Proportion Of CD4 Research Articles

Abstract 15: Single-cell Immune Landscape Of Human Clot Retrieved At Mechanical Thrombectomy. Association With Stroke Origin.

Background: Mechanical thrombectomy (MT) has significantly improved clinical outcomes in acute ischemic stroke. We used single-cell RNA sequencing (scRNA-seq) to describe the landscape of human clots at single cell resolution seeking to provide greater mechanistic insight into stroke pathophysiology and to uncover an association with stroke origin. Methods: scRNA-seq was performed on 14 clots retrieved from 14 patients undergoing MT between October 2020-July 2022. We followed standard molecular biology and bioinformatics workflows for sample processing and data to characterize differences in cellular composition between clots of different stroke origin (atrial fibrillation (AF) and carotid atherosclerosis (CA)). Results: Clots revealed a significant heterogeneity of immune cells with major constituents including cells of the mononuclear phagocyte system (MPS) and neutrophils. The cellular populations most enriched in CA clots were B-cells and CD4+ T-cells. The cellular population most enriched in AF clots was red blood cells. When looking at cellular subpopulations of the MPS component, we found that CD16+ monocytes (intermediate and non-classical) were significantly more abundant than CD14+CD16- (classical) monocytes in all clots. Moreover, the proportions of CD16+ monocytes varied with stroke origin: the proportion of non-classical (CD14-CD16+) monocytes was higher in CA clots (p=0.05) whereas the proportion of intermediate monocytes (CD14+CD16+) was higher in FA clots (p=0.03). AF clots had upregulated biomarkers and pathways involved with AF or contractile dysfunction (IFI30, FABP4, PPAR, HOTAIR, ILK, Integrin). CA clots had upregulated biomarkers and pathways associated with cardiovascular diseases and atherogenesis (RNSE1, POLR2J3.1, PLTP, Sirtuin, FAK). (Figure) Conclusions: These results reveal a significant heterogeneity of immune cells and point to important differences in cellular composition associated with stroke etiology.

Topography of immune cell infiltration in different stages of coronary atherosclerosis revealed by multiplex immunohistochemistry.

Aim of this study was to investigate immune cells and subsets in different stages of human coronary artery disease with a novel multiplex immunohistochemistry (mIHC) technique. Human left anterior descending coronary artery specimens were analyzed: eccentric intimal thickening (N=11), pathological intimal thickening (N=10), fibroatheroma (N=9), and fibrous plaque (N=9). Eccentric intimal thickening was considered normal, and pathological intimal thickening, fibroatheroma, and fibrous plaque were considered diseased coronary arteries. Two mIHC panels, consisting of six and five primary antibodies, autofluoresence, and DAPI, were used to detect adaptive and innate immune cells. Via semi-automated analysis, (sub)types of immune cells in whole plaques and specific plaque regions were quantified. Increased numbers of CD3+ T cells (P<0.001), CD20+ B cells (P=0.013), CD68+ macrophages (P=0.003), CD15+ neutrophils (P=0.017), and CD31+ endothelial cells (P=0.024) were identified in intimas of diseased coronary arteries compared to normal. Subset analyses of T cells and macrophages showed that diseased coronary arteries contained an abundance of CD3+CD8- non-cytotoxic T cells and CD68+CD206- non-M2-like macrophages. Proportions of CD3+CD45RO+ memory T cells were similar to normal coronary arteries. Among pathological intimal thickening, fibroatheroma, and fibrous plaque, all immune cell numbers and subsets were similar. The type of immune response does not differ substantially between different stages of plaque development and may provide context for mechanistic research into immune cell function in atherosclerosis. We provide the first comprehensive map of immune cell subtypes across plaque types in coronary arteries demonstrating the potential of mIHC for vascular research.

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.

