Lymphocyte Subsets Research Articles

Exploring lymphocyte subsets in COVID-19 patients: insights from a tertiary academic medical center with a high proportion of patients on immunosuppression

IntroductionSevere COVID-19 is associated with reduced absolute lymphocyte counts, suggesting that lymphocyte subsets may serve as predictors of clinical outcomes in affected patients. Early identification of patients at risk for severe disease is crucial for optimizing care, accurately informing patients and their families, guiding therapeutic interventions, and improving patient flow in the ED. Given that immunosuppressive drugs significantly impact lymphocyte profiles, we aimed to determine the association between prior use of immunosuppressive drugs, lymphocyte subsets, and COVID-19 severity in our population with a high prevalence of immunosuppression.MethodsIn 2021, suspected COVID-19 patients were included in the ED. Lymphocyte subsets were determined in peripheral blood within 24 hours after presentation and comparative analyses was performed between SARS-CoV-2 negative and positive patients, mild versus severe disease and patients with and without prior immunosuppressive drug use. Mild cases were patients discharged home or admitted to a general ward, severe cases were patients with COVID-19-related mortality or necessitating ICU admission. Logistic regression analysis was performed to assess the association between lymphocyte subsets and COVID-19 severity, and between prior immunosuppressive drug use and COVID-19 severity.ResultsTwenty-five SARS-CoV-2 negative and 77 SARS-CoV-2 positive patients were included, whereof 57 (74%) had mild and 20 (26%) severe COVID-19. No significant differences were observed in the absolute counts of CD3+, CD4+, and CD8+ T-lymphocytes, B-lymphocytes, and NK-cells between SARS-CoV-2 negative and positive patients or between mild and severe cases. The 36 patients with prior use of immunosuppressive drugs had significantly lower CD4+ T-lymphocytes (p<0.01). Prior use of immunosuppressive drugs was not associated with COVID-19 severity (adjusted OR 1.074, 0.355-3.194).ConclusionLymphocyte subsets were not significantly different between SARS-CoV-2 negative and positive patients and between mild versus severe cases. Neither lymphocyte subsets nor prior immunosuppressive drug use were associated with COVID-19 severity.

Nomogram for the prediction of relapse factors in patients with neuromyelitis optica spectrum disorder during rituximab treatment.

Develop a nomogram to analyse the factors influencing the relapse of neuromyelitis optica spectrum disorder (NMOSD) during rituximab (RTX) treatment. A retrospective analysis of 214 NMOSD patients identified 181 with AQP4-IgG-seropositive. 32 patients who relapsed during RTX treatment were included, and 122 sets of lymphocyte subset monitoring data were collected. 110 sets of data were finally included and divided into relapse (n = 30) and nonrelapse (n = 80) groups depending on whether a relapse occurred between two adjacent RTX treatments. Logistic and LASSO regressions were used to identify the relevant factors influencing NMOSD relapse, and a nomogram was constructed. Receiver operating characteristic (ROC) curves were generated to evaluate the nomogram's ability to differentiate, and the bootstrap method was utilized for internal validation. Calibration curve and decision curve analysis were also conducted. Comparing baseline data revealed differences in the RTX administration interval, CD3-CD19+ B lymphocyte and CD3-CD56+ NK cell levels. The RTX administration interval and the level of CD3-CD19+ B lymphocytes were independent variables influencing relapse. The nomogram had an area under the curve (AUC) of 0.71 and a 95% confidence interval (CI) of 0.58-0.83. The Hosmer-Lemeshow (H-L) goodness-of-fit test yielded a χ2 = 11.80 (p = 0.16). Decision curve analysis revealed that the model provided greater net benefits within the threshold probability range of 0.18-0.98. The nomogram developed in this study showed that the RTX administration interval and CD3-CD19+ B lymphocyte levels independently influence NMOSD relapse, indicating good discriminative ability, consistency, and clinical benefits.

