Cell Lymphocyte Subsets Research Articles

Expression and Significance of PD-1/PD-L1 in MDS Blast Cells, T Lymphocyte Cell Subsets and Treg Cells

To investigate the expression and significance of PD-1/PD-L1 in MDS blast cells, T lymphocyte cell subsets and Treg cells. Eighty-eight MDS patients and 19 AML patients were collectd as the study subjects, and Iron deficiency anemia and healthy bone marrow donors were used as control group. The expression of PD-1/PD-L1 in MDS/AML blast cells, T lymphocyte cell subsets and Treg cells was detected by flow cytometry, and the expression level of Th1/Th2/Th17-related cytokines in peripheral serum was detected. The expression of PD-1/PD-L1 in blast cells, T lymphocyte cell subsets and Treg cells in low risk MDS group were lower than that in control group, medium and high risk MDS group and AML group（all P < 0.01）, and Th1/Th17 type cytokines were dominant. The expression of PD-1/PD-L1 in blast cells, T lymphocyte cell subsets and Treg cells of intermediate and high risk MDS group and AML group were higher than that of control group and low risk MDS group (all P < 0.01), and Th2 type and Treg type (IL-10、TGF-β) cytokines were dominant. After treatment, the differences of PD-1/PD-L1 expression were not statisticatly significant in blast cells, T lymphocyte cell subsets and Treg cells between the MDS remission group and the control group (all P >0.05). The expression of PD-1/PD-L1 in blast cells, T lymphocyte cell subsets and Treg cells in MDS non-remission group were significantly higher than that in remission group and control group (all P < 0.01). The high expression of PD-1/PD-L1, dominance of Treg (IL-10、TGF-β) and Th2-related cytokines and inhibition of effector T lymphocyte cells in patients with MDS is conducive to tumor cell proliferation and immune escape, which may promote the progression of MDS disease.

Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice

Although immunotherapy has not yet been as successful in ovarian cancer (OC), it remains a potential therapeutic strategy. Preclinical models of OC are necessary to evaluate the efficacy of immuno-oncology (IO) drugs targeting human immune components but have been underutilized. Developing mouse models with a humanized (Hu) immune system can help understand the human immune response to IO drugs which have demonstrated limited effectiveness in OC patients. We established OC xenograft Hu-mouse models by intraperitoneally injecting luciferase-expressing SKOV-3 Luc and OVCAR-3 Luc OC cells into CD34+ Hu-mice. Tumor growth was monitored through bioluminescence imaging (BLI). In the SKOV-3 Luc Hu-mouse model, we assessed the efficacy of PD-1 blockade with pembrolizumab. We observed the presence of human lymphocyte and myeloid cell subsets within the tumors, lymph nodes, blood, and spleens in these models. Notably, these tumors exhibited a high prevalence of tumor-infiltrating macrophages. Furthermore, we identified HDAC class I target genes, and genes associated with epithelial-mesenchymal transition (EMT) and fibroblasts in the tumors of Hu-mice treated with pembrolizumab. Our xenograft Hu-mouse model of OC provides a valuable tool for investigating the efficacy of IO drugs. The insights gained from this model offer useful information to explore potential mechanisms associated with unresponsive anti-PD-1 treatment in OC.

Abstract 4187: Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice

Abstract Background: Although immunotherapy has not yet been as successful in ovarian cancer (OC), it remains a potential therapeutic strategy. Preclinical models of OC are necessary to evaluate the efficacy of immuno-oncology (IO) drugs targeting human cancer and immune components but have been underutilized. Developing mouse models with a humanized (Hu) immune system can help understand the human immune response to IO drugs, including immune checkpoint inhibitors (ICIs), which have demonstrated limited effectiveness in OC patients. Methods: We established OC xenograft Hu-mouse models by intraperitoneally injecting luciferase-expressing SKOV-3 Luc and OVCAR-3 Luc OC cells into CD34+ Hu-mice. Tumor growth was monitored through bioluminescence imaging (BLI). We assessed the efficacy of PD-1 blockade with pembrolizumab in the SKOV-3 Luc Hu-mouse model. The immune profiles of the tumors were characterized using colorimetric immunostaining and flow cytometry. Additionally, we analyzed RNA-seq data to investigate the gene expression signature of pembrolizumab refractory tumors. Results: We confirmed tumor development in both OC cell lines within CD34+ Hu-mice. In these models, human lymphocyte and myeloid cell subsets were present in the tumors, draining lymph nodes, blood, and spleens. The SKOV-3 Luc tumor-bearing Hu-mice did not respond to pembrolizumab monotherapy. These tumors exhibited a high presence of tumor-infiltrating macrophages. Tumors in Hu-mice unresponsive to pembrolizumab showed a lower abundance of CD8+ T-cells, memory B cells, plasma cells, and a higher proportion of naïve M0 macrophages and mast cells compared to the PBS control. Furthermore, we identified 43 significantly enriched gene sets in these tumors. The differentially expressed genes (DEGs) were predominantly enriched in HDAC class I, RB1, KLF1/3, TCF21, MYD88, SMARCE1 target genes, and genes associated with epithelial-mesenchymal transition (EMT) and fibroblasts. Conclusion: Our xenograft Hu-mouse model of OC provides a valuable tool for investigating the efficacy of IO drugs. The insights gained from this model offer potential avenues to explore mechanisms of resistance to PD-1/PD-L1 blockade in OC. Citation Format: Sung Wan Kang, Ji-young Lee, Ok-Ju Kang, Yong-Man Kim, Eun Kyung Choi, Shin-Wha Lee. Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice [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 4187.