Analysis on Cellular Immune Function Recovery after HLA Mismatched Allogeneic Hematopoietic Stem Cell Micro-Transp- lantation in Elderly AML Patients

To investigate the recovery of cellular immunity in elderly patients with acute myeloid leukemia (AML) after micro-transplantation (MST) and the changes of cellular immunity during relapse, as well as their clinical significance. A total of 41 elderly AML patients who received MST treatment in a single center and 25 healthy elderly people were included. Immune function among different age groups in normal population was compared. Furthermore, immune fuction was compared between elderly AML patients of different age groups who achieved continuous complete remission (CR) after MST treatment and normal controls, between high risk group and medium-low risk group, as well as among before diagnosis, after CR, and relapse. Peripheral blood of patients and normal controls was collected, and the percentage of lymphocyte subsets was detected by multi-color flow cytometry. Thirty-five patients achieved CR after MST treatment while six patients did not. After MST treatment, CD3+ T cells, CD8+T cells and activated T cells in all age groups were higher than normal. Significant recovery of CD3+ and CD8+T cells was observed in both high risk and medium-low risk groups, and the overall recovery of immune cells in medium-low risk group was better. It was also observed that B lymphocytes and NK cells could not return to normal levels within 1 year after MST treatment. The proportion of CD3+ T cells, CD4+ T cells, and CD4/CD8 ratio were significantly decreased during relapse compared with continuous CR after MST (P<0.05). MST treatment can promote the recovery of CD3+T cells, CD8+T cells and other killer cells, so as to improve the cellular immune function of elderly patients, which provides a new immune cell therapy for elderly AML.

Circulating CD4+ Treg, CD8+ Treg, and CD3+ γδ T Cell Subpopulations in Ovarian Cancer.

Background and Objectives: Regulatory T cells (Tregs) are usually enriched in ovarian cancer (OC), and their immunosuppressive function plays a key role in tumorigenesis and progression. We mainly explored the phenotypical characterization of Treg-related markers on αβ and γδ T cell subsets in patients with OC. Materials and Methods: Thirty-six untreated patients with OC at the Women's Hospital of Nanjing Medical University from September 2019 to August 2021 were enrolled. Phenotypical characterization of Tregs-related markers were detected by flow cytometry (FCM). Enzyme-linked immunosorbent assay was used to detect the levels of carbohydrate antigen (CA125) and transforming growth factor β (TGF-β). The level of human epididymis protein 4 (HE4) was detected by electrochemiluminescence immunoassay. Results: Circulating CD4+ Tregs, CD8+ Tregs, and CD3+γδ T cell subpopulations from OC patients have elevated Foxp3, CD25, CD122, Vδ1, and reduced CD28 expression compared to benign ovarian tumor (BOT) patients and healthy controls (HC). The upregulation of Foxp3 and Vδ1 and the downregulation of CD28 were highly specific for maintaining the immunosuppression function of CD4+ Tregs, CD3+γδ T cells, and CD8+ Tregs in OC patients. These Treg subpopulations were able to discriminate OC from BOT and HC. The levels of CA125, HE4, and TGF-β were increased in OC patients. A significant positive correlation between Treg subpopulations and CA125, HE4, and TGF-β was revealed. Conclusions: Proportions of CD4+ Tregs, CD8+ Tregs, and CD3+γδ T cell subsets were significantly increased in OC patients and were positively correlated with FIGO stage/metastasis status, CA125, HE4, and TGF-β. These indicators have the potential to be used as immunosurveillance biomarkers for OC.

Immune infiltration analysis reveals immune cell signatures in salivary gland tissue of primary Sjögren's syndrome.

Mouse models are the basis for primary Sjögren's syndrome (pSS) research. However, the depth of comparisons between mice and humans in salivary gland (SG) immune cells remains limited. The gene expression profiles of SGs from normal subjects and pSS patients were downloaded from the Gene Expression Comprehensive Database. The proportion of infiltrating immune cell subsets was then assessed by cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT). An experimental Sjögren's syndrome (ESS) mouse model was successfully constructed using SG protein. Based on mouse SG tissue RNA-Seq data, the seq-ImmuCC model was used to quantitatively analyze the compositional ratios of 10 immune cells in pSS patients and mouse model SG tissues. Computed and obtained 31 human data samples using the CIBERSORT deconvolution method. The immune cell infiltration results showed that, compared to normal human SG tissue, the content of gamma delta T cells was significantly different from naive CD4+ T cells and significantly increased, while the plasma cell content decreased. Principal component analysis indicated differences in immune cell infiltration between pSS patients and normal subjects. Meanwhile, for ESS model mouse data analysis, we found that the proportion of macrophages increased, while the proportion of CD4+ T cells, B cells, and monocytes decreased. Furthermore, we found that the proportion of monocytes was decreased, while the proportion of macrophages was increased in the SG tissues of pSS patients and model mice. The infiltration of CD4+ T, CD8+ T, and B cells also showed some differences. We comprehensively analyzed SG immune infiltration in pSS patients and model mice. We demonstrated conserved and nonconserved aspects of the immune system in mice and humans at the level of immune cells to help explain the primary regulation of immune mechanisms during the development of Sjögren's syndrome.