Circulating B Lymphocyte Subsets in Patients with Systemic Lupus Erythematosus

Background/Objectives: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the abnormal activation of autoreactive T and B cells, autoantibody production, complement activation, and immune-complex deposition, resulting in tissue damage. However, data on immunologic disturbances in SLE, particularly regarding flares, are scarce. Methods: We investigated 35 patients with SLE: 12 (34.3%) with disease exacerbation (SLE disease activity index [SLEDAI] ≥ 5 points) and 23 (65.7%) in remission (SLEDAI < 5 points). All patients met the 2019 EULAR/ACR SLE criteria. Flow cytometry was used to identify B cell subsets, including memory B cells. Results: In the whole patient group, SLEDAI was positively related to the percentage of transitional/regulatory B cells (r = 0.38, p = 0.034). Some lymphocyte subsets correlated with complement levels, e.g., the percentage of naïve and memory B cells showed associations with C3c complement (r = 0.43, p = 0.018 and r = −0.45, p = 0.016, respectively). Furthermore, regarding inflammatory markers, we found associations between C-reactive protein and the percentage of plasmablasts (r = 0.40, p = 0.026) and plasmocytes (r = 0.44, p = 0.017). Finally, the percentage of plasmablasts correlated with SLE duration (r = 0.42, p = 0.016). In the follow-up analysis, during a median observation of 5 years, 5 out of the initially 23 inactive SLE patients developed a disease flare. They were characterized by longer disease duration stated in the beginning compared to patients who remained in remission (p = 0.019). Conclusions: Our study highlights significant associations between various B cell subsets and SLE disease activity. A more personalized approach to indicate patients with SLE at a higher risk of lupus flares is crucial for better management.

Two cases of deficiency in ELF4 gene X-linked and literature review

Objective: To summarize the clinical phenotype and genetic characteristics of deficiency in ELF4 gene X-linked (DEX). Methods: A case series study was conducted to retrospectively analyze the clinical data and genetic testing results of 2 cases of DEX treated at Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and the First Hospital of Jilin University from January 2023 to April 2024. And literature up to April 2024 was searched from the PubMed database, as well as CNKI and Wanfang databases, using keywords such as "ELF4 deficiency" "deficiency in ELF4, X-linked""ELF4 gene". The main clinical manifestations and genotypes of DEX were summarized. Results: Both patients were male, with onset ages of 3 months and 3 years, respectively. Both patients presented with recurrent oral ulcers and abdominal pain. And the laboratory examination showed a significant increase in inflammatory markers. Intestinal examinations showed multiple intestinal ulcers, and both patients developed intestinal fistulas. Whole exome sequencing found ELF4 c.799C>T, p Arg267Trp and ELF4 c. 248-7G>A, both maternal variants. Based on clinical and genetic results, DEX were diagnosed. In terms of treatment, both patients underwent surgical treatment during the acute phase of the disease and received anti-tumor necrosis factor α therapy, but recurrent gastrointestinal symptoms were still observed in Patient 1, while the clinical effect in Patient 2 was still acceptable. However, the inflammatory markers in both patients were not normal even after treatment. Literature review found 18 patients including 2 patients in this study, reported in 5 English articles and no Chinese reports. Thirteen patients had disease onset age before 5. The main clinical manifestations were fever (12/17), oral ulcers (14/18), abdominal pain (8/18), diarrhea (6/18), perianal ulcers (5/17), ileum ulcers (6/16), colon ulcers (7/16), skin involvement (7/17) and recurrent infections (7/18); laboratory examinations found increased erythrocyte sedimentation rate (13/15) as well as C-reactive protein (9/9), and anemia (13/15); in terms of immunological function, there is a decrease in natural killer cells (9/15) as well as a decrease in class switching memory B cells (8/9). The main types of gene variantions were missense variantions (6/18), nonsense variantions (4/18) or frameshift variantions (3/18). Conclusions: DEX should be considered when an early-onset male patient manifested with recurrent fever, oral ulcers or mucosal ulcers, with elevated inflammatory markers, with or without recurrent infection. It is recommended to perform lymphocyte subsets analysis, gastrointestinal endoscopy and genetic testing to support the diagnosis.

Associations between blood markers of glucose metabolism and characteristics of circulating lymphocytes