Early Life Stress Is Associated with Alterations in Lymphocyte Subsets Independent of Increased Inflammation in Adolescents.

Early life stress (ELS) is linked to an elevated risk of poor health and early mortality, with emerging evidence pointing to the pivotal role of the immune system in long-term health outcomes. While recent research has focused on the impact of ELS on inflammation, this study examined the impact of ELS on immune function, including CMV seropositivity, inflammatory cytokines, and lymphocyte cell subsets in an adolescent cohort. This study used data from the Early Life Stress and Cardiometabolic Health in Adolescence Study (N = 191, aged 12 to 21 years, N = 95 exposed to ELS). We employed multiple regression to investigate the association between ELS, characterized by early institutional care, cytomegalovirus (CMV) seropositivity (determined by chemiluminescent immunoassay), inflammation (CRP, IL-6, and TNF-a determined by ELISA), and twenty-one immune cell subsets characterized by flow cytometry (sixteen T cell subsets and five B cell subsets). Results reveal a significant association between ELS and lymphocytes that was independent of the association between ELS and inflammation: ELS was associated with increased effector memory helper T cells, effector memory cytotoxic T cells, senescent T cells, senescent B cells, and IgD- memory B cells compared to non-adopted youth. ELS was also associated with reduced percentages of helper T cells and naive cytotoxic T cells. Exploratory analyses found that the association between ELS and fewer helper T cells and increased cytotoxic T cells remained even in cytomegalovirus (CMV) seronegative youth. These findings suggest that ELS is associated with cell subsets that are linked to early mortality risk in older populations and markers of replicative senescence, separate from inflammation, in adolescents.

Beyond αβ T cells: NK, iNKT, and γδT cell biology in leukemic patients and potential for off-the-shelf adoptive cell therapies for AML.

Acute myeloid leukemia (AML) remains an elusive disease to treat, let alone cure, even after highly intensive therapies such as stem cell transplants. Adoptive cell therapeutic strategies based on conventional alpha beta (αβ)T cells are an active area of research in myeloid neoplasms given their remarkable success in other hematologic malignancies, particularly B-cell-derived acute lymphoid leukemia, myeloma, and lymphomas. Several limitations have hindered clinical application of adoptive cell therapies in AML including lack of leukemia-specific antigens, on-target-off-leukemic toxicity, immunosuppressive microenvironments, and leukemic stem cell populations elusive to immune recognition and destruction. While there are promising T cell-based therapies including chimeric antigen receptor (CAR)-T designs under development, other cytotoxic lymphocyte cell subsets have unique phenotypes and capabilities that might be of additional benefit in AML treatment. Of particular interest are the natural killer (NK) and unconventional T cells known as invariant natural killer T (iNKT) and gamma delta (γδ) T cells. NK, iNKT, and γδT cells exhibit intrinsic anti-malignant properties, potential for alloreactivity, and human leukocyte-antigen (HLA)-independent function. Here we review the biology of each of these unconventional cytotoxic lymphocyte cell types and compare and contrast their strengths and limitations as the basis for adoptive cell therapies for AML.

Immune cell subsets in patients with bipolar disorder or schizophrenia with history of childhood maltreatment

