Blood Lymphocyte Subsets Research Articles

The value of NLR versus MLR in the short-term prognostic assessment of HBV-related acute-on-chronic liver failure.

Systemic inflammation is associated with the development and progression of hepatitis B-associated acute-on-chronic liver failure (HBV-ACLF). The neutrophil-to-lymphocyte ratio (NLR) has been reported to be a prognostic biomarker in patients with HBV-ACLF. However, the role of the monocyte-to-lymphocyte ratio (MLR) as a prognostic inflammatory biomarker in multiple diseases is rarely mentioned in HBV-ACLF. We included a total of 347 patients with HBV-ACLF who met the definition of the Chinese Guidelines for the Diagnosis and Treatment of Liver Failure (2018 edition). Among them, 275 cases were included retrospectively, and 72 cases were collected prospectively. Clinical characteristics and laboratory examination data were collected from medical records within 24h after diagnosis to calculate MLR and NLR levels, and lymphocyte subpopulation counts were collected in prospectively included patients. Of the 347 patients with HBV-ACLF, 128 patients in the non-surviving group had a mean age of 48.87±12.89years; 219 patients in the survival group had a mean age of 44.80±11.80years and a combined 90-day mortality rate of 36.9%. The median MLR was higher in the non-survivors than in the survivors (0.690 vs 0.497, P<0.001). MLR values were significantly associated with 90-day mortality in HBV-ACLF (OR 6.738; 95% CI 3.188-14.240, P<0.001). The AUC for the predictive power of the combined MLR and NLR analysis for HBV-ACLF was 0.694, and the calculated MLR threshold was 4.495. In addition, in the analysis of peripheral blood lymphocyte subsets in HBV-ACLF, a significant decrease in the number of circulating lymphocytes was found in HBV-ACLF patients in the non-surviving group (P<0.001), with a predominant decrease in the number of CD8+T cells and no significant difference in the number of CD4+T cells, B cells or NK cells. Increased MLR values are associated with 90-day mortality in patients with HBV-ACLF, and the MLR may serve as a potential prognostic indicator for patients with HBV-ACLF. Decreased CD8+T-cell counts may be associated with poor survival in patients with HBV-ACLF.

Th17 / Treg ratio: A prospective study in a group of pregnant women with preeclampsia and fetal growth restriction

IntroductionDuring pregnancy, the maternal immune system is challenged to tolerate a semi-allogenic fetus. A proinflammatory profile has been linked to adverse pregnancy outcomes and poor placental development. In this study, the authors evaluated the number of circulating Tregs and Th17 cells in a group of patients diagnosed with preeclampsia(PE) and fetal growth restriction(FGR). MethodsProspective longitudinal observational study where peripheral blood lymphocyte subsets were analyzed in a cohort of pregnant patients with PE, FGR, and a control group of healthy pregnant women. ResultsThe diagnosis of PE was associated with a significative higher number of circulating Th17 cells and a significative relative reduction in the Treg cell count. This proinflammatory profile was also expressed in the evolution of the Th17/ CD4+CD25highFOXP3+ Treg ratio. In the FGR group, the Th17 cell count was significantly higher during the third trimester of pregnancy. This proinflammatory profile was also expressed in the evolution of the Th17/ CD4+CD25highFOXP3+ Treg ratio. When we compare the immunological profiles of patients with PE and FGR we observed a higher number of proinflammatory Th17 cells and a significative lower number of Treg cells in PE patients. This is particularly expressed in the differences found between the Th17/ CD4+CD25highFOXP3+ Treg ratios of these two groups.Discussion/ConclusionOur data showed a that a proinflammatory profile and a relative excess of Th17 cells was associated with the diagnosis of PE and FGR. A more exuberant systemic proinflammatory profile present in the PE patients is absent in patients with FGR without preeclampsia

Changes in Peripheral Immune Cells after the Third Dose of SARS-CoV-2 mRNA-BNT162b2 Vaccine and Disease Outcomes in Cancer Patients Receiving Immune Checkpoint Inhibitors: A Prospective Analysis of the Vax-on-Third-Profile Study.

Anti-SARS-CoV-2 mRNA vaccines can deeply affect cell-mediated immune responses in immunocompromised recipients, including cancer patients receiving active treatments. The clinical implications of changes in peripheral blood lymphocyte subsets following the third dose of mRNA-BNT162b2 vaccination (tozinameran) in patients on immune checkpoint blockade are not fully understood. We conducted a prospective analysis of the Vax-On-Third-Profile study to evaluate the impact of circulating lymphocyte dynamics on disease outcomes in this subgroup of patients. Recipients of booster dosing who had received before vaccination at least one course of an anti-PD-1/PD-L1 treatment for an advanced solid tumor were eligible. Immunophenotyping of peripheral blood was performed before the third dose of tozinameran (timepoint-1) and four weeks later (timepoint-2) to quantify the absolute counts of lymphocyte subpopulations, including CD3+CD4+ T cells, CD3+CD8+ T cells, B cells, and NK cells. Logistic regression was used to analyze the relationship between lymphocyte subsets and durable clinical benefit (DCB). The log-rank test and Cox regression model were applied to evaluate the relationship between lymphocyte subpopulations and both vaccine-related time-to-treatment failure (V-TTF) and overall survival (OS). We included a total of 56 patients with metastatic disease who were given a third dose of tozinameran between 23 September and 7 October 2021 (median age: 66 years; male: 71%). Most recipients had a diagnosis of lung cancer and were being treated with pembrolizumab or nivolumab. Compared to baseline, the third immunization resulted in an incremental change in the median counts of all lymphocyte subpopulations, which was statistically significant only for NK cells (p < 0.001). A significant correlation was found between NK cell counts and DCB at timepoint-2 (p < 0.001). Multivariate logistic regression analysis of DCB confirmed the predictive significance of high-level NK cell counts (p = 0.020). In multivariate Cox regression analysis, high-level NK cell counts independently predicted longer V-TTF [HR 0.34 (95% CI 0.14-0.80), p = 0.014] and OS [HR 0.36 (95% CI 0.15-0.89), p = 0.027]. Our data suggest expansion of NK cell counts as the most noteworthy change in circulating lymphocytes after the third dose of tozinameran in cancer patients receiving PD-1/PD-L1-targeted agents. This change correlated with enhanced therapeutic efficacy, improving the rate of disease control, and prolonging survival outcomes. Similar findings have not been previously reported, implying that they have proof-of-concept value and warrant further confirmation.