AimsThe pathophysiology of diabetes is not fully understood; recent research indicates close relations with immunological alterations. Therefore, the aim of this study was to investigate the associations between markers of glucose metabolism and characteristics of blood lymphocytes in a population-based cohort. MethodsThe analysis was based on data from 219 non-diabetic participants of the MEGA study in Augsburg, Germany, who were recruited between 2018 and 2021. The majority of participants were examined two different times with a time lag of 9 months. Fasting venous blood samples were taken and oral glucose tolerance tests (OGTT) were performed at both visits. Immune cells were analyzed from fresh blood using flow cytometry. The associations between fasting blood glucose levels, glucose levels at 2 hours after oral glucose bolus and glycated hemoglobin (HbA1c) concentrations and the quantity of different lymphocyte subsets were analyzed using linear mixed regression models with random intercept. P values were FDR-adjusted. ResultsHbA1c was negatively associated with the marginal zone B cells (IgD+ CD27+ B cells). Fasting glucose was positively associated with natural killer (NK) cells and 2-hour OGTT glucose was positively associated with NKT cells. Finally, HbA1c showed significantly negative associations with the CD57-PD1-NKT cell subset. ConclusionMarkers of glucose metabolism showed significant associations with B cell, NK cell and NKT cell subsets, which clearly indicates a relation between glucose metabolism and the adaptive immune system.

Establishment and evaluation of a risk prediction model for coronary heart disease in primary Sjögren’s syndrome based on peripheral blood IL-6 and Treg percentages

ObjectiveThis study aims to establish and evaluate a risk prediction model for coronary heart disease (CHD) in patients with primary Sjögren’s syndrome (pSS) based on peripheral blood levels of interleukin-6 (IL-6) and the percentage of regulatory T cells (Treg%). This model is intended to facilitate the timely identification of high-risk patients and the implementation of preventive measures.MethodsClinical data were collected from 120 pSS patients who visited the Second Hospital of Shanxi Medical University between November 2021 and September 2023. Patients were classified into pSS and pSS-CHD groups according to CHD diagnostic criteria. Peripheral blood lymphocyte subsets and cytokine levels were assessed using flow cytometry. Univariate and multivariate logistic regression analyses were employed to identify independent risk factors, and a nomogram was constructed based on these factors. The model’s discriminatory ability, calibration, and clinical utility were evaluated using receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis.ResultsThe univariate and multivariate logistic regression analyses identified several independent risk factors for CHD in pSS patients: erythrocyte sedimentation rate (ESR) (OR=1.10, P=0.019), triglycerides (TG) (OR=3.67, P=0.041), IL-6 (OR=1.29, P=0.048), and Treg% (OR=0.25, P=0.004). A nomogram incorporating these factors demonstrated an area under the curve (AUC) of 0.96, indicating excellent predictive performance, and showed good calibration (P=0.599), suggesting significant clinical applicability. Furthermore, Treg% exhibited a negative correlation with cholesterol (CHOL) and low-density lipoprotein cholesterol (LDL-C) levels, while IL-6 showed a positive correlation with CHOL and LDL-C levels. TG was positively correlated with C-reactive protein (CRP).ConclusionThis study successfully developed a risk prediction model based on peripheral blood IL-6 and Treg% levels, providing critical evidence for the early identification and personalized prevention of CHD in pSS patients, with potential clinical implications.

Immunophenotyping characteristics and clinical outcome of COVID-19 patients treated with azvudine during the Omicron surge

BackgroundLittle is known about immunophenotyping characteristics and clinical outcomes of COVID-19 patients treated with azvudine during the Omicron variant surge.MethodsThis study enrolled patients diagnosed with COVID-19 from December 2022 to February 2023. The primary outcome was defined as all-cause mortality, along with a composite outcome reflecting disease progression. The enrolled patients were followed for a period of 60 days from their admission.ResultsA total of 268 COVID-19 patients treated with azvudine were enrolled in this retrospective study. The study found that the counts of lymphocyte subsets were significantly reduced in the composite outcome and all-cause mortality groups compared to the non-composite outcome and discharge groups (all p < 0.001). Correlation analysis revealed a negative association between lymphocyte subsets cell counts and inflammatory markers levels. The receiver operating characteristic (ROC) curve analysis identified low CD4+ T cell count as the most significant predictor of disease progression and all-cause mortality among the various lymphocyte subsets. Additionally, both the Kaplan-Meier curve and multivariate regression analysis demonstrated that low CD4+ T cell count level (< 156.00 cells/μl) was closely associated with all-cause mortality in COVID-19 patients treated with azvudine.ConclusionsA low CD4+ T cell count may serve as a significant predictive indicator for identifying COVID-19 patients receiving azvudine treatment who are at an elevated risk of experiencing adverse outcomes. These findings may offer valuable insights for physicians in optimizing the administration of azvudine.