A history of Childhood Maltreatment (CM) has been repeatedly associated with an increased risk of developing bipolar disorders (BD) or schizophrenia (SZ). The impact of severe stress induced by CM has been proposed to be mediated by elevated inflammation reflected by dysregulated inflammatory processes. Little is known about the potential impact of CM on lymphocyte subpopulations or the role of pre-existing infections on CM physiological consequences. This study therefore explored the role of CM and past infection exposure impact on lymphocyte subpopulations to give an indication of their relevance as stressors in the pathoetiology of major mood and psychotic disorders.118 adult patients with SZ, and 152 with BD were included in the analysis. CM history was assessed by the Childhood Trauma Questionnaire (CTQ), with current and past psychiatric symptomatology also evaluated. Circulating immune cell subsets were analyzed using flow cytometry-based analysis. Past exposure to common infectious stigma including toxoplasma, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) were measured by solid phase-enzyme microplate and ELISA immunoassays. The relationship between CM, biological phenotypes (including immune cell subsets distribution and past infectious status) and clinical phenotypes were analyzed using univariate and multivariate analyses.BD patients with, versus without, CM had higher levels of CD3+CD8+ cytotoxic T cells and CMV antibodies along with decreased levels of CD45RA+CCR7+CD8+ naïve CD8+ T cells, and a more severe clinical profile. CMV antibody levels were inversely associated with the CD3 + CD8 + lymphocyte subset level. SZ patients with, versus without, CM, showed lower levels of CD14 + monocytes and no specific clinical characteristics. The accumulation of different types of maltreatment associated with increased body mass index and CMV autoantibodies as well as decreased levels of CD14 + monocytes. In both BD and SZ, further analysis according to the type and the number of CM subtypes showed association with specific changes in lymphocyte cell subsets, clinical profile, and infectious stigma.Adults with BD or SZ exposed to CM exhibit specific immune cell subset profiles, clinical features, and stigma of past infections. In BD, our data indicate an interplay between CM and CMV infections, which may possibly contribute to premature aging and cellular senescence, both of which have previously been shown to associate with mood disorders. Longitudinal studies of CM-exposed patients are required to clarify the interactions of CM and viral infections, including as to the pathophysiological processes driving patient symptomatology.

Detection of humoral and cellular immune response to anti-SARS-CoV-2 BNT162b2 vaccine in breastfeeding women and naïve and previously infected individuals

This study explored humoral and cellular responses to anti-SARS-CoV-2 BNT162b2 mRNA vaccine in breastfeeding women and naïve and seropositive individuals in the first six months after vaccination.Sixty-one volunteers vaccinated with two doses of the BNT162b2 mRNA vaccine were enrolled in the study. In-house developed ELISA was used for the quantification of SARS-CoV-2 RBD-specific antibodies. Cell surface marker expression and intracellular IFN-γ analysis were carried out by flow cytometry. The concentrations of IFN-γ, IL-6 and TNF were determined by ELISA. A significant rise in anti-RBD IgG antibody levels was observed 14 days after the first vaccine dose (p < 0.0001) in serum and milk. The expression of CD28 on CD4+ T cells was significantly higher compared to baseline (p < 0.05). There was a significant increase (p ≤ 0.05) in B cell lymphocyte subset after revaccination, and increased percentage of CD80+ B cells. The expression of IFN-γ in peripheral blood lymphocytes, CD3+ T cells and serum was significantly increased (p < 0.05). No significant difference in immune response was observed between breastfeeding women and other study participants. The anti-SARS-CoV-2 BNT162b2 mRNA vaccine-induced measurable and durable immune response in breastfeeding women and in naïve and previously infected individuals.

Immune response to BNT162b2 SARS-CoV-2 vaccine in patients living with HIV: The COVIH-DAPT study.

Data on immune response to SARS-CoV-2 vaccine in patients living with HIV (PLWH) over a period longer than 3 months are currently limited. We measured the immune response after BNT162b2 vaccination against SARS-CoV-2 in this population. We prospectively enrolled PLWH on successful antiretroviral therapy, initiating vaccination with two doses of the BNT162b2 SARS-CoV-2 vaccine administered at six-week interval. SARS-CoV-2 humoral and cellular responses and lymphocyte cell subsets were recorded at inclusion and 6 weeks (W6), 3 months (M3) and 6 months (M6) later. Humoral, humoral strong and cellular responders were defined by IgG titers >10, ≥264BAU/mL and IFN-γ T cell release, respectively. Nineteen subjects without SARS-CoV-2 infection were included (74% men, mean age 51 years, CD4 nadir 399/mm3). All subjects were humoral responders, their antibody titer peak reached at M3. Strong responders' rates were 63% and 21% at M3 and M6, respectively. CD19+CD10+ B cells had increased significantly at W6 then decreased at M3, while CD19+CD27+ B cells remained unchanged. Rates of patients with a cellular response increased from 39% at W6 to 69% at M6. Cellular responders had significantly higher CD3+, CD4+ and CD8+ Effector Memory cells at inclusion (p=0.048, p=0.024, p=0.012, respectively) and CD4+ Terminally Differentiated Effector Memory cells at M3 (p=0.044). PLWH have a robust immune response after SARS-CoV-2 vaccination, but a rapid decline in humoral response from 3 months onwards, due to a blunted memory B cell response. Analysis of lymphocyte subsets may help identify optimal times for vaccine boosters.