Peripheral lymphocyte subsets as predicting factors for molecular recurrence after imatinib discontinuation in a phase 2 imatinib discontinuation trial in patients with chronic myeloid leukemia

IntroductionTreatment-free remission (TFR) is successful in half of the patients with chronic myeloid leukemia who discontinue Imatinib (IM) after sustained molecular response. MethodsIn a prospective trial, we used pioglitazone for 3 months before stopping IM in 30 patients. Percentages of peripheral blood lymphocyte subsets were assessed before and after treatment. The relation of these data with duration of IM treatment and TRF were examined. ResultsThe median time of IM treatment was 117.6 months. After discontinuation, 11 patients had molecular recurrence after 5.2 months (2.4 – 30). The observation time for those remaining in TFR was 46 (26 – 56) months. The independent factors for the maintenance of TFR were the duration of IM treatment and the percentage of double-positive T cells at IM stop. ConclusionA longer treatment with imatinib was associated with a longer TFR after discontinuation. Pioglitazone could act as an immunomodulator, increasing DP T cells which may contribute to prevent relapse.

Analysis of lymphocyte subsets in patients with sepsis and its impact on prognosis

To explore the characteristics of changes in peripheral blood lymphocyte subsets in patients with sepsis in intensive care unit (ICU) and analyze their predictive value for prognosis. The clinical data of sepsis patients admitted to the surgical intensive care unit (SICU) of the First Affiliated Hospital of Zhengzhou University from January 2020 to December 2021 were analyzed retrospectively. The patients met the diagnostic criteria of Sepsis-3 and were ≥ 18 years old. Peripheral venous blood samples were collected from all patients on the next morning after admission to SICU for routine blood test and peripheral blood lymphocyte subsets. According to the 28-day survival, the patients were divided into two groups, and the differences in immune indexes between the two groups were compared. Logistic regression analysis was used to analyze the risk factors of immune indexes that affect prognosis. (1) A total of 279 patients with sepsis were enrolled in the experiment, of which 198 patients survived at 28 days (28-day survival rate 71.0%), and 81 patients died (28-day mortality 29.0%). There were no significant differences in age (years old: 57.81±1.71 vs. 54.99±1.05) and gender (male: 60.5% vs. 63.6%) between the death group and the survival group (both P > 0.05), and the baseline data was comparable.(2) Acute physiology and chronic health evalution II (APACHE II: 22.06±0.08 vs. 14.08±0.52, P < 0.001), neutrophil percentage [NEU%: (88.90±1.09)% vs. (84.12±0.77)%, P = 0.001], procalcitonin [PCT (μg/L): 11.97±2.73 vs. 5.76±1.08, P = 0.011], platelet distribution width (fL: 16.81±0.10 vs. 16.57±0.06, P = 0.029) were higher than those in the survival group, while lymphocyte percentage [LYM%: (6.98±0.78)% vs. (10.59±0.86)%, P = 0.012], lymphocyte count [LYM (×109/L): 0.70±0.06 vs. 0.98±0.49, P = 0.002], and platelet count [PLT (×109/L): 151.38±13.96 vs. 205.80±9.38, P = 0.002], and thrombocytocrit [(0.15±0.01)% vs. (0.19±0.07)%, P = 0.012] were lower than those in the survival group. (3) There was no statistically significant difference in the percentage of lymphocyte subsets between the death group and the survival group, but the absolute value of LYM (pieces/μL: 650.24±84.67 vs. 876.64±38.02, P = 0.005), CD3+ absolute value (pieces/μL: 445.30±57.33 vs. 606.84±29.25, P = 0.006), CD3+CD4+ absolute value (pieces/μL: 239.97±26.96 vs. 353.49±18.59, P = 0.001), CD19+ absolute value (pieces/μL: 111.10±18.66 vs. 150.30±10.15, P = 0.049) in the death group was lower than those in the survival group. Other lymphocyte subsets in the death group, such as CD3+CD8+ absolute value (pieces/μL: 172.40±24.34 vs. 211.22±11.95, P = 0.112), absolute value of natural killer cell [NK (pieces/μL): 101.26±18.15 vs. 114.72±7.64, P = 0.420], absolute value of natural killer T cell [NKT (pieces/μL): 33.22±5.13 vs. 39.43±2.85, P = 0.262], CD4-CD8- absolute value (pieces/μL: 41.07±11.07 vs. 48.84±3.31, P = 0.510), CD4+CD8+ absolute value (pieces/μL: 3.39±1.45 vs. 3.47±0.36, P = 0.943) were not significantly different from those in the survival group. (4)Logistic regression analysis showed that lymphocyte subsets were not selected as immune markers with statistical significance for the prognosis of sepsis. The changes of immune indexes in sepsis patients are closely related to their prognosis. Early monitoring of the above indexes can accurately evaluate the condition and prognosis of sepsis patients.

