Polyclonal but not monoclonal circulating memory CD4+ T cells attenuate the severity of Staphylococcus aureus bacteremia.

Staphylococcus aureus bacteremia causes significant morbidity and mortality. Treatment of staphylococcal infections is hindered by widespread antibiotic resistance, and attempts to develop an S. aureus vaccine have failed. Improved S. aureus treatment and infection prevention options require a deeper understanding of the correlates of protective immunity. CD4+ T cells have been identified as key orchestrators in the defense against S. aureus, but uncertainties persist regarding the subset, polarity, and breadth of the memory CD4+ T-cell pool required for protection. Here, using a mouse model of systemic S. aureus infection, we discovered that the breadth of bacterium-specific memory CD4+ T-cell pool is a critical factor for protective immunity against invasive S. aureus infections. Seeding mice with a monoclonal bacterium-specific circulating memory CD4+ T-cell population failed to protect against systemic S. aureus infection; however, the introduction of a polyclonal and polyfunctional memory CD4+ T-cell pool significantly reduced the bacterial burden. Our findings support the development of a multi-epitope T-cell-based S. aureus vaccine, as a strategy to mitigate the severity of S. aureus bacteremia.

Association between intracellular adenosine triphosphate content of CD4+ T lymphocytes and mortality in sepsis patients: A prospective observational study.

OBJECTIVE: This study aimed to link intracellular adenosine triphosphate content in CD4+ T lymphocytes (CD4+ iATP) with sepsis patient mortality, seeking a new predictive biomarker for outcomes and enhanced management. METHODS: 61 sepsis patients admitted to the Intensive Care Unit between October 2021 and November 2022 were enrolled. iATP levels were gauged using whole blood CD4+ T cells stimulated with mitogen PHA-L. Based on CD4+ iATP levels (<132.24 and ≥132.24 ng/mL), patients were categorized into two groups. The primary endpoint was all-cause mortality. To identify factors associated with mortality, both univariate and multivariate Cox proportional hazard analyses were conducted. RESULTS: Of the patients, 40 had high CD4+ iATP levels (≥132.24 ng/mL) and 21 had low levels (<132.24 ng/mL). In a 28-day follow-up, 21 (34.4%) patients perished. Adjusting for confounders like SOFA score, APACHE II score, lactic acid, and albumin, those with low CD4+ iATP had three- to fivefold higher mortality risk compared to high CD4+ iATP patients (61.9% vs. 20.0%; hazard ratio [95% confidence interval], Model 1: 4.515 [1.276-15.974], p = .019, Model 2: 3.512 [1.197-10.306], p = .022). CD4+ iATP correlated positively with white blood cell and neutrophil counts but not with lymphocytes, CD3, and CD4 counts. CONCLUSIONS: Low CD4+ iATP levels were associated with a higher risk of mortality in sepsis patients. Measurement of CD4+ iATP may serve as a useful tool for identifying patients at a higher risk of mortality and could potentially provide a basis for clinical treatment. Further research is warranted to fully elucidate the underlying mechanisms of this association.

Changes upon the gluten-free diet of HLA-DQ2 and TRAFD1 gene expression in peripheral blood of celiac disease patients.

Background: Celiac disease (CD) is a chronic immuno-mediated enteropathy caused by dietary gluten in genetically susceptible individuals carrying HLA (Human Leukocytes Antigen) genes that encode for DQ2.5 and DQ8 molecules. TRAFD1 (TRAF-type zinc finger domain 1) is a gene recently found associated with CD and defined as a master regulator of IFNγ signalling and of MHC class I antigen processing/presentation. There is no specific drug therapy and the only effective treatment is the gluten-free diet (GFD). The great majority of celiac patients when compliant with GFD have a complete remission of symptoms and recovery of gut mucosa architecture and function. Until now, very few studies have investigated molecular differences occurring in CD patients upon the GFD therapy. Methods: We looked at the expression of both HLA DQ2.5 and TRAFD1 risk genes in adult patients with acute CD at the time of and in treated patients on GFD. Specifically, we measured by qPCR the HLA-DQ2.5 and TRAFD1 mRNAs on peripheral blood mononuclear cells (PBMC) from the two groups of patients. Results: When we compared the HLA-DQ mRNA expression, we didn't find significant variation between the two groups of patients, thus indicating that GFD patients have the same capability to present gliadin antigens to cognate T cells