The Recovery of the Absolute Lymphocyte Subpopulation Count in Cervical Cancer Patients after Radiotherapy

<h3>Purpose/Objective(s)</h3> Radiotherapy (RT) results in varying degrees of lymphopenia. However, to date, there is still no consensus on the persistence of lymphopenia after RT treatment for cervical cancer. Therefore, this study aimed to evaluate the dynamic changes of absolute lymphocyte subset count (ALSC) in cervical cancer patients after receiving RT. In addition, the study also explored the diagnostic value of the lymphocyte count (LC) in patients with declining ALSC <h3>Materials/Methods</h3> The peripheral blood ALSC was tested by flow cytometry before RT (baseline) and at regular follow-up intervals after completion of RT. The blood cell counts were also collected on the same day. Patient characteristics, tumor variables, and treatment factors were gathered for risk assessment. The primary endpoint was the degree of reduction in the lymphocyte subpopulation and the secondary endpoint was the predictive value of LC for declining ALSC. Logistic regression was used to identify the risk factors of lymphopenia, and the receiver operating characteristic (ROC) curve was used to assess the predictive value of LC. <h3>Results</h3> A total of 260 consecutive cervical cancer patients met the study criteria, among which 228 (87.7%) patients had a reduction in either one or some of the lymphocyte subpopulations. The median percentage decrease in ASLC ranged from 22.52% to 58.32%. When compared with the baseline the lymphocyte subpopulations dropped immediately after initiation of RT, to eventually reach their minimum levels at 3 to 6 months after completion of treatment. The B-lymphocyte and CD4+ to CD8+ ratios recovered within the first 6 months and the CD8+ T cell count recovered to normal levels within one year after completion of RT. In comparison, the CD3+ and CD4+ T cell counts persisted at low levels for one year and recovered five years after RT. The natural killer (NK) cells rarely decreased after RT. Multivariate logistic regression analysis identified advanced tumor stage and age above 60 years as independent risk factors affecting the count of lymphocyte cell subsets after RT. The ROC analysis confirmed that the LC obtained from a routine blood examination could be used to predict the count for each lymphocyte subtype except for the NK cell subtype. <h3>Conclusion</h3> The lymphocyte subpopulation counts decreased immediately after initiation of RT, to eventually reach their minimum levels after 3 to 6 months following completion of treatment, and it took several months or years to recover. Advanced tumor stage and age above 60 were identified as independent risk factors for lymphopenia in cervical cancer patients. The LC was a good predictor of ALSC decline. Therefore, this test could be used as a cheaper alternative to complex ALSC monitoring with flow cytometry.

Seroprevalence of Toxoplasma gondii among hemodialysis patients: A possible link to main T-lymphocyte subsets levels and dialysis adequacy

BackgroundA high prevalence of Toxoplasma gondii infection has been reported in patients with hemodialysis (HD). However, a lack of data exists on the relationship between T lymphocyte subsets and dialysis adequacy. This study aimed to investigate the seroprevalence of Toxoplasma gondii infection among HD patients and its relation to T lymphocyte cells subsets, CD3+, CD4+, CD8+, CD4+/CD8+ ratio, and HD adequacy. MethodsThis cross-sectional comparative study was conducted on 92 subjects. Of them, 42 HD patients and 50 were control participants. Anti-Toxoplasma gondii IgG, IgM antibodies, the T lymphocyte cells subset CD3+, CD4+, CD8+, CD4+/CD8+ ratio, and adequacy of dialysis via calculation of Kt/V were detected for all subjects. ResultsThe seropositivity for anti-Toxoplasma gondii IgG antibodies was significantly higher in HD patients 66.7% (28/42) compared to 34% in controls (17/50), (p = 0.0003). The main T lymphocyte subsets was significantly lower in HD compared to controls (p < 0.05). Seropositive HD patients exhibited statistically significantly lower T lymphocyte cell subsets and CD4+/CD8+ ratio compared to seronegative patients (p < 0.05). There was a negative correlation between anti-Toxoplasma gondii IgG level and T lymphocyte subsets and the CD4/CD8+ ratio. Binary logistic regression showed that CD4+ T cell significantly predicts Toxoplasma gondii susceptibility among HD patients (p = 0.03). The mean Kt/V index is significantly lower among seropositive HD patients compared to seronegative HD patients (1.05 ± 0.46, 1.41 ± 0.53, respectively, p = 0.03) with a significant negative correlation with anti-Toxoplasma gondii IgG level (p < 0.05). ROC curve analysis showed the CD4+ T cell % had the highest AUC value among HD patients (AUC= 0.88, p < 0.001). The Kt/V value of ≤ 0.8 significantly predicted susceptibility to Toxoplasma gondii infection among HD patients (AUC = 0.68, p = 0.03). ConclusionThe current study reinforces previous reports of a high prevalence of Toxoplasma gondii infection among HD patients. CD4+ T cell and Kt/V showed a good diagnostic performance in predicting susceptibility for Toxoplasma gondii infection in HD patients. Considering the clinical consequences caused by Toxoplasma gondii infection in these patients, regular screening and adequate HD are recommended.