The decreased of peripheral blood natural killer cell is associated with serum IL-2 level in the renal tubular acidosis in patients with primary sjogren’s syndrome

BackgroundPrimary Sjogren’s Syndrome (pSS) is a lymphoproliferative disease with autoimmune characteristics, which is characterized by lymphocyte infiltration of exocrine glands and involvement and dysfunction of extraglandular organs. Renal tubular acidosis (RTA) is a common renal involvement in pSS. This study investigated the phenotypic characteristics of peripheral blood lymphocyte subsets and cytokines in pSS patients complicated with RTA (pSS-RTA).MethodThis retrospective study included 25 pSS patients complicated with RTA and 54 pSS patients without RTA (pSS-no-RTA). To examine the level of peripheral lymphocytes subsets, flow cytometry analysis was used. The level of serum cytokines were detected by flow cytometry bead array(CBA). The influencing factors related to the occurrence of pSS-RTA were identified through logistic regression analyze.ResultsThe absolute number of CD4 + T cells and Th2 cells in peripheral blood were decreased in pSS-RTA patients than pSS-no-RTA patients. Moreover, the absolute number of NK cells and Treg cells were also decreased in pSS-RTA patients than pSS-no-RTA. The level of serum IL-2 was higher in pSS-RTA patients than pSS-no-RTA patients, and is negatively correlated with the number of NK cells, the number and percentage of Th17 cells, and Th17/Treg. Serum IL-2 level is also correlated with various cytokines. Multivariate logistic analysis proved that elevated ESR and ALP were risk factors for pSS complicated with RTA, while Treg was a protective factor.ConclusionThe increase of serum IL-2 level and the decrease of peripheral blood NK cells and Treg cells may be the immune mechanism of the development of pSS-RTA disease.

#5903 CLINICAL OUTCOME CORRELATION AMONG NEWLY DIAGNOSED LUPUS NEPHRITIS IN RELATION TO PERIPHERAL BLOOD LYMPHOCYTE SUBSET: A PROSPECTIVE STUDY

Abstract Background and Aims Given the heterogeneity in presentation, treatment practices and the outcome of lupus nephritis, there is a need for new protocols to predict outcome and rationalize treatment. Aim To prospectively study the correlation between peripheral blood lymphocyte subset, inflammatory markers and clinical outcomes among newly diagnosed lupus nephritis patients. Method Lymphocyte subset typing by flow cytometry method done at diagnosis and end of 6 months. SLEDAI -2k score. Intensive phase of Immunosuppression individualized by treating physician. Urine protein excretion was used to assess the final treatment outcome. Statistical analyses done using SPSS software. Two-sided P value of &amp;lt; 0.05 was considered significant. Pearson correlation coefficient formula used for correlations. Logistic regression done to identify factors responsible for clinical remission achieved at six months. Inclusion: All newly diagnosed renal biopsy proven Class 3, class 4, Class 5 or a combination class of lupus nephritis patients seen at Kasturba Hospital and Medical College, Manipal. Exclusion: Patients with active/recent infection, active malignancy, those with prior explosion to immunosuppression. Outcome measures, 1. To detect correlation if any, between peripheral blood lymphocyte subset, serum inflammatory markers and disease activity score at presentation. 2. To determine correlation if any, between peripheral blood lymphocyte subset and clinical remission achieved following immunosuppressive regimens at the end of six months of initial intensive treatment phase. Results 22 patients were taken for study. Relationship between SLEDAI score, lymphocyte subset characters, and clinical outcome given in Table 1 and 2. There was good correlation between lymphocyte subset characters, inflammatory markers and SLEDAI score, indicating ability of lymphocyte subset characters to characterize the severity of lupus nephritis. We found significant difference in lymphocyte subset characters between baseline and at the end of 6 months treatment. But there was no significant correlation for remission attainment with lymphocyte subset characters and SLEDAI score. Conclusion 1. Correlation found between peripheral blood lymphocyte subset, serum inflammatory markers and disease activity score at presentation and 6 months of treatment of lupus nephritis. 2. Peripheral blood Lymphocyte subset analysis was not found useful in predicting clinical outcome in lupus nephritis at the end of 6 months of intensive treatment.

Satisfaction and quality of life of patients treated with tobramycin-dexamethasone suspension in daytime surgery managed by integrated nursing model

Purpose: To investigate the effect of an integrated nursing model in day surgeries, treated with tobramycin-dexamethasone suspension, and its influence on patient satisfaction, quality of life, and indicators of immunity recovery.&#x0D; Methods: Ninety-eight patients with ophthalmic diseases admitted to Anhui No. 2 Provincial People’s Hospital, Anhui, China from January 2021 to June 2022 were scheduled for surgery and treated with tobramycin-dexamethasone. They were randomly divided into two groups (control and study groups, with 49 cases in each group). All patients received routine nursing intervention, and an integrated nursing model was applied to the study group on this basis. Improvement in ocular symptoms, SF-36, changes in immune-related lymphocyte subsets in peripheral blood, and nursing satisfaction were compared.&#x0D; Results: Ocular symptom score at 24 h after operation in the study group was lower than in control group (p &lt; 0.05). The SF-36 scores (physical status, cognitive ability, social function, emotional and mental status), immune-related lymphocyte subsets, and overall satisfaction rate after intervention in the study group were higher than those in the control group (p &lt; 0.05).&#x0D; Conclusion: Integrated nursing model achieves ideal nursing effect in day surgery managed with tobramycin-dexamethasone&gt; It also facilitates the recovery of patient’s eyes and plays a significant role in improving the quality of life, restoration of immunity, and nursing satisfaction of patients. A multicenter investigation will, however, be required prior to its full-scale clinical application.

