providing a rationale for subtype-specific combination immune therapies.

Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib Show Expansion of T-Cell Clonotypes Composed of Antitumor Cytotoxic CD8+ T-Cells

We recently showed that ibrutinib induced a more oligoclonal T-cell receptor (TCR) repertoire in patients with chronic lymphocytic leukemia (CLL). Using next generation sequencing (NGS), we found that overrepresented TCRβ clonotypes were mostly patient-specific and that the degree of oligoclonal expansion correlated with CD8+ T-cell counts. Here, we tested the hypothesis that overrepresented clonotypes are CD8+ T-cells that respond to tumor antigens. Flow cytometric profiling of TCR Vβ (TRBV) chain usage, T-cell subset distribution, and granzyme B (GrB) expression was performed on cryopreserved peripheral blood mononuclear cells obtained at baseline and during response to single-agent ibrutinib (n = 5). Antibodies against TRBV were selected to investigate the ten most abundant TCRβ clonotypes identified by NGS. The cumulative frequency of these clonotypes evaluable by flow cytometry increased at the time of response (mean 31.2% ± SEM 3.9% of all T-cells) compared to baseline (mean 24.1% ± SEM 2.3% of all T-cells). Within each TRBV subpopulation, the proportion of CD4+ T-cells decreased (mean fold-change 0.78 ± SEM 0.04), while the proportion of CD8+ T-cells increased (mean fold-change 1.79 ± SEM 0.36). We further observed that the TRBV clonotypes expanding the most during treatment were comprised exclusively of CD8+ T-cells in every patient. These highly expanded CD8+ clonotypes expressed GrB, consistent with a cytotoxic phenotype. To evaluate whether expanding CD8+ clonotypes are tumor-specific, T-cells were expanded ex vivo in two conditions: (1) co-culture with autologous CLL cells, CD40L, IL-2, and IL-7 or (2) with anti-CD3/CD28/CD137 beads, IL-2, and IL-7. Overall, T-cells expanded with autologous CLL cells had a higher proportion of CD8+ GrB+ cells than T-cells expanded with anti-CD3/CD28/CD137 beads. The dominant CD8+ clonotypes detected in each patient at the time of response were preferentially expanded by co-culture with autologous CLL cells compared to expansion with beads. The biased expansion of cytotoxic T-cell clonotypes in the CLL-primed T-cell product supports a TCR mediated response to tumor antigens. Next, cytotoxicity assays were performed by mixing the expanded T-cell products described above with autologous CLL cells at an effector to target ratio of 3:1, 1:1 and 1:3. CLL-primed T-cells killed CLL cells in a dose-dependent fashion. In contrast, bead-expanded T-cells were ineffective at killing CLL cells or even protected tumor cells from spontaneous apoptosis. Thus, tumor-specific cytotoxic T-cells expand during treatment with ibrutinib and are further enriched ex vivo by co-culture with autologous CLL cells. In summary, increased TCR oligoclonality in patients responding to ibrutinib is driven by the expansion of specific CD8+ clonotypes. These clonotypes expand in co-culture with, and are cytotoxic against, autologous CLL cells. Tumor-specific CD8+ T-cell clonotypes may contribute to the overall treatment response with ibrutinib and supports the investigation of therapeutic strategies combining ibrutinib with T-cell directed immunotherapy. Disclosures Baskar: NIH: Patents &amp; Royalties: ROR1 mAb 2A2. Wiestner:Nurix: Research Funding; Acerta: Research Funding; Merck: Research Funding; Pharmayclics: Research Funding.

P930Inflammatory leukocyte phenotypes are associated with increased mortality after transfemoral transcatheter aortic valve implantation

Abstract Background Systemic inflammatory response syndrome (SIRS) was shown to be a strong predictor of mortality in patients undergoing transcatheter aortic valve implantation (TAVI). However, given the rather non-specific nature of the SIRS criteria and their limited applicability in the modern era of TAVI, including lower periprocedural complication rates and shorter hospitalization periods in experienced competence centers, there is a need for defining novel prognostic inflammatory signatures for improved patient risk stratification. Thus, the objective of the present study was to characterize and assess the prognostic relevance of circulating leukocyte subsets, including phenotypical heterogeneity of monocytes and effector T cells, before and at various times after transfemoral TAVI. Methods and results 129 consecutive patients (59% male, mean age 82.3±5.6 years) with severe symptomatic aortic stenosis (Pmean 44.2±17mmHg), and high or prohibitive operative risk (mean EuroSCORE II 5.9; STS score 4.1) admitted to our clinic for TAVI were included into the study. Peripheral blood samples were obtained pre-procedurally (baseline, BL), directly after the intervention, and at 24h and 3 days after TAVI, and analyzed for inflammatory and cardiac biomarkers, including hs-CRP, IL-6, hs-TropT, and NT-proBNP. Differential myeloid and T-cell subset (Th1, Th2, Th17, Th1/Th17, Th22, Tregs) distribution and kinetics were analyzed using multiparameter flow cytometry. Neutrophil (P&lt;0.001 vs. BL) as well as classical and intermediate monocyte counts were significantly elevated at 24h (both p&lt;0.0001 vs. BL), whereas non-classical monocytosis developed 3 days after TAVI (P&lt;0.0001 vs. BL). Among CD4+ T-cell subsets, the percentage of Tregs and Th17 significantly increased (both P&lt;0.0001 at 24h vs. BL) after valve implantation. Remarkably, these changes were independent on the valve type (balloon- vs. self-expandable) and no significant effects of predilatation were observed (p&gt;0.05 for all cell subsets). Univariate analysis showed that elevated levels of NT-proBNP (HR: 3.4, 95% CI: 1.7–6.8; P=0.0005), hsCRP (HR: 1.4, 95% CI: 1.2–1.7; P=0.0003), and IL-6 (HR: 1.0, 95% CI: 1.0–1.03; P=0.0007), lower counts of Th2 cells (HR: 0.94, 95% CI: 0.90–0.94; P=0.0045), as well as increased percentages of Th17 cells (HR: 1.2, 95% CI: 1.0–1.4; P=0.023), and of non-classical monocytes (HR=1.019, 95% CI: 1.001–1.039; P=0.049) were independently associated with 12-month all-cause mortality. When included in the regression model with STS score, these inflammatory biomarkers provided higher area under ROC curve and category-free net reclassification improvementof 59% at 1 year (P=0.0001). ROC curves inflammation markers add STS Conclusions Our findings demonstrate for the first time an association of inflammatory leukocyte phenotypes with increased mortality after TAVI. Specific monocytic and T-cell signatures might therefore provide novel additive biomarkers to improve individual risk stratification in patients with severe aortic stenosis.