Altered compositions of monocyte, T lymphocyte and NK cell subsets in heart failure of adult congenital heart disease

BackgroundAdults with congenital heart disease (ACHD) display changes in adaptive immunity related to early open-heart surgery and subsequent incidental thymectomy. In acquired heart failure (HF) systemic inflammation, innate and adaptive immune cells play an important role. However, cellular immune alterations of monocyte, T lymphocyte and natural killer (NK) cell subsets have not been related to HF in ACHD. Methods and resultsThis cross-sectional study included 209 ACHD outpatients (mean age: 35.3 ± 11.0 years; NYHA class I/II/III-IV 64.1%/21.5%/14.4%; 59.8% male) and 21 healthy controls (29.8 ± 12.6 years; 47.6% male). Patients with clinical signs of infection, inflammatory diseases or malignancies were excluded. Flow cytometry of fresh whole blood revealed significantly elevated levels of CD14++CD16+ (Mon2) and CD14+CD16++ (Mon3) monocytes that increased in advanced NYHA class. Immature CD14+HLA-DRneg/low monocytes were significantly higher in ACHD, but did not relate to NT-proBNP or NYHA class. Frequencies of Mon2, CD14+HLA-DRneg/low and neutrophils related to plasma S100A8/A9 levels. Furthermore, ACHD had decreased T helper and cytotoxic T cell counts, however NKT and CD3bright T cells remained unchanged compared to control. The cytokine-producing CD56bright NK subset related to NT-proBNP and expanded in advanced HF stages. Patients with elevated Mon2 and CD14+HLA-DRneg/low monocytes exhibited a worse outcome regarding all-cause mortality/cardiac decompensations after a mean follow-up of 5.5 years. High Mon2, low total lymphocyte and low T helper cell counts were independent prognostic markers for the onset of adverse cardiac events. ConclusionsAltered immune cell compositions related to heart failure and exhibited prognostic relevance. This suggests an important link between immunity, inflammation and HF in ACHD bearing potential for refined clinical risk stratification.

Compassionate open-label use of rituximab following a randomised clinical trial against neuromyelitis optica (RIN-2 study): B cell monitoring-based administration

ObjectiveThe aim of the RIN-2 study was a compassionate use of rituximab (RTX) for patients who completed the RIN-1 study, a multicentre, randomised, double-blind, placebo-controlled trial of RTX. We also investigated the long-term safety and efficacy of RTX. MethodsA study design was a prospective open-label extension study following the RIN-1 study. RTX was infused repeatedly under monthly monitoring of CD19-positive and CD 20-positive B cell lymphocyte subsets from 24 weeks after an infusion. ResultsThirty-three (87%) of 38 patients of the RIN-1 study were enrolled from February 2016 to March 2019 at six sites in Japan. In RIN-2, RTX was administered three times (median, range 1–5 times), and the interval of RTX administrations were 9.5 [2.5] months (mean [SD]). The observation period was 20.5 [10.1] months. During the trial, three patients dropped out due to two withdrawals and one adverse event. During the study, 28 (90%) of 31 patients were treated with RTX monotherapy. Neuromyelitis optica (NMO) relapses were observed in two patients. The annualized relapse rate (ARR) was 0.035 counts per person-years, ∼1/10th compared with 0.321 in the placebo arm of the RIN-1 study. We observed 14 severe adverse events in six (18%) and 156 adverse events, of which 135 were grade 1, 11 were grade 2 and 10 were grade 3. ConclusionsUnder B cell monitoring, the interval of RTX re-infusion was elongated to nine months, and NMO relapses were suppressed with 0.035 of ARR.

CD4+ T-cell activation of bone marrow causes bone fragility and insulin resistance in type 2 diabetes.

Type 2 diabetes mellitus (T2DM) causes an increased risk of bone fractures. However, the pathophysiology of diabetic bone fragility is not completely understood. It has been proposed that an inflammatory microenvironment in bone could be a major mechanism by inducing uncontrolled bone resorption, inadequate bone formation and consequently more porous bones. We propose that activated T-cells in the bone marrow cause a pro-inflammatory microenvironment in bone, and cause bone fragility in T2DM. We induced T2DM in C57BL/6 male mice through a hypercaloric diet rich in carbohydrates and low doses of streptozocin. In T2DM mice we inhibited systemic activation of T-cells with a fusion protein between the extracellular domain of Cytotoxic T-Lymphocyte Antigen 4 and the Fc domain of human immunoglobulin G (CTLA4-Ig). We analysed the effects of T2DM or CTLA4-Ig in lymphocyte cell subsets and antigen-presenting cells in peripheral blood and femoral bone marrow; and their effect on the metabolic phenotype, blood and bone cytokine concentration, femoral bone microarchitecture and biomechanical properties, and the number of osteoblast-like cells in the femoral endosteum. We performed a Pearson multiple correlation analysis between all variables in order to understand the global mechanism. Results demonstrated that CTLA4-Ig decreased the number of activated CD4+ T-cells in the femoral bone marrow and consequently decreased TNF-α and RANK-L concentration in bone, notably improved femoral bone microarchitecture and biomechanical properties, increased the number of osteoblast-like cells, and reduces osteoclastic activity compared to T2DM mice that did not receive the inhibitor. Interestingly, we observed that blood glucose levels and insulin resistance may be related to the increase in activated CD4+ T-cells in the bone marrow. We conclude that bone marrow activated CD4+ T-cells cause poor bone quality and insulin resistance in T2DM.