The initial report of phase I trial of VG2025, a non-attenuated HSV-1 oncolytic virus expressing IL-12 and IL-15/RA payloads, in patients with advanced solid tumors.

2580 Background: VG2025 is a non-attenuated HSV-1 Oncolytic virus (OV). The wildtype neurovirulence is controlled by the Transcription and Translation Dual Regulation (TTDR) of ICP27 and ICP34.5 proteins. IL-12, IL-15, IL-15Ra payloads are controlled by tumor promoter CxCR4. The native promoter of ICP27 was replaced with a promoter for Carcinoembryonic Antigen (CEA). Here we report a first-in-human, open label, study to evaluate the safety, pharmacokinetics (PK), and biologic effects of VG2025 in patients (pts) with advanced solid tumors progressed after standard of care from China and United States. The study is actively recruiting. NCT05266612. Methods: Dose escalation follows a 3+3 design at 4 dose levels as intratumoral, and image guided injections into deep lesions on days 1 and 15 of each 28 days treatment cycle. PK and viral shedding (DNA), samples from biopsies, blood, urine, and swabs from injection site and other anatomical locations were analyzed by PCR. Changes of cytokines and lymphocyte subsets in blood were also observed as pharmacodynamic parameters. Results: As of 10 Jan 2023, 10 pts received doses of 1.0x10^8 and 2.0x10^8 PFU. 5 males and 5 females with the median age of 58 years were enrolled. 20% were PD(L)1 refractory, 20% had 2 Prior lines of therapy and 60% ≥3. The Median CEA level was 7.4. Tumor types were: 2 Duodenal Adeno Ca., 1 Pancreatic, 1 Ovarian, 1 Leiomyosarcoma, 1 Salivary Duct Ca., 1 Hepatocellular Ca (HCC), 1 Intrahepatic Cholangiocarcinoma (ICC), 1 Colorectal Ca (CRC) and 1 Colon Ca. No Dose Limiting Toxicities were observed. Any grade treatment-related AEs (TRAEs) was 70%, the most common TRAEs was fever 60%. Grade ≥3 TRAEs were 50% [3 pts had grade 4 Lymphocyte decreased (all recovered)], 1 had grade 3 COVID-19 and 1 had grade 3 nausea and vomiting. 4 pt had SAEs (2 cases of COVID-19, 1 Ascites and 1 Nausea and Vomiting). No TRAEs leading to dose reduction and treatment discontinuation. No pts had positive viral shedding. 4 pts were efficacy evaluable; ORR 25% (1 pt with ICC had confirmed PR with -45%) and DCR 100% based on RECIST v1.1. The PR pt had meaningful reductions in CEA levels. Tumor shrinkage was also observed in non-injected lesions demonstrating an abscopal effect. Nanostring analysis of paired biopsies from the PR pt demonstrate upregulation of genes for integrin signaling pathway, activation of CD8+ T cells and T cell-based immunity, upregulation of antigen-presenting MHC (HLA-DQA1 and HLA-DRB1) and chemokine receptor (CMKLR1) genes. Cellular immunity was activated by upregulation of Th1 cells, macrophage-associated genes and downregulation of Treg-associated genes. The results are consistent with the mechanism of action of VG2025. Analysis is ongoing with other subjects. Conclusions: Monotherapy activity was observed with an acceptable safety profile in heavily pretreated pts with advanced solid tumors. Clinical trial information: NCT05266612 .

Early peripheral blood lymphocyte subsets and cytokines in predicting the severity of influenza B virus pneumonia in children.

Children with influenza B virus infection have a higher susceptibility and higher severity of illness. The activation and disorder of immune function play an important role in the severity of influenza virus infection. This study aims to investigate whether early lymphocyte count and cytokines can provide predictive value for the progression in children with influenza B virus pneumonia. A retrospective cohort study was conducted to analyze the clinical data of children with influenza B virus pneumonia from December 1, 2021, to March 31, 2022, in the National Children's Regional Medical Center (Shengjing Hospital of China Medical University). According to the severity of the disease, the children were divided into a mild group and a severe group, and the clinical characteristics, routine laboratory examination, lymphocyte subsets, and cytokines were compared. A total of 93 children with influenza B virus pneumonia were enrolled, including 70 cases in the mild group and 23 cases in the severe group. Univariate analysis showed that drowsiness, dyspnea, white blood cell (WBC), lymphocytes, monocytes, procalcitonin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), fibrinogen (FIB), Immunoglobulin M (IgM), lung consolidation, total T cell count, CD4+ T cell count, CD8+ T cell count, NK cell count, NK cell % and B cell % had statistical differences between the mild and severe groups (P<0.05). In multivariate logistic regression analysis, reduced ALT (OR = 1.016), FIB (OR = 0.233), CD8+ T cell count (OR = 0.993) and NK cell count (OR = 0.987) were independently associated with the development of severe influenza B virus pneumonia. The levels of T lymphocytes and NK cells were related to the progression of influenza B virus pneumonia in children, and the reduction of CD8+ T cell count and NK cell count can be used as independent risk factors for predicting the severity of influenza B virus pneumonia.