Abstract B012: Advanced epigenetic aging in non-AIDS-defining cancer patients living with HIV

Abstract Background. With widespread use of antiretroviral therapy, people with HIV (PWH) are living longer and are now at risk for age-related co-morbidities, including non-AIDS defining cancers (NADCs). Given this epidemiological trend, it is important to understand how prolonged HIV infection may relate to molecular features of aging that can impact cancer risk or outcomes. Methyl groups naturally accumulate on human DNA over time, and methylation patterns can be translated into metrics of accelerated aging. We compared epigenetic measures of aging between PWH receiving treatment for an NADC and matched HIV-uninfected NADC patients at Moffitt Cancer Center in Tampa, Florida. Methods. We identified PWH diagnosed with a non-AIDS defining solid tumor from 2004 to 2021 were identified. To be included, patients needed to have an archived specimen amenable for DNA extraction (whole blood, buffy coat, or PBMC) collected within 1 year of cancer diagnosis. HIV-uninfected matches were identified in a similar manner and matched by tumor site, reported age (+/- 5 years), tumor sequence (primary vs. non-primary), and treatment status. Extracted DNA was assayed using the Illumina MethylationEPIC BeadChip, which incorporates data from 850,000 CpG sites across the genome. Data were translated into a biological age using the epiTOC2 clock, which estimates the number of total number of stem cell divisions, and GrimAge, associated with mortality. Methylation data were also deconvoluted to determine immune cell composition (CD4+ T-cells, CD8+ T-cells, B-cells, neutrophils, natural killer cells, monocytes, basophils, eosinophils). Epigenetic age and immune cell proportions were compared across HIV status using Wilcoxan rank sum testing. Age acceleration was calculated as the difference (residuals from a linear model) in the observed age for PWH and a NADC diagnosis, compared to the predicted age based on HIV-uninfected NADC patients, using both unadjusted and immune-cell adjusted linear models. Results. DNA methylation was assessed in 65 NADC PWH and HIV and 64 HIV-uninfected NADC patients. Epigenetic age assessed using the epiTOC2 and GrimAge clocks was significantly elevated in PWH (p&amp;lt;0.001 and p=0.02, respectively). When methylation data was used to de-convolute immune cell composition, the proportion of CD4+ T-cells and neutrophils was significantly (P&amp;lt;0.05) lower in PWH, while the proportions of memory B-cells and CD8+ T-cells were higher. Epigenetic age was accelerated in PWH compared to HIV-uninfected patients using both epiTOC2 and GrimAge clocks, with the difference in the GrimAge clock remaining significant after adjustment for immune cell composition (P-value&amp;lt;0.01). Conclusions. The results of this study demonstrated age acceleration in PWH with a NADC diagnosis compared to their HIV-uninfected counterparts. As cancer is now the leading cause of non-AIDS death in PWH, research into the prognostic implications of these differences is warranted. Citation Format: Anna E. Coghill, Brittney Dickey, Ryan Putney, Anders Berglund. Advanced epigenetic aging in non-AIDS-defining cancer patients living with HIV [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B012.

Acute exercise mobilizes CD8+ cytotoxic T cells and NK cells in lymphoma patients.

Background: Studies have shown that acute exercise can mobilize several leukocyte subpopulations in healthy individuals. Our aim was to investigate whether a 10-min acute exercise has an effect on immune cell proportions in lymphoma patients. Methods: This study included seven lymphoma patients referred to curative oncologic therapy. Three had Hodgkin and four non-Hodgkin lymphoma, one was female, and their mean age was 51. Patients underwent a 10-min acute exercise on a bicycle ergometer at moderate exercise intensity. Whole blood samples were taken at rest, immediately after exercise, and 30min after exercise. Leukocyte subpopulation levels were determined using flow cytometry. Results: Proportions of total NK cells and CD56+CD16+ NK cells of total leukocytes increased immediately after exercise and decreased back to baseline at 30min post-exercise. Proportion of CD8+ T cells of total T cells increased and proportion of CD4+ T cells of total T cells decreased immediately after exercise, and both returned to baseline at 30min post-exercise. There was no change in the proportions of B cells, granulocytes, or monocytes. Exercising diastolic blood pressure correlated positively with changes in total NK cell and CD56+CD16+ NK cell proportions, and exercising mean arterial pressure correlated positively with change in CD56+CD16+ NK cell proportion. Conclusion: Our findings indicate that a single acute exercise bout of only 10min can cause leukocytosis in lymphoma patients, particularly on cytotoxic T cells and NK cells, which are the most important immune cells fighting against cancer.