The Role of PD-1/PD-L1 and IL-7 in Lymphocyte Dynamics and Sepsis Progression: A Biomarker Study in Critically Ill Patients

Sepsis pathophysiology involves a dysregulated immune response to infection, excessive inflammation, and immune paralysis. This study explores the relationships between cell death biomarkers (serum-soluble levels of programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and interleukin-7 (IL-7)) and the percentages of various lymphocyte subsets in relation to the severity and progression of sepsis. This prospective, observational study included 87 critically ill patients. We monitored parameters on days 1 (sepsis was diagnosed according to the Sepsis-3 Consensus) and 5. We established an IL-7 cutoff value of 1.94 pg/mL by comparing levels between a healthy control group and patients with sepsis (p < 0.0001). Lymphopenia was observed in all patients, with negative correlations between helper T lymphocytes and cytotoxic and B lymphocytes, and positive correlations involving cytotoxic lymphocytes across all groups. We found correlations between PD-1/PD-L1 and lymphocyte subsets. IL-7 showed a statistical correlation with PD-1 in non-survivors. Assessing lymphocyte levels shows potential as a biomarker for evaluating the progression of sepsis. Monitoring IL-7 levels could help assess survival, as low levels are associated with higher mortality risk. Monitoring IL-7 levels could help assess survival, as low levels are associated with higher mortality risk. Elevated PD-1/PD-L1 expression impairs costimulatory signalling, reducing T cell responses and lymphopenia, which increases the risk of nosocomial infections.

Immune Signatures of Solid Tumor Patients Treated With Immune Checkpoint Inhibitors: An Observational Study.

Our study aimed to comprehensively describe the features of peripheral blood multiple immune cell phenotypes in solid tumor patients during pretreatment and after immunotherapy, providing a more convenient approach for studying the prognosis of immunotherapy in different solid tumor patients. We prospectively recruited patients with advanced solid tumors from Peking Union Medical College Hospital (PUMCH) between February 2023 and April 2024. Using multicolor flow cytometry, our study comprehensively observed and described the signatures of peripheral blood lymphocyte subsets including activation, proliferation, function, naïve memory, and T cell exhaustion immune cell subsets in this population of pretreatment and after immunotherapy. Our study enrolled 59 advanced solid tumor patients with immunotherapy and 59 healthy controls were matched by age and gender. The results demonstrated a marked upregulation in the expression of lymphocyte activation markers CD38 and HLA-DR, as well as exhaustion and proliferation markers PD-1 and Ki67, in solid tumor patients compared to healthy controls. After immune checkpoint blockade (ICB) treatment, mainly the expression of Ki67CD4+T and HLA-DRCD38CD4+T, was significantly upregulated compared to pretreatment levels (p = 0.017, p = 0.019, respectively). We further found that gynecological tumors with better prognoses had higher baseline activation levels of CD4+ T cells compared to other solid tumors with poorer prognoses. Our study elucidated the characteristics of different lymphocyte subsets in the peripheral blood of solid tumor patients. Further research revealed changes in the phenotypes of different lymphocyte subsets after ICIs treatment, with the activated phenotype of CD4+ T cells playing a crucial role in the antitumor effect. This lays the groundwork for further exploration of prognostic biomarkers and predictive models for cancer patients with immunotherapy.

Real-World Safety and Effectiveness of Dimethyl Fumarate in Patients with MS: Results from the ESTEEM Phase4 and PROCLAIM Phase3 Studies with a Focus on Older Patients.

Real-world studies in the USA report that 41-56% of patients with multiple sclerosis (MS) are ≥ 50years old, yet data on their response to disease-modifying therapies (DMTs) is limited. Dimethyl fumarate (DMF) is an oral DMT approved for treating relapsing MS. This analysis evaluated the safety, efficacy, and immunophenotype changes of DMF in patients ≥ 50years compared with patients < 50years. ESTEEM, a 5-year, real-world, observational phase4 study, assessed the safety and effectiveness of DMF, including treatment-emergent serious adverse events (SAEs) and adverse events (AEs) leading to treatment discontinuation. Absolute lymphocyte counts (ALCs) were recorded from a subset of patients. The PROCLAIM study, a phase3b interventional study, reported safety outcomes and lymphocyte subset changes in patients with relapsing-remitting MS (RRMS) treated with DMF. The study evaluated safety outcomes by analyzing the incidence of SAEs and detailed changes in CD4+ and CD8+ Tcell compartments over 96weeks of DMF treatment. ESTEEM included 4020 patients aged < 50years and 1069 aged ≥ 50years. AEs leading to discontinuation were reported by 19.6% patients < 50years and 29.6% of patients ≥ 50years, with gastrointestinal disorders being the most common. SAEs were reported by 5.2% of patients < 50years and 8.9% those ≥ 50years. In PROCLAIM, SAEs were reported in 13% of patients < 50years and 10% of those ≥ 50years. Median ALC decreased by 35% in patients < 50years and 50% in those ≥ 50years in ESTEEM, with similar patterns observed in PROCLAIM. ESTEEM found no unexpected safety signals in older patients and annualized relapse rates (ARRs) were significantly reduced in both age groups. Both studies indicated that DMF is efficacious and has a favorable safety profile in patients with RRMS aged ≥ 50years. ESTEEM (NCT02047097), PROCLAIM (NCT02525874).