Peripheral blood lymphocytes influence human papillomavirus infection and clearance: a retrospective cohort study

BackgroundThere is a close correlation between HPV infection and systemic immune status. The purpose of this study was to determine which lymphocytes in peripheral blood influence human papillomavirus (HPV) infection and to identify whether peripheral blood lymphocyte (PBL) subsets could be used as biomarkers to predict HPV clearance in the short term.MethodsThis study involved 716 women undergoing colposcopy from 2019 to 2021. Logistic and Cox regression were used to analyze the association of PBLs with HPV infection and clearance. Using Cox regression, bidirectional stepwise regression and the Akaike information criterion (AIC), lymphocyte prediction models were developed, with the C-index assessing performance. ROC analysis determined optimal cutoff values, and their accuracy for HPV clearance risk stratification was evaluated via Kaplan‒Meier and time-dependent ROC. Bootstrap resampling validated the model and cutoff values.ResultsLower CD4 + T cells were associated with a higher risk of HPV, high-risk HPV, HPV18 and HPV52 infections, with corresponding ORs (95% CI) of 1.58 (1.16–2.15), 1.71 (1.23–2.36), 2.37 (1.12–5.02), and 3.67 (1.78–7.54), respectively. PBL subsets mainly affect the natural clearance of HPV, but their impact on postoperative HPV outcomes is not significant (P > 0.05). Lower T-cell and CD8 + T-cell counts, as well as a higher NK cell count, are unfavorable factors for natural HPV clearance (P < 0.05). The optimal cutoff values determined by the PBL prognostic model (T-cell percentage: 67.39%, NK cell percentage: 22.65%, CD8 + T-cell model risk score: 0.95) can effectively divide the population into high-risk and low-risk groups, accurately predicting the natural clearance of HPV. After internal validation with bootstrap resampling, the above conclusions still hold.ConclusionsCD4 + T cells were important determinants of HPV infection. T cells, NK cells, and CD8 + T cells can serve as potential biomarkers for predicting natural HPV clearance, which can aid in patient risk stratification, individualized treatment, and follow-up management.

Assessment of circulating blood lymphocytes in adult patients on rituximab to treat immune thrombocytopenia: Circulating number of NK cells is associated with the response at 6 months.

Immune thrombocytopenia (ITP) is defined by a low platelet count that can trigger potentially life-threatening haemorrhages. Three-quarters of adult patients exhibit persistent or chronic disease and require second-line treatments. Among these, rituximab, an anti-CD20 antibody, has yielded valuable results, with global responses in 60% of patients at 6 months and complete responses in 30% at 5 years. Factors predictive of response to ITP therapy would help physicians choose optimal treatments. We retrospectively analysed clinical courses, biological markers and blood lymphocyte subset numbers of 72 patients on rituximab to treat persistent/chronic ITP followed-up in our department between 2007 and 2021, divided into three groups according to the platelet count at 6 months: complete, partial or no response. Among all studied parameters, a low number of CD3- CD16+ CD56+ circulating NK cells was associated with the complete response to rituximab. We also found that, after rituximab therapy, complete responders exhibited increased NK and decreased activated CD8+ T cell percentages. These results emphasize that the role played by NK cells in ITP remains incompletely known but that factors predictive of response to rituximab can be easily derived using blood lymphocyte subset data.

Abstract 41: Subcutaneous growth and immune cell profiling of BT-474 human breast carcinoma in four strains of triple immunodeficient mice

Abstract Triple immunodeficient mouse models, that lack functional T cells, B cells, and natural killer cells, are necessary when evaluating human cancer cell growth and cell-based therapy activity, allowing for assessment without innate mouse immune system involvement. Due to limited colony sizes at any given vendor, study timelines can sometimes become compromised unless a sufficient alternative is validated. Site location can also be a factor, as licensing restrictions may be an issue for international corporations. In an effort to prove overall similarities across four strains of triple immunodeficient mice, the growth characteristics of BT-474 were compared in female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, NOD.CB17-Prkdcscid IL2rgtm1/BcgenHsd (B-NDG) mice, NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl (NCG) mice, and NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (CIEA NOG) mice. An immune cell profile was also performed across these four mouse strains and evaluated via flow cytometry to observe any immune cell subset differences in blood, spleen, and tumor samples. Samples analyzed post implant were collected when subcutaneous tumors were ~500 mm3. Model development studies were completed previously to optimize tumor growth kinetics. Historic growth conditions were BT-474 cells implanted at 1.0E+07 cells/implant in the presence of an extracellular matrix into the high right axilla of female NSG mice. Using the same optimized growth conditions, NSG, B-NDG, NCG, and CIEA NOG mice were implanted with BT-474 cells. BT-474 grew well in all the tested mouse strains, producing 100% take rate. Compared to NSG mice, median tumor volume doubling times differed by 1-5 days, median times to an evaluation size of 150 mm3 (typical study enrollment tumor volume) differed by +/- ~4 days and median time to an evaluation size of 1250 mm3 (tumor volume at the end of life) differed by +/- ~10 days. BT-474 did not induce body weight loss in any of the tested mouse strains and all clinical observations were similar. Flow cytometry was performed to quantify lymphocyte and myeloid immune subsets in blood, spleen, and tumor. Overall, compared to NSG mice, the absolute counts were similar for all immune subsets measured except for regulatory T cells (Tregs). B-NDG, NCG, and CIEA NOG mice had reduced Treg numbers in the spleen of tumor-bearing mice. In the tumor, Treg numbers were lower in the B-NDG and CIEA NOG mice. No differences were observed for any immune subset in the blood. Overall, the tumor growth kinetics of BT-474 in B-NDG mice was most similar to historical data using NSG mice. The flow data also confirmed that each strain’s immune cell population generally looked very similar, with limited differences between the four strains of triple immunodeficient mice. Citation Format: Justin Snider, Derrik Germain, Lauren Kucharczyk, Anita Zaitouna, Erin Trachet, Scott Wise. Subcutaneous growth and immune cell profiling of BT-474 human breast carcinoma in four strains of triple immunodeficient mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 41.