P313 Within, 6 months from COVID-19 BNT162b2 vaccine patients with Inflammatory Bowel Diseases treated with Anti-TNFα have significantly lower serologic responses

BackgroundWhile vaccines against COVID-19 are effective in healthy individuals, we reported significantly lower serologic responses to BNT162b2 in patients with inflammatory bowel diseases (IBD) treated with anti-tumor necrosis factor (TNF) α agents. As this was apparent already, 4 weeks post vaccination, vaccine longevity is concerning. Aim: to assess long-term serologic responses to BNT162b2 in patients with IBD stratified according to medical treatment.MethodsA prospective, observational multi-center Israeli study. Patients with IBD (anti-TNFα treated versus non-anti-TNFα treated) and healthy controls (HC) were followed from before the, 1st BNT162b2 dose until, 6 months after vaccination. COVID-19 spike (S) and nucleocapsid (N) antibodies (Abs) concentrations were analyzed by ELISA, followed by neutralization studies. Specific anti-receptor binding domain (RBD) memory B-cells response, serologic responses against variants of concern (VOCs), Beta, Gamma and Delta, immunoglobulin levels and lymphocyte cell subsets were evaluated as well. Safety was assessed using questionnaires, clinical and laboratory data.ResultsOf, 193 subjects, 130 had IBD (45 and, 85 in the anti-TNFα and non-anti-TNFα groups, respectively), 63 HC. Serologic response assessed, 176 (median) days (IQR, 166–186) and compared to, 4 weeks after, 1st dose significantly declined in all three groups, but was lowest in the anti-TNFα group:, 6 months anti-S Abs titer geometric means:, 193 (95%CI:, 128–292), 703 (520–951), and, 1253 (1023–1534) in anti-TNFα, non- anti-TNFα and HC groups, respectively, p<0.001, Figure, 1. This was further supported by neutralization and inhibition studies. Importantly, significantly decreased memory B-cell response towards RBD was detected only in the anti-TNFα group, with the most significant reduction in response to Beta VOC (p<0.0008 and p<0.0001, vs. non-anti-TNFα and HC, respectively). Older age was an additional predictor of lower serologic response. Immunoglobulin levels and lymphocyte cell subsets were comparable between the study groups. Infection rate reflected by anti-N Abs was ~1% in all groups. Safety was comparable in all groups.ConclusionThe, 6-months serologic response to BNT162b2 vaccine, evaluated prospectively, decreased in all subjects, most prominently in patients with IBD treated with anti-TNFα. Importantly, the latter also had the sharpest decline in serologies, the lowest functional activity and lowest RBD specific memory B-cells. Older age is an additional predictor of decreased serologic response. Altogether, waning of COVID-19 serologic and functional response over, 6 months, specifically in patients with IBD treated with anti-TNFα, supports the need for an early third vaccine dose.

Comparison of the lymphocyte response to interval exercise versus continuous exercise in recreationally trained men

The purpose of this investigation was to compare changes in circulating lymphocyte subset cell counts between high-intensity interval exercise (HIIE), sprint interval exercise (SIE), and moderate-intensity continuous exercise (MICE). Recreationally active men (n ​= ​11; age: 23 ​± ​4 ​yr; height: 179.9 ​± ​4.5 ​cm; body mass: 79.8 ​± ​8.7 ​kg; body fat %:12.6 ​± ​3.8%; V̇O2max: 46.6 ​± ​3.9 ​ml⋅kg−1⋅min−1) completed a maximal graded exercise test to determine maximal oxygen uptake (V̇O2max) and three duration-matched cycling trials (HIIE, SIE, and MICE) in a randomized, counterbalanced fashion. HIIE consisted of fifteen 90-s bouts at 85% V̇O2max interspersed with 90-s active recovery periods. SIE consisted of fifteen 20-s bouts at 130% maximal power and 160-s active recovery periods. MICE was a continuous bout at 65% V̇O2max. Total exercise duration was 53 ​min in all three trials, including warm-up and cool-down. Blood was collected before, immediately post, 30 ​min, 2 ​h, 6 ​h, and 24 ​h post-exercise. Changes in lymphocyte subset counts, and surface expression of various markers were analyzed via flow cytometry. Changes were assessed using mixed model regression analysis with an autoregressive first order repeated measures correction. Significant decreases were observed in absolute counts of CD56dim NK cells, CD19+ B cells, CD4+ T cells, and CD8+ T cells 30 ​min and 24-h post-exercise in all three trials. Despite resulting in greater total work and oxygen consumption, MICE elicited similar changes in lymphocyte subset counts and receptor expression compared to both SIE and HIIE. Similarly, while the two interval trials resulted in differing oxygen consumption and total work, no differences in the lymphocyte response were observed. Though both forms of exercise resulted in declines in circulating lymphocyte cell counts, neither exercise type provides an immune-related advantage when matched for duration.