Peripheral blood lymphocyte subsets in children with nephrotic syndrome: a retrospective analysis

BackgroundNephrotic syndrome (NS) in children is widely believed to be associated with severe changes in the immune system. Based on lymphocyte subset analysis, we examined the pathogenesis of immune deficiencies in children with NS with varying steroid sensitivity.Methods Our study utilized flow cytometry to retrospectively analyze the ratios of lymphocyte subsets in 204 children with nephrotic syndrome and 19 healthy children.ResultsCompared with healthy children, the ratio of CD4 + /CD8 + in onset and remission was decreased in SRNS group (p < 0.05), and CD19 + B lymphocytes were increased in onset (p < 0.05). Compared with onset, the proportion of CD19 + B lymphocytes decreased in SRNS, while the proportion of CD19 + B lymphocytes increased in SDNS, p < (0.01). The ratio of CD8 + T/CD19 + B in onset in SDNS group was significantly higher than that in SSNS and SRNS groups (p < 0.01) and healthy control group (p < 0.05). Compared with onset, the ratio of CD8 + T/CD19 + B in SDNS group decreased significantly (p < 0.01), while the ratio of CD8 + T/CD19 + B in SRNS group increased significantly (p < 0.01). The proportion of CD56 + CD16 + NK cells was significantly reduced in children with INS (p < 0.01).ConclusionCD8 + T lymphocytes may be involved in the mechanism of lymphocyte subsets disorder during onset of SDNS, while CD19 + B lymphocytes may be involved in the mechanism of lymphocyte subsets disorder during relapse of SDNS. The CD8 + T/CD19 + B ratio may predict the degree of frequent recurrence. There is a certain degree of lymphoid subsets disorder in children with NS.

Quantities of CD3+, CD8+ and CD56+ lymphocytes decline in breast cancer recurrences while CD4+ remain similar

BackgroundMuch is known about tumor infiltrating lymphocytes (Tils) in primary breast cancer, as this has been the focus of much research in recent years, but regarding recurrent breast cancer, only few studies have been done. Our aim was to compare the quantities of Tils in primary breast carcinomas and their corresponding recurrences and to analyze the differences in the tumor Tils compositions in correlations with recurrence-free times and the clinicopathology of the tumor.MethodsOne hundred thirty-seven breast cancer patients self-paired for primary- tumor-recurrence were divided into three groups based on the length of the recurrence-free interval. H&E-staining and immunohistochemical staining with antiCD3, antiCD4, antiCD8 and antiCD56 were performed. Differences in Tils between primaries and recurrences, between the recurrence-free interval groups, and between different clinicopathologic parameters were statistically analyzed.ResultsFewer stromal CD3+, CD8+ and CD56+ lymphocytes were found at recurrences compared to the primaries. No significant change in the percentage of CD4+ stromal lymphocytes. ER-negative primaries, PR-negative or HER2-positive tumors had more Tils in some subgroups. Ductal primaries had more Tils than lobular primaries and G3 tumors had more Tils than lower-grade tumors. The corresponding differences at recurrences could either not be detected or they were reversed. The fastest recurring group had generally more Tils than the slower groups.ConclusionsCD4+ cell numbers did not decline from primary to recurrence in contrast to all other subclasses of lymphocytes. The proportion of CD4+ cells was higher in recurrences than in primaries. Tumors with a higher grade and proliferation rate had higher percentages of Tils. HER2+ and hormone receptor negative tumors tended to have higher Tils scores. In recurrences these differences were not seen or they were reversed.

Уровни регуляторных Т-лимфоцитов и B-клеток у пациенток с болезнью Грейвса после тиреоидэктомии

Aim: To study the levels of regulatory T lymphocytes (Treg) and B-cells in peripheral blood in patients with Graves' disease in dynamics after thyroidectomy. Design: Single center, observational, prospective, cohort, open, controlled study. Materials and methods. The study included 96 patients with Graves' disease, mean age 42.86 ± 10.81 years. Clinical, hormonal and immunological examinations were performed against the background of persistent drug-induced euthyroidism, before surgery, and also 1, 3, and 6 months after thyroidectomy. The levels of Treg and B-cells in the blood was examined by flow cytometry using direct immunofluorescence and monoclonal antibodies. The level of antibodies to thyroid-stimulating hormone receptors (rTSH) was assessed by enzyme immunoassay. The control group consisted of 85 healthy women of the same age. Results. In patients with Graves' disease in the dynamics of the postoperative period, there was a consistent statistically significant decrease in the titer of antibodies to rTSH: from 14.69 (8.67–19.81) to 0.81 (0.59–0.93) IU/l. The absolute amount of Treg in their blood was reduced relative to the control values already in the preoperative period and decreased even more at 6 months after thyroidectomy. The proportion of CD19+CD5+- and CD19+CD5–-cells in the examined patients before surgery was significantly higher than in the control group. The content of CD19+CD5+ cells decreased as early as 1 month after thyroidectomy and remained at the level of control values. The сontent of CD19+CD27–-cells in patients with Graves' disease in the preoperative period was higher than in the control group, decreased to the control values 1 month after thyroidectomy and remained in this range until the end of the follow-up. The content of CD19+CD27+ cells in patients before thyroidectomy was lower compare to control, in the period of 1–3 months it corresponded to the control values, but decreased again on the 6th month after surgery. Conclusion. The reduction of Treg and B-memory cells in the peripheral blood in patients with Graves' disease six months after thyroidectomy remains of immunosuppressive regulatory mechanisms with an increase in the migration activity of immune cells for a long time. Keywords: Graves' disease, thyroidectomy, regulatory T lymphocytes, memory B cells, target therapy.

A novel fluoropolymer as a protein delivery vector with robust adjuvant effect for cancer immunotherapy.

The inefficient treatment using protein-based nanovaccines is largely attributed to their inadequate immunogenicity. Herein, we developed a novel fluoropolymer (PF) via ring-opening polymerization and constructed a fluoropolymer-based nanovaccine for tumor immunotherapy. Due to the existence of fluoroalkyl chains, PF not only played a crucial role in tumor antigen delivery but also exhibited a remarkable adjuvant effect in enhancing the immunogenicity of nanovaccines. The nanovaccines formed by mixing PF with a model antigen ovalbumin (OVA) enhanced the uptake of antigen proteins by dendritic cells (DCs) and promoted the maturation and antigen presentation of DCs. Compared with free OVA, PF/OVA showed better efficacy in both pre-cancer prevention and tumor treatment. Furthermore, the proportion of CD4+ T and CD8+ T cells was significantly increased in lymph nodes and tumors of mice immunized with PF/OVA. Additionally, there was a great enhancement in the levels of key anti-tumor cytokines (TNF-α and IFN-γ) in the serum of the PF/OVA immunized mice. Our research has shown that fluoropolymer PF applied as a protein vector and adjuvant has great potential for the development of nanovaccines with robust immunogenicity.

Results of flow cytometric detection of γδT cells in peripheral blood of patients with ankylosing spondylitis: a pilot study

Previous studies have suggested that γδT cells play an important role in the pathogenesis of ankylosing spondylitis (AS). In this pilot study, the peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (AS) and healthy volunteers were stained and analyzed by flow cytometry to distinguish γδ T cells and its subtypes, and then to report the distribution of γδ T cells and iyts subtypes and their correlation with ankylosing spondylitis. A total of 17 patients with active AS and 10 age- and gender- matched healthy volunteers were enrolled in this study, and their peripheral blood were drawn to collect mononuclear cells (PBMCs). Flow cytometry was used to analyze γδ T cell subpopulations by measuring the surface and intracellular expressions of phenotypic markers. Serum levels of inflammatory and bone turnover markers were measured, and their correlations with subpopulations of γδ T cells were evaluated. In patients with AS, the Vδ2 fractions within γδ T cells and CD3+ T cells decreased significantly, in particular, the proportions of CD27+ Vδ2 T cells, CD86+CD80+ Vδ1 T cells, and IL17A-secreting and TNFα-secreting Vδ1 T cells within the parental cells decreased significantly. γδ T cells/PBMCs, Vδ2 cells/γδ T cells, and Vδ2 cells/CD3+ T cells were negatively correlated with CRP, whereas Vδ1 cells/CD3+ T cells were negatively correlated with ESR. Vδ1 cells/γδ T cells were positively correlated with CRP, γδ T cells/PBMCs were positively correlated with β-CTx, CD69+CD25+ and IL-17A-secreting Vδ1 cells were positively correlated with TP1NP, and CD69+CD25+ Vδ1 and Vδ2 cells were positively correlated with osteocalcin. Decreases in peripheral Vδ2, CD27+ Vδ2, CD86+CD80+ Vδ1, and IL17A or TNFα-secreting Vδ1 T cells are associated with AS. The correlations between γδ T cell subpopulations and CRP and the CD69+CD25+ subpopulation with TP1NP or osteocalcin suggest that an imbalance in peripheral γδ T cell subpopulations contributes to the pathogenesis of AS.