Stratified distribution of Th17 and Treg cells in patients with multi-stage rheumatoid arthritis

ObjectiveRheumatoid arthritis (RA) is a typical, progressive autoimmune disease. Its occurrence and development are associated with dysregulation of T and B cell numbers. However, the specific immune characteristics of different RA courses remain incompletely defined. Here, we describe the peripheral blood lymphocyte subsets, particularly CD4 + T subsets, of different RA courses with a focus on early RA (Ea-RA).MethodsIn all, 131 patients with Ea-RA, 117 with advanced RA (Ad-RA), and 109 with treated RA (Tr-RA) were enrolled. We collected general clinical data. Whole blood samples obtained from the patients and 97 healthy controls (HCs) were analysed via flow cytometry.ResultsDecreased absolute NK cell numbers and increased CD4/CD8 T cell ratios were observed in different RA groups, including Ea-RA, compared to healthy controls. In Ea-RA patients, the Th17 and Treg cell numbers were similar to those in HCs. We performed k-means clustering based on the profiles of Th17 and Treg cells for patients with multi-stage of RA. We identified three patient types: type A characterised by relatively low Treg and Th17 cell numbers, type B with moderate levels of Treg cells and levels of Th17 cells similar to that of type C patients, and type C with high levels of Treg cells and levels of Th17 cells similar to that of type B patients.ConclusionThe immune characteristics of Ea-RA patients differ from those of HCs; an immune system disorder is apparent although no differences in Th17 and Treg levels were evident between Ea-RA patients and HCs. We found distributional heterogeneities of Th17 and Treg cells in patients with multi-stage of RA. Stratified management based on such heterogeneity may serve as a useful novel immunotherapy allowing of early intervention.

Immunomodulatory effects of umbilical mesenchymal stem cell-derived exosomes on CD4+ T cells in patients with primary Sjögren's syndrome

BackgroundPrimary Sjögren's syndrome (pSS) is an autoimmune disease that leads to the destruction of exocrine glands and multisystem lesions. Abnormal proliferation, apoptosis, and differentiation of CD4+ T cells are key factors in the pathogenesis of pSS. Autophagy is one of the important mechanisms to maintain immune homeostasis and function of CD4+ T cells. Human umbilical cord mesenchymal stem cell-derived exosomes (UCMSC-Exos) may simulate the immunoregulation of MSCs while avoiding the risks of MSCs treatment. However, whether UCMSC-Exos can regulate the functions of CD4+ T cells in pSS, and whether the effects via the autophagy pathway remains unclear.MethodsThe study analyzed retrospectively the peripheral blood lymphocyte subsets in pSS patients, and explored the relationship between lymphocyte subsets and disease activity. Next, peripheral blood CD4+ T cells were sorted using immunomagnetic beads. The proliferation, apoptosis, differentiation, and inflammatory factors of CD4+ T cells were determined using flow cytometry. Autophagosomes of CD4+ T cells were detected using transmission electron microscopy, autophagy-related proteins and genes were detected using western blotting or RT-qPCR.ResultsThe study demonstrated that the peripheral blood CD4+ T cells decreased in pSS patients, and negatively correlated with disease activity. UCMSC-Exos inhibited excessive proliferation and apoptosis of CD4+ T cells in pSS patients, blocked them in the G0/G1 phase, inhibited them from entering the S phase, reduced the Th17 cell ratio, elevated the Treg ratio, inhibited IFN-γ, TNF-α, IL-6, IL-17A, and IL-17F secretion, and promoted IL-10 and TGF-β secretion. UCMSC-Exos reduced the elevated autophagy levels in the peripheral blood CD4+ T cells of patients with pSS. Furthermore, UCMSC-Exos regulated CD4+ T cell proliferation and early apoptosis, inhibited Th17 cell differentiation, promoted Treg cell differentiation, and restored the Th17/Treg balance in pSS patients through the autophagy pathway.ConclusionsThe study indicated that UCMSC-Exos exerts an immunomodulatory effect on the CD4+ T cells, and maybe as a new treatment for pSS.

Effect of Huangdisan grain on improving cognitive impairment in VD rats and its mechanism in immune inflammatory response.