Hematopoietic Stem Cell Transplantation (HSCT) with Omidubicel Is Associated with Robust Immune Reconstitution and Lower Rates of Severe Infection Compared to Standard Umbilical Cord Blood Transplantation

IntroductionOmidubicel is an advanced cell therapy for allogeneic hematopoietic stem cell transplantation (HSCT), derived from appropriately HLA-matched umbilical cord blood (UCB) and comprised of ex-vivo expanded CD133+ cells and a non-cultured lymphocyte-containing fraction. Recent results of a global phase III trial of omidubicel vs standard UCB showed more rapid hematopoietic recovery, reduced rates of infection, and shorter hospitalizations in patients (pts) randomized to omidubicel (Horwitz et al, Blood, 2021). We now report results of correlative immune reconstitution (IR) studies in this trial (NCT02730299).MethodsA total of 125 pts aged 13-65 with hematologic malignancies were randomized to allogeneic transplantation with omidubicel or standard UCB following myeloablative conditioning; 108 pts were transplanted per protocol. An optional IR sub-study was conducted and blood was collected at intervals from Day 7 through one year post-transplant. Cryopreserved samples were analyzed in a central laboratory (Covance) using 16-color and 14-color panels and flow cytometric assays to explore T cell, NK cell, B cell, monocyte, and dendritic cell (DC) subsets. Means, medians, ranges, and standard errors were used to summarize cell counts, and one-tailed t-tests were used to compare counts in the two treatment arms.ResultsA total of 37 pts from 15 sites consented to the IR sub-study, representing 34% of the per protocol population; 17 pts were transplanted with omidubicel and 20 pts with control (15 [75%] of control with double UCB). Median age was 30 (range: 13-62) years for omidubicel pts and 43 (range: 19-55) years for controls in the sub-study. Median CD3+ content of omidubicel prior to cryopreservation was lower (180 x 10^6; range: 71-580 cells) than that of controls post-thaw (516 x 10^6, range: 183-990 cells).Omidubicel pts achieved neutrophil engraftment at a median of 10 (range: 6-28) days post-transplant compared to a median of 18.5 (range: 14-40) days in controls. Omidubicel pts had fewer BMT-CTN Grade 3 viral infections in the first-year post-transplant than controls (6% vs. 25%, respectively).At Day 7 post-transplant, CD4+ T cell counts were significantly higher in omidubicel pts (37x10^3 cells/ml) than in controls (17x10^3 cells/ml, p=0.011). B cells (12x10^3 vs 1x10^3 cells/ml, p=0.013) and NK cells (6x10^3 vs 3 x10^3 cells/ml, p=0.016), as well as monocyte and DC subsets, were also significantly higher in omidubicel pts (Table). Day 14 results similarly demonstrated higher counts of circulating immune cell subsets in omidubicel pts than in controls (Table). Higher B cell counts were observed in omidubicel pts than in controls at 6 months ([863±463] x10^3 vs. [543±221] x10^3 cells/ml, p=0.03) and one year ([1492±370] x10^3 vs [763±150] x10^3, p=0.02) following transplant (Figure).ConclusionsCirculating immune cell subsets were consistently higher in omidubicel pts than controls as early as one week after transplant, and higher B cell counts persisted through one year. These findings correlated with the clinical observation of fewer severe bacterial, fungal, and viral infections in pts treated with omidubicel compared to standard UCB. These results demonstrate that rapid hematopoietic recovery in pts transplanted with omidubicel is accompanied by the early and robust appearance of a broad array of lymphocyte, monocyte, DC, and NK cell subsets, despite substantially fewer numbers of these cells infused, suggesting a facilitator effect of omidubicel on their in vivo expansion. [Display omitted] DisclosuresSzabolcs: Gamida Cell: Consultancy; Prevail Therapeutics: Consultancy; Sotiria/Forge Biologics: Current equity holder in publicly-traded company. Levy: Gamida Cell: Current Employment. Yackoubov: Gamida Cell: Current Employment. Pato: Gamida Cell: Current Employment. Galamidi-Cohen: Gamida Cell, Ltd: Current Employment. Horwitz: Gamida Cell: Research Funding.

Primary vulvar squamous cell carcinomas with high T cell infiltration and active immune signaling are potential candidates for neoadjuvant PD-1/PD-L1 immunotherapy

BackgroundA profound insight into the immune landscape of vulvar squamous cell carcinoma (VSCC) is lacking. Here, an in-depth interrogation of T cell infiltration, local immune contexture, signaling pathways and checkpoint...

Immune Response of Elite Enduro Racers to Laboratory and Racing Environments: The Influence of Training Impulse and Vibration.