Unveiling the hidden link: elevated platelets and T cell subsets in 5% of moderate COVID-19 patients 48 days post-onset.

Platelets are hyperactived during acute COVID-19, promoting clotting and modulating immune-cell responses. Immune thrombocytopenia in adults can manifest as an uncommon complication resulting from various viral infections or as a rare adverse event associated with vaccination. However, their role in convalescent COVID-19 patients remains underexplored. This study examines platelet dynamics early in the pandemic, 48 days post-symptom onset, in unvaccinated patients. This longitudinal study included 298 unvaccinated COVID-19 patients (17 mild, 281 moderate) from multiple centers. Clinical evaluations and peripheral lymphocyte subset analyses via flow cytometry were conducted upon admission and on day 48 post-symptom onset (DPSO 48). At DPSO 48, 5.3% of moderate COVID-19 patients exhibited high platelet counts (>300×109/L), associated with elevated total T-cells (26.4%), CD4 T-cells (24.4%), CD8 T-cells (36.9%), and Tregs (33.9%) compared to patients with normal platelet counts. However, the CD4/CD8 T-cell ratio and T-cell subset frequencies remained unaffected, indicating ongoing T-cell homeostasis restoration. Additionally, a significant positive correlation (r=0.636, p=0.03) was found between platelet counts and B cells in patients with elevated platelet counts. Platelets may play a pivotal role in immune regulation during the recovery phase of COVID-19. Targeting platelets and their secreted mediators could improve immune balance in patients with immune disorders, highlighting a potential therapeutic approach for enhancing recovery in post-COVID-19 patients.

The diagnostic value of peripheral blood lymphocyte testing in children with infectious mononucleosis

ObjectiveTo investigate the diagnostic value of peripheral blood lymphocyte testing in children with infectious mononucleosis (IM).MethodsA total of 135 children with IM as the IM group and 100 healthy volunteers as the healthy group were included in this retrospective study. Peripheral blood lymphocyte subsets marked as CD3+, CD4+, CD8+, CD16 + CD56+, and CD19 + in the peripheral blood were quantified using flow cytometry. Statistical analysis was performed using the chi-square test, Kruskal-Wallis test, AUROC curve, and Kappa consistency test to assess the diagnostic value of these markers in IM.ResultsThe AUROC curve for CD8 + cells and for CD4+/CD8 + ratios both achieved a value of 1 with the sensitivity and specificity of 100% (P<0.001). The Kappa coefficients were 1 for CD8+, CD4+/CD8 + ratios and the combined EBV analysis, indicating a 100% consistency with the clinical diagnosis. Significant differences were also observed in the CD3+, CD4+, CD16 + CD56+, and CD19 + lymphocyte subsets between the IM group and the healthy group (P<0.05).ConclusionThe evaluation of CD8 + and CD4+/CD8 + ratios in peripheral blood lymphocytes represents a significant advancement in the diagnosis of IM. Peripheral blood lymphocyte testing offers a reliable, sensitive, and specific diagnostic tool to enhance the clinical management of children with IM.

Changes in T Lymphocyte Subsets Following COVID-19 Infection and Vaccination

Changes in T Lymphocyte Subsets Following COVID-19 Infection and Vaccination

Infants < 90 days of age with late-onset sepsis display disturbances of the microbiome-immunity interplay.