Vascular dementia (VD) is the second most common type of dementia after Alzheimer's disease (AD). Although the incidence rate is very high, there is no definitive treatment for VD. And it has serious impact on the quality of life of VD patients. In recent years, more and more studies about the clinical efficacy and pharmacological effects of traditional Chinese medicine (TCM) in the treatment of VD have been conducted. And Huangdisan grain has been used to treat VD patients with a good curative effect in clinic. This study was designed to investigate the effect of Huangdisan grain on the inflammatory response and cognitive function of VD rats modeled by bilateral common carotid artery occlusion (BCCAO), that aimed to improve the treatment methods for VD. 8-week-old healthy SPF male Wistar rats (280±20g) were randomly divided into the normal group (Gn, n=10), sham operated group (Gs, n=10), and operated group (Go, n=35). The VD rat models in Go group were established by BCCAO. 8weeks after surgery, the operated rats were screened by the hidden platform trail of Morris Water Maze (MWM), and the rats with cognitive dysfunction were further randomly divided into the impaired group (Gi, n=10) and TCM group (Gm, n=10). The VD rats in Gm group were given the intragastric administration of Huangdisan grain decoction once a day for 8weeks, and the other groups were given intragastric administration of normal saline. Then the cognitive ability of rats in each group was detected by the MWM Test. The lymphocyte subsets in peripheral blood and hippocampus of rats were measured by flow cytometry. The levels of cytokines (IL-1β, IL-2, IL-4, IL-10, TNF-α, INF-γ, MIP-2, COX-2, iNOS) in peripheral blood and hippocampus were measured by ELISA (enzyme linked immunosorbent assay). The number of Iba-1+ CD68+ co-positive cells in the CA1 region of hippocampus was measured by immunofluorescence. Compared with the Gn group, the escape latencies of the Gi group were prolonged (P<0.01), the time spent in the former platform quadrant was shortened (P<0.01), and the number of times of crossing over the former platform location was reduced (P<0.05). But compared with the Gi group, the escape latencies of Gm group were shortened (P<0.01), the time spent in the former platform quadrant was prolonged (P<0.05), and the number of times of crossing over the former platform location was increased (P<0.05). The number of Iba-1+ CD68+ co-positive cells in the CA1 region of hippocampus of VD rats in Gi group was increased (P<0.01) compared with the Gn group. And the proportions of T Cells, CD4+ T Cells, CD8+ T Cells in the hippocampus were increased (P<0.01). The level of pro-inflammatory cytokines in the hippocampus was increased significantly, such as IL-1β (P<0.01), IL-2 (P<0.01), TNF-α (P<0.05), IFN-γ (P<0.01), COX-2 (P<0.01), MIP-2 (P<0.01) and iNOS (P<0.05). And the level of IL-10 (P<0.01), a kind of anti-inflammatory cytokine, was decreased. The proportions of T Cells (P<0.05), CD4+ T Cells (P<0.01) and NK Cells (P<0.05) in the peripheral blood of the VD rats in Gi group were decreased, and the level of IL-1β, IL-2, TNF-α, IFN-γ, COX-2, MIP-2 and iNOS was increased significantly (P<0.01) compared with the Gn group. Meanwhile, the level of IL-4 and IL-10 was decreased (P<0.01). Huangdisan grain could reduce the number of Iba-1+ CD68+ co-positive cells in the CA1 region of hippocampus (P<0.01), decrease the proportions of T Cells, CD4+ T Cells, CD8+ T Cells and the level of IL-1β, MIP-2 in hippocampus (P<0.01) of VD rats. Moreover, it could rise the proportion of NK Cells (P<0.01) and the level of IL-4 (P<0.05), IL-10 (P<0.05), and decrease the level of IL-1β (P<0.01), IL-2 (P<0.05), TNF-α (P<0.01), IFN-γ (P<0.01), COX-2 (P<0.01) and MIP-2 (P<0.01) in peripheral blood of VD rats. This study indicated that Huangdisan grain could decrease the activation of microglia/macrophages, regulate the proportions of lymphocyte subsets and the level of cytokines, which could adjust the immunologic abnormalities of VD rats, and ultimately improve cognitive function.

The profile and prognostic value of circulating lymphocyte subsets in metastatic colon cancer.

Colon cancer (CC) are the most common malignant cancer in human digestive system, however, the profile and prognostic value of circulating lymphocyte subsets in CC patients has not been systemically clarified. In this study, 158 patients with metastatic CC were enrolled. Chi-square test was used to analyze the relationship between baseline peripheral blood lymphocyte subsets and clinicopathological parameters. Kaplan-Meier and Log-rank tests were used to analyze the relationship between clinicopathological parameters and baseline peripheral lymphocyte subsets and overall survival (OS) of patients with metastatic CC. Univariate/multivariate COX regression analysis was used to identify the independent factors in metastatic CC. The baseline peripheral blood CD3+T cells, CD4+T cells, NK cells and B cells of BRAF mutant patients were significantly lower than those in BRAF wild-type patients; The baseline CD8+T cells of KRAS mutation group was lower than that in KRAS wild type group. Peripheral blood CA19-9>27, left-sided colon cancer (LCC), KRAS and BRAF mutation were poor prognostic factors, and ALB>40, NK cells were protective prognostic factors for metastatic CC. In patients with liver metastases subgroup, higher NK cells also indicated a longer OS. Finally, LCC (HR=0.56), CA19-9 (HR=2.13), ALB (HR=0.46) and circulating NK cells (HR=0.55) were independent prognostic factors for metastatic CC. LCC, higher level of ALB and NK cells at baseline are protective factors, and higher CA19-9, KRAS/BRAF gene mutation are adverse prognostic factors. Sufficient circulating NK cells are independent prognostic factor for metastatic CC patients.

Changes in T lymphocyte subsets in peripheral blood of patients with middle-advanced cervical cancer before and after nimotuzumab combined with concurrent chemoradiotherapy