Introduction: Understanding the sport-specific immune response elicited during both training and competition is imperative to maximise athlete health and performance. Despite a growing population of professional enduro mountain bike athletes, little is known about the recovery of the immune system following enduro racing events. Methods: Nine international level elite enduro mountain bike athletes (age 24.3 ± 2.4 years, height 178.5 ± 8.7 cm, mass 76.5 ± 12.5 kg) completed a laboratory-based maximal exercise test (LAB) on a cycle ergometer and competed in an international mountain bike enduro race event (RACE). Blood samples were taken before, immediately after, and 1 h after LAB and before, 1 h after, and 17 h after RACE. Leukocyte subsets were enumerated using seven-colour flow cytometry. Lucia’s training impulse (LuTRIMP) and vibration exposure (VIB) were quantified during RACE. Results: Seven participants were included in the final analyses. There was a significant (p < 0.05) increase in neutrophil count alongside a reduction of cytotoxic lymphocyte cell subsets of both the innate (CD3−/CD56+ NK-cells and CD3−/CD56dim NK-cells) and adaptive (CD8+/CD62L−/CD45RA− T-cells and CD8+/CD27+/CD28− T-cells) components of the immune system one hour after RACE. All cell counts returned to baseline values 17 h afterwards (p > 0.05). Cell subset redistribution from pre- to post-one-hour time points (%Δpre-post1h) in cell subsets with potent effector functions (Neutrophils, CD3−/CD56+ NK-cells, CD8+/CD62L−/CD45RA− T-cells, CD8+/CD27+/CD28− T-cells, and CD3−/CD56dim/CD57− NK-cells) was significantly greater at RACE than LAB (p < 0.05). VIB was shown to be a superior predictor of %Δpre-post1h CD4+ T-cells, CD4+ early T-cells, CD4+ naïve T-cells, and NK cells as compared with LuTRIMP on its own (ΔR2 = 0.63 − 0.89, p < 0.05). Conclusions: The race event offers a greater challenge to the immune system than LAB, and potentially, whole body vibration is a key component of training load measurement in mountain bike applications.

Global immune characterization of HBV/HCV-related hepatocellular carcinoma identifies macrophage and T-cell subsets associated with disease progression

Diverse immune cells in the tumor microenvironment form a complex ecosystem, but our knowledge of their heterogeneity and dynamics within hepatocellular carcinoma (HCC) still remains limited. To assess the plasticity and phenotypes of immune cells within HBV/HCV-related HCC microenvironment at single-cell level, we performed single-cell RNA sequencing on 41,698 immune cells from seven pairs of HBV/HCV-related HCC tumors and non-tumor liver tissues. We combined bio-informatic analyses, flow cytometry, and multiplex immunohistochemistry to assess the heterogeneity of different immune cell subsets in functional characteristics, transcriptional regulation, phenotypic switching, and interactions. We identified 29 immune cell subsets of myeloid cells, NK cells, and lymphocytes with unique transcriptomic profiles in HCC. A highly complex immunological network was shaped by diverse immune cell subsets that can transit among different states and mutually interact. Notably, we identified a subset of M2 macrophage with high expression of CCL18 and transcription factor CREM that was enriched in advanced HCC patients, and potentially participated in tumor progression. We also detected a new subset of activated CD8+ T cells highly expressing XCL1 that correlated with better patient survival rates. Meanwhile, distinct transcriptomic signatures, cytotoxic phenotypes, and evolution trajectory of effector CD8+ T cells from early-stage to advanced HCC were also identified. Our study provides insight into the immune microenvironment in HBV/HCV-related HCC and highlights novel macrophage and T-cell subsets that could be further exploited in future immunotherapy.

Lymphocyte activation gene 3 expression on T lymphocyte cell subsets in patients with myelodysplastic syndrome

Objective: To investigate the expression of Lymphocyte activation gene 3 (LAG3) in myelodysplastic syndromes (MDS) patients. Methods: A total of 16 MDS patients newly diagnosed in Hematology Department of Tianjin Medical University were enrolled from January to November 2019. The healthy control (HC) group includes 16 cases of healthy adults. The expression levels of LAG3 on CD8(+)T cells, CD4(+)T cells and regulatory T cells (Treg) in MDS patients and healthy controls were detected by flow cytometry. Results: A total of 16 patients with MDS were included, including 5 males and 11 females, with a median age of 56 (18-80) years old. HC group includes 16 healthy adults, 8 men and 8 women, with a median age of 40 (17-69) years. There was no statistically significant difference in gender and age composition between the two groups (both P>0.05). The expression of LAG3 on CD8(+)T cells in MDS patients (74.45%±22.31%) was significantly higher than that in HC group (58.78%±14.82%, P<0.05). The LAG3 expression on Treg in MDS patients (64.91%±10.32%) were significantly higher than that of HC group (49.09%±13.58%, P<0.05). There was no statistical difference in LAG3 expression on CD4(+)T cells between the two groups. Conclusion: The expression of LAG3 on CD8(+)T cells and Treg increases in MDS patients than that of healthy people.