Biological Features of CD5+ CD19+ B1 Cell and Natural IgM (VH4-34) in Chronic Lymphocytic Leukemia vs. Multiple Sclerosis.

Chronic lymphocytic leukemia (CLL) is the clonal expansion of mature CD5+ B cells and the most common lymphoproliferative disease in adults (B1-CLL). B1 cells' anti-inflammatory effects include the production of natural IgM (nIgM) by the spleen and bone marrow, decreased inflammatory cytokines as a primary response to maintaining tissue homeostasis, and enhanced release of transforming growth factor β (TGFβ). We used the flow cytometry technique in peripheral blood from patients with CLL and multiple sclerosis (MS) to immunophenotype B cells and their subpopulations. Whole blood from CLL and MS patients, as well as healthy controls, was used to detect nIgM using the VH4-34 gene copy number and real-time RT-PCR. We found that the proportion of CD5+ B cells was significantly lower in MS patients than in the control group and that CD5+ B lymphocytes were significantly higher in CLL patients than in the control group. Compared to the control group, CLL patients had significantly higher levels of the VH4-34 gene copy number. On the contrary, MS patients had significantly lower VH4-34 gene copy number levels compared to the control group. As the number of antibodies in CLL patients increases due to the high number of B1 cells, we propose a new way to treat MS by extracting this natural antibody from the sera of CLL patients and injecting it into MS patients.

Differential Activity of Human Leukocyte Extract on Systemic Immune Response and Cyst Growth in Mice with Echinococcus Multilocularis Infection After Oral, Subcutaneous and Intraperitoneal Routes of Administration.

Alveolar echinococcosis (AE) caused by the larval stage of Echinococcus multilocularis is serious parasitic diseases associated with the host´s immunosuppression. The effects of human non-immune dialyzable leukocyte extract (DLE) on immune cells in blood and spleen and parasitic cysts weight in Balb/c mice after oral (PO), subcutaneous (SC) and intraperitoneal administration (IP) were compared. The reduction in cysts weight (p < 0.01) was recorded after PO route, whereas moderate reduction was found after SC and IP routes. The elevation of lymphoid populations in blood and spleen was found after PO administration (p < 0.01) in parallel with reduced myeloid population. Infection-elicited decline in B220+B cells was partially abolished by PO route, but DLE routes did not influence the CD3+ T cells. The proportions of CD3+CD4+Th lymphocytes were moderately upregulated, whereas CD3+CD8+Tc populations were reduced after all DLE routes (p < 0.01). PO administration increased CD11b+MHCIIhigh blood monocytes, CD11b-SigleF+ cell, but not CD11b+Si-glecF+ eosinophils in the blood, stimulated after SC and IP routes. DLE induced downregulation of NO production by LPS-stimulated adherent splenocytes ex vivo. Con A-triggered T lymphocyte proliferation was associated with the elevated IFN-γ production and transcription factor Tbet mRNA expression. The alleviation of Th2 (IL-4) and Treg (TGF-β) cytokine production by lymphocytes ex vivo paralleled with downregulation of gene transcription for cytokines, GATA and FoxP3. Reduction of myeloid cells with suppressive activity was found. The SC and IP routes affected partially the cysts weights, diminished significantly gene transcription, NO levels and Th2 and Treg cytokines production. Results showed that PO route of DLE administration was the most effective in ameliorating immunosuppression via stimulation of Th1 type, reducing Th2 and Treg type of immunity and CD3+CD8+Tc lymphocytes in the blood and spleens during E. multilocularis infection in mice.

Comparative characterization of frozen-thawed CD146+ and CD146- subsets of CD73+CD90+CD105+CD34+ human ASCs.

Mesenchymal stem cells are currently used to treat several diseases. Populations of putative stem cells found in the adipose tissue (ASCs) have been shown to possess particularly enhanced functionalities. Nonetheless, there is lack of evidence that evaluates the effects of cryopreservation techniques on well-defined functional ASC populations characterized by immunophenotypical repertoire. We therefore embarked a study to compare the frozen-thawed ASC subsets: CD73+CD90+CD105+CD34+CD146-(CD34+CD146), CD73+CD90+CD105+CD34+CD146+(CD34+CD146+), and CD73+CD90+CD105+CD34+(CD34+). We assessed their characterization in different functional assays. The ASC immunophenotypical subsets-purified by a flow cytometry sorting technique-were frozen in liquid nitrogen. After a period, they were thawed to examine their differentiation ability, colony-forming units, viability, and growth rate. We confirmed that inside the primary cell culture system, the proportion of CD34+, CD34+CD146-, and CD34+CD146+ took up 80%, 62%, and 19% on average, respectively. All populations could be frozen and stored in liquid nitrogen with retention of more than 85% of cell viability and displayed comparable stemness characteristics. Most importantly, the CD34+CD146+ subpopulation displayed a higher proliferation rate than other groups. Our data demonstrated that the frozen-thawed CD34+CD146+ cells might represent a promising source for autologous cellular-based therapy. These findings set the basis for ASC subpopulations-based application in future potential clinical settings.

Higher Frequencies of T-Cells Expressing NK-Cell Markers and Chemokine Receptors in Parkinson’s Disease

Immune cells are thought to be involved in a destructive cycle of sterile cerebral inflammatory responses in neurodegenerative diseases such as Parkinson’s Disease (PD). Despite their peripheral origin, immune cells may enter the CNS due to impaired blood–brain barrier function and may potentially contribute to neuronal damage. Hence, specific characteristics of peripherally activated immune cells could help in understanding neurodegeneration in PD and could potentially serve as accessible disease markers. To investigate immune cell activation status, the expression of receptors for cell surface molecules CD161, NKG2A, NKG2C and NKG2D as well as chemokine receptors CCR6, CXCR2, CXCR3 and CCR5 associated with neurodegenerative diseases was investigated. The frequencies of peripheral CD8+ T-cells expressing the inhibitory and activating receptors NKG2A and NKG2C, and the activating receptor NKG2D were higher in PD patients than in healthy matched controls. The frequencies of NKG2C+CD8− cells were also higher, whereas the frequencies of CD161+ cells were not significantly different. Of the chemokine receptor-expressing cells, only the proportion of CD4−CD56+CCR5+ T-cells was higher in PD patients than in the controls. These observations support the hypothesis that an imbalance in the activation state of T-cells plays a role in the pathological processes of PD and suggest that peripheral blood immune cell phenotypes could be specific early markers for inflammation in PD.

Study of epirubicin sustained-release chemoablation in tumor suppression and tumor microenvironment remodeling.

Although intratumoral chemoablation can obtain an impressive therapeutic effect, there is still incomplete ablation and tumor recurrence in some patients. This could be due to the short retention time of the drug in the tumor, the limited distribution of intratumoral drugs, and, beyond that, the immunotolerance caused by the tumor microenvironment (TME). There is still an urgent need to find an optimal drug sustained-release carrier and figure out the impact of regional injection to TME. In this study, we supposed to use polyethylene glycol (PEG) hydrogel as a drug carrier to improve the retention time of the drug to extend the exposure of tumor cells and investigate the feasibility of combination local Epirubicin injection with anti-PD-L1. The results revealed obvious tumor suppression based on the tumor volume and the inhibition time of tumor growth in the A549 lung cancer mouse model after local injection. Furthermore, the enhanced antitumor effects of the combination of systematic anti- programmed death ligand 1 (PD-L1) therapy with local chemoablation (EPI-GEL/PD-L1) for abscopal tumor reduction in the 4T1 breast model were also observed. Flow cytometry analysis of the tumor and blood samples showed significant variations in the proportions of PD-L1+ and CD3+CD8+PD-1+ cells before and after anti-PD-L1 therapy. On day 4 after local injection of the EPI gel, the expression of PD-L1 in abscopal tumors was upregulated, while the expression of PD-L1 in bilateral tumors in mice was significantly reduced after anti-PD-L1 treatment. The proportion of CD3+CD8+PD-1+ cells in the tumor and circulating blood in the EPI-GEL/PD-L1 group was decreased compared with that in the EPI-GEL (single injection of epirubicin) group. The combination of local injection of the chemoablation agent with anti-PD-L1 monoclonal antibody (mAb) therapy may strengthen the antitumor activity, and the use of PEG hydrogel as the drug carrier can extend the retention time of the chemoablation agent around the tumor, maintaining a long-term tumor-killing activity.

Piperlongumine alleviates corneal allograft rejection via suppressing angiogenesis and inflammation.

Neovascularization and inflammatory response are two essential features of corneal allograft rejection. Here, we investigated the impact of Piperlongumine (PL) on alleviating corneal allograft rejection, primarily focusing on pathological angiogenesis and inflammation. A murine corneal allograft transplantation model was utilized to investigate the role of PL in preventing corneal allograft rejection. PL (10 mg/kg) or vehicle was intraperitoneally injected daily into BALB/c recipients from day -3 to day 14. The clinical signs of the corneal grafts were monitored for 30 days. Corneal neovascularization and inflammatory cell infiltration were detected by immunofluorescence staining and immunohistochemistry. The proportion of CD4+ T cells and macrophages in the draining lymph nodes (DLNs) was examined by flow cytometry. In vitro, HUVECs were cultured under hypoxia or incubated with TNF-α to mimic the hypoxic and inflammatory microenvironment favoring neovascularization in corneal allograft rejection. Multiple angiogenic processes including proliferation, migration, invasion and tube formation of HUVECs in hypoxia with or without PL treatment were routinely evaluated. The influence of PL treatment on TNF-α-induced pro-inflammation in HUVECs was investigated by real-time PCR and ELISA. In vivo, PL treatment effectively attenuated corneal allograft rejection, paralleled by coincident suppression of neovascularization and alleviation of inflammatory response. In vitro, PL distinctively inhibited hypoxia-induced angiogenic processes in HUVECs. Two key players in hypoxia-induced angiogenesis, HIF-1α and VEGF-A were significantly suppressed by PL treatment. Also, TNF-α-induced pro-inflammation in HUVECs was hampered by PL treatment, along with a pronounced reduction in ICAM-1, VCAM-1, CCL2, and CXCL5 expression. The current study demonstrated that PL could exhibit both anti-angiogenic and anti-inflammatory effects in preventing corneal allograft rejection, highlighting the potential therapeutic applications of PL in clinical strategy.

Immune repertoire sequencing reveals an abnormal adaptive immune system in COVID-19 survivors.

Accumulating evidence suggests that severeacute respiratory syndrome coronavirus 2 (SARS-CoV-2) impairs the adaptive immune system during acute infection. Still, it remains largely unclear whether the frequency and functions of T and B cells return to normal after the recovery of CoronavirusDisease 2019 (COVID-19).Here, we analyzed immune repertoires and SARS-CoV-2-specific neutralization antibodies in a prospective cohort of 40 COVID-19 survivors with a 6-month follow-up after hospital discharge. Immune repertoire sequencing revealed abnormal T- and B-cell expression and function with large T cell receptor/B cell receptorclones, decreased diversity, abnormal class-switch recombination, and somatic hypermutation. A decreased number of B cells but an increased proportion of CD19+ CD138+ B cells were found in COVID-19 survivors. The proportion of CD4+ T cells, especially circulating follicular helper T (cTfh) cells, was increased, whereas the frequency of CD3+ CD4- T cells was decreased. SARS-CoV-2-specific neutralization IgG and IgM antibodies were identified in all survivors, especially those recorded with severe COVID-19 who showed a higher inhibition rate of neutralization antibodies. All severe cases complained of more than one COVID-19 sequelae after 6 months of recovery. Overall, our findings indicate that SARS-CoV-2-specific antibodies remain detectable even after 6 months of recovery. Because of their abnormal adaptive immune system with a low number of CD3+ CD4- T cells and high susceptibility to infections, COVID-19 patients might need more time and medical care to fully recover from immune abnormalities and tissue damage.