The current study sought to investigate the effect of nimotuzumab combined with concurrent chemoradiotherapy (CCRT + Nim) on T lymphocyte subsets in middle-advanced CC. Firstly, patients with middle-advanced CC were administered CCRT or CCRT + Nim. Next, levels of T lymphocytes in peripheral blood of CC patients pre- or post-treatment and healthy females were determined by flow cytometry. The short-term efficacy was evaluated, and overall survival (OS) and progression-free survival (PFS) of patients were recorded. In addition, the correlation of T lymphocyte subsets post-treatment with OS/PFS was assessed with Pearson analysis. CC patients exhibited decreased total T cells/T helper cells/CD4+/CD8+ ratio and increased T suppressor cells/Tregs in peripheral blood. Meanwhile, CCRT and CCRT + Nim improved T lymphocyte subset imbalance, with CCRT + Nim exhibiting better efficacy. CCRT + Nim exhibited better short-term efficacy and higher PFS than CCRT, with no evident difference in OS. The levels of total T cells/T helper cells/T suppressor cells/Tregs were not significantly-correlated with OS/PFS, and the CD4+/CD8+ ratio was correlated with PFS but not OS. Collectively, CCRT + nimotuzumab ameliorate the imbalance of T lymphocyte subsets in peripheral blood of middle-advanced CC patients, and the CD4+/CD8+ ratio after therapy is correlated with PFS. IMPACT STATEMENT What is already known on this subject? The utilisation of Nimotuzumab targeting epidermal growth factor receptor (EGFR) combined with concurrent chemoradiotherapy (CCRT) as an efficient treatment for middle-advanced cervical cancer (CC) has garnered the attention of numerous researchers over the years. T cells represent a major immune cell type in the tumour microenvironment and serve as the basis for maintaining cellular immune functions. What do the results of this study add? Our findings revealed that nimotuzumab combined with CCRT improves the abnormality of T lymphocyte subsets in peripheral blood of patients with middle-advanced CC, such that the CD4+/CD8+ ratio after treatment was significantly correlated with progression-free survival (PFS). What are the implications of these findings for clinical practice and/or further research? CCRT of CC may have a short-term negative impact on the peripheral T-cell immune micro-environment, and the combination of nimotuzumab, cisplatin-based chemotherapy, and radiotherapy enhances the frequency of Tregs in peripheral blood. Our findings illustrated that nimotuzumab combined with CCRT can improve the imbalance of T lymphocyte subsets in peripheral blood of patients with middle-advanced CC. A better understanding of the mechanisms of these therapies will optimise the selection of patients most likely to benefit from treatment, serving as a reference for further research on the relationship between EGFR-specific T cells and clinical benefit in patients treated with nimotuzumab in combination with CCRT.

Peripheral CD8+CD28+ T lymphocytes predict the efficacy and safety of PD-1/PD-L1 inhibitors in cancer patients.

Programmed cell death protein-1/programmed cell death ligand-1 (PD-1/PD-L1) inhibitors works by reactivating immune cells. Considering the accessibility of noninvasive liquid biopsies, it is advisable to employ peripheral blood lymphocyte subsets to predict immunotherapy outcomes. We retrospectively enrolled 87 patients with available baseline circulating lymphocyte subset data who received first-line PD-1/PD-L1 inhibitors at Peking Union Medical College Hospital between May 2018 and April 2022. Immune cell counts were determined by flow cytometry. Patients who responded to PD-1/PD-L1 inhibitors had significantly higher circulating CD8+CD28+ T-cell counts (median [range] count: 236 [30-536] versus 138 [36-460]/μL, p < 0.001). Using 190/μL as the cutoff value, the sensitivity and specificity of CD8+CD28+ T cells for predicting immunotherapy response were 0.689 and 0.714, respectively. Furthermore, the median progression-free survival (PFS, not reached versus 8.7 months, p < 0.001) and overall survival (OS, not reached versus 16.2 months, p < 0.001) were significantly longer in the patients with higher CD8+CD28+ T-cell counts. However, the CD8+CD28+ T-cell level was also associated with the incidence of grade 3-4 immune-related adverse events (irAEs). The sensitivity and specificity of CD8+CD28+ T cells for predicting irAEs of grade 3-4 were 0.846 and 0.667, respectively, at the threshold of CD8+CD28+ T cells ≥ 309/μL. High circulating CD8+CD28+ T-cell levels is a potential biomarker for immunotherapy response and better prognosis, while excessive CD8+CD28+ T cells (≥ 309/μL) may also indicate the emergence of severe irAEs.

Deciphering Hypoplastic Myelodysplastic Syndrome and Aplastic Anemia via In-Depth Analysis of Lymphocyte Subsets

To explore the difference of lymphocyte subsets in peripheral blood (PB) between aplastic anemia (AA) and hypoplastic myelodysplastic syndrome (hypo-MDS) patients, meanwhile to compare the clinical parameters obtained from PB and bone marrow (BM). The lymphocyte subsets in hypo-MDS (n=25) and AA (n=33) patients were investigated by flow cytometry. Meanwhile, the differences in PB cell counts, biochemical indicators, BM cell counts and abnormal chromosomes between the two groups were analyzed. The percentage of CD8+T cells in AA group was significantly higher than that in hypo-MDS group (P=0.001), while the percentage of CD4+ T cells and the CD4+/CD8+ ratio in AA group were obviously lower than those in hypo-MDS group (P=0.015 and 0.001, respectively). Furthermore, the proportion of CD4+ and CD8+ activated T (TA) cells, and memory Tregs in AA group was distinctly lower than those in hypo-MDS group (P=0.043, 0.015 and 0.024, respectively). Nevertheless, the percentage of CD8+ naive T (TN) cells in AA patients was remarkably higher (P=0.044). And hypo-MDS patients had declined lymphocyte counts (P=0.025), increased levels of total bilirubin (TBil), lactate dehydrogenase (LDH), vitamin B12 and proportion of BM blasts than AA patients (P=0.019, 0.023, 0.027 and 0.045, respectively). In this study it was confirmed that the percentages of CD4+ and CD8+ TA cells, memory Tregs and CD8+ TN cells were significantly different between AA and hypo-MDS patients, which provide an essential basis for the identification of these two diseases.