We hypothesized that previously healthy infants < 90 days of age with late-onset sepsis (LOS) have disturbances of the gut microbiome with yet undefined specific immunological patterns. We performed a prospective single-center convenience sample study between January 2019 and July 2021 in a case-control design. Routine diagnostics included conventional cultures (blood, cerebrospinal fluid, urine), PCRs and inflammatory markers in infants aged < 90 days with clinical LOS. We additionally analyzed blood lymphocyte subsets including CD4 + CD25 + forkhead box protein (FoxP3)+ Tregs and performed 16S rRNA sequencing of stool samples, both compared to age-matched healthy controls. Results were adjusted for potential confounders that may influence microbial composition. 51 infants with fever and clinical LOS were enrolled. Bacterial sepsis was diagnosed in n = 24 (47.1%) and viral infection in n = 13 (25.5%) infants, whereas in 14 (27.3%) infants the cause of fever remained undetermined. When compared to healthy controls, the gut microbiome of LOS infants at disease onset was characterized by a shift in community composition, specifically, decreased abundance of B. longum and an increase of Bacteroidia spp. Intriguingly, the abundance of B. longum negatively correlated with the frequency of blood CD4-positive cells in healthy controls but not in infants with LOS. At one year of age, we observed microbiome differences in infants with history of LOS when compared to healthy controls, such as an increased gut microbial diversity. Our data suggest potential signatures of the microbiome-immunity interplay in infants with LOS, which should be investigated further as possible targets for prevention.

Sex differences and immune correlates of Long Covid development, symptom persistence, and resolution.

Sex differences have been observed in acute coronavirus disease 2019 (COVID-19) and Long Covid (LC) outcomes, with greater disease severity and mortality during acute infection in males and greater proportions of females developing LC. We hypothesized that sex-specific immune dysregulation contributes to LC pathogenesis. To investigate the immunologic underpinnings of LC development and symptom persistence, we performed multiomic analyses on blood samples obtained during acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and 3 and 12 months after infection in a cohort of 45 participants who either developed LC or recovered. Several sex-specific immune pathways were associated with LC. Males who would later develop LC exhibited increases in transforming growth factor-β (TGF-β) signaling during acute infection, whereas females who would go on to develop LC had reduced TGFB1 expression. Females who developed LC demonstrated increased expression of XIST, an RNA gene implicated in autoimmunity, during acute infection compared with females who recovered. Many immune features of LC were also conserved across sexes, such as alterations in monocyte phenotype and activation state. Nuclear factor κB (NF-κB) transcription factors were up-regulated in many cell types at acute and convalescent time points. Those with ongoing LC demonstrated reduced ETS1 expression across lymphocyte subsets and elevated intracellular IL-4 in T cell subsets, suggesting that ETS1 alterations may drive aberrantly elevated T helper cell 2-like responses in LC. Altogether, this study describes multiple innate and adaptive immune correlates of LC, some of which differ by sex, and offers insights toward the pursuit of tailored therapeutics.

EXTH-48. ADENO-ASSOCIATED VIRUS TARGETING OF THE TUMOR MICROENVIRONMENT TO IMPROVE LYMPHOCYTE RECRUITMENT IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is notoriously devoid of lymphocytes driven in part by a paucity of lymphocyte trafficking factors necessary to prompt their recruitment, infiltration, and activation. We have developed a novel recombinant adeno-associated virus (AAV) gene therapy strategy that enables focal and stable reconstitution of the GBM tumor microenvironment (TME) with C-X-C motif ligand 9 (CXCL9), a powerful call-and-receive chemokine for cytotoxic T lymphocytes (CTLs). By increasing lymphocyte recruitment in GBM, we HYPOTHESIZE that these tumors will be more responsive to immunotherapy. METHODS 3D fluorescence microscopy was used to characterize the distribution, tropism, and durability of AAV6 transgene expression in preclinical GBM. Flow cytometry and 3D fluorescence microscopy was used to quantify T cell infiltration and visualize their biodistribution in preclinical GBM treated with AAV-CXCL9 in combination with anti-PD-1 immune checkpoint blockade (ICB). Single-cell RNA sequencing was used to characterize intratumor lymphocyte subsets following treatment. Survival studies with and without lymphocyte depletion were done to evaluate immunogenic responsiveness of therapy. RESULTS Focal delivery of CXCL9 with AAV6 results in stable transduction of peri-tumoral astrocytes. When combined with anti-PD-1 ICB, lymphocyte infiltration was improved, with cells found disseminated across the tumor stroma and within the tumor parenchyma. CXCR3, the chemokine receptor for CXCL9, was expressed across lymphocyte subsets and NKT cells, validating treatment selectivity of AAV6-CXCL9 for lymphocytes. Combination treatment considerably improved overall survival in two distinct GBM models, with durable responses dependent on CD8 T-lymphocyte infiltration. CONCLUSIONS By manipulating local chemokine directional guidance, AAV-CXCL9 increases tumor infiltration by CD8-postive cytotoxic lymphocytes, sensitizing GBM to anti-PD-1 ICB. These effects are accompanied by immunologic signatures evocative of an inflamed and responsive TME. These findings support targeted AAV gene therapy as a promising adjuvant strategy for reconditioning GBM immunogenicity given its excellent safety profile, TME-tropism, modularity, and off-the-shelf capability.

Correlation Analysis of Serum 25-Hydroxyvitamin D Levels With Immune Function and Calcium-Phosphate Metabolism in Patients With Bronchial Asthma Treated With Combination Therapy

It was to investigate the clinical efficacy of the combination therapy of fluticasone propionate inhalation aerosol and vitamin D (VD) in pediatric bronchial asthma (BA) and analyze the correlation between serum 25-(OH)-D3 levels and immune function, as well as calcium-phosphorus metabolism. A total of 110 patients with BA were recruited. Regarding treatment plan, patients were randomly rolled into a single-drug treatment group (SDT, treated with fluticasone propionate inhalation aerosol alone) and a dual-drug treatment group (TDT, treated with the combination of fluticasone propionate inhalation aerosol and VD). The changes in serum 25-(OH)-D3 levels, immunoglobulins, T lymphocyte subsets, and inflammatory cytokine levels in children with BA under different treatment modalities were compared. Clinical symptom disappearance, asthma control, and quality of life (QoL) were assessed, and the total effective rate and adverse reactions (ARs) were compared. A control group consisting of 60 healthy children who underwent concurrent physical examinations was included. The differences in serum 25-(OH)-D3 levels, immunoglobulins, and T lymphocyte subset levels between children with BA and healthy controls were compared, and their correlations were analyzed. The TDT group showed a drastic reduction in the disappearance time of lung wheezing and dyspnea relative to the SDT group. Furthermore, the TDT group exhibited notable improvements in lung function parameters, including forced vital capacity (FVC), forced expiratory volume at one second (FEV1), FEV1/FVC, and peak expiratory flow (PEF). Blood gas analysis revealed a great decrease in PaCO2 and an increase in PaO2. The Childhood Asthma Control Test (C-ACT) scores for asthma control and Pediatric Asthma Quality of Life Questionnaire (PAQLQ) scores for QoL showed marked increases in the TDT group. Moreover, the TDT group demonstrated notable increases in serum 25-(OH)-D3 levels, immunoglobulins (IgA, IgG, and IgM), T lymphocyte subsets (CD4+ and CD8+), as well as blood calcium and phosphorus levels. Additionally, the TDT group exhibited a prominent increase in the anti-inflammatory cytokine interleukin (IL)-10 level and a drastic decrease in the pro-inflammatory cytokines IL-6 and tumor necrosis factor alpha (TNF-α) levels (all P&lt;0.05). The total effective rates of treatment in the SDT group and TDT group were 83.64 % and 96.36 %, respectively, with AR rates of 16.36 % and 7.27 %. The TDT group exhibited a superior total effective rate and an inferior incidence of ARs to the SDT group (both P&lt;0.05). Additionally, in contrast to the control group, the BA group showed notable decreases in serum 25-(OH)-D3 levels, immunoglobulins (IgA, IgG, and IgM), T lymphocyte subsets (CD4+, CD8+, and CD4+/CD8+), as well as blood calcium and phosphorus levels (all P&lt;0.05). Prior to treatment, there was a positive correlation between serum 25-(OH)-D3 levels and immunoglobulins (IgA, IgG, and IgM), T lymphocyte subsets (CD4+ and CD8+), as well as blood calcium and phosphorus levels in children with BA (P&lt;0.05). In patients with BA, combined treatment with inhaled fluticasone propionate aerosol and VD may have a regulatory effect on serum 25-hydroxyVD levels, immune function, and calcium-phosphate metabolism. The correlation between serum 25-(OH)-D3 levels and immune function, as well as calcium-phosphate metabolism, suggested that VD may play a crucial role in the immune regulation and calcium-phosphate metabolism of BA.

Tumor-Infiltrating B Cells and Tissue-Resident Memory T Cells as Prognostic Indicators in Brain Metastases Derived from Gastrointestinal Cancers.

Tumor-infiltrating B cells (TIBs) and tissue-resident memory T cells (TRMs) play significant roles in antitumor immunity. However, their prognostic relevance in brain metastases (BMs) derived from gastrointestinal (GI) cancers remains unclear. This study aimed to investigate the prognostic significance of TIBs and TRMs in GI cancer-derived BMs (GIBMs). Retrospective histopathological analyses were performed on surgically resected GIBM tissues from 13 patients. The densities of tumor-infiltrating lymphocytes (TIL) subsets (TIBs, CD4+ T cells, CD8+CD103+ TRMs, and CD8+CD103- non-TRMs) were quantified and correlated with clinical parameters and overall survival (OS) including the Graded Prognostic Assessment (GPA). TIBs and CD4+ T cells were predominantly accumulated in the tumor stroma, particularly around blood vessels, where they formed lymphocyte clusters without characteristics of tertiary lymphoid structures (TLSs). In contrast, TRMs more deeply infiltrated into the tumor epithelium than their counterpart non-TRMs. Positive correlations were found between TIB density and both the prognostic prediction of GPA and overall survival (OS) after BM diagnosis or surgery. Furthermore, increased densities of TIBs and TRMs were associated with enhanced survival after BM diagnosis. TIB and TRM densities in BM tissues could serve as reliable prognostic indicators for survival in patients with GIBMs. This study provides crucial insights for the development of novel immunotherapeutic strategies against this lethal disease.

Consensus of experts on clinical quality management for flow cytometry-based detection of lymphocyte subsets

Flow cytometry (FCM) is used to detect lymphocyte subsets and evaluate the level of cellular immunity in the body, which is one of the commonly used FCM detection projects in clinical practice. To date, numerous institutions and academic organizations, both domestically and internationally, have published guidelines and consensus documents covering various aspects, including the establishment of cytometry laboratories, standardized procedures for detecting lymphocyte subsets, and their clinical applications across different fields, each with a distinct focus. However, in clinical practice, laboratories continue to face challenges in establishing standardized quality management systems, implementing quality assurance activities, developing appropriate detection protocols and gating strategies for various scenarios, and identifying and resolving common issues encountered during detecting. Currently, there is no unified consensus on these issues. To standardize the development of quality management systems for lymphocyte subsets detecion via FCM, enhance laboratory quality management practices, and ensure the accuracy and reliability of results, the Laboratory Medicine Committee of Chinese Association of Integrative Medicine (LMC-CAIM) convened experts to formulate this expert consensus.

Dynamic Changes in Lymphocyte Populations and Their Relationship with Disease Severity and Outcome in COVID-19.

Studies suggest that the dynamic changes in cellular response might correlate with disease severity and outcomes in SARS-CoV-2 patients. The study aimed to investigate the dynamic changes of lymphocyte subsets in patients with COVID-19. In this regard, 53 patients with COVID-19 were prospectively included, classified as mild, moderate, and severe. The peripheral lymphocyte profiles (LyT, LyB, and NK cells), as well as CD4+/CD8+, CD3+/CD19+, CD3+/NK and CD19+/NK ratios, and their dynamic changes during hospitalization and correlation with disease severity and outcome were assessed. We found significant differences in CD3+ lymphocytes between severity groups (p < 0.0001), with significantly decreased CD3+CD4+ and CD3+CD8+ in patients with severe disease (p < 0.0001 and p = 0.048, respectively). Lower CD3+/CD19+ and CD3+/NK ratios among patients with severe disease (p = 0.019 and p = 0.010, respectively) were found. The dynamic changes of lymphocyte subsets showed a significant reduction in NK cells (%) and a significant increase in CD3+CD4+ and CD3+CD8+ cells in patients with moderate and severe disease. The ROC analysis on the relationship between CD3+ cells and fatal outcome yielded an AUC of 0.723 (95% CI 0.583-0.837; p = 0.007), while after addition of age and SpO2, ferritin and NLR, the AUC significantly improved to 0.927 (95%CI 0.811-0.983), p < 0.001 with a sensitivity of 90.9% (95% CI 58.7-99.8%) and specificity of 85.7% (95% CI 69.7-95.2%). The absolute number of CD3+ lymphocytes might independently predict fatal outcomes in COVID-19 patients and T-lymphocyte subset evaluation in high-risk patients might be useful in estimating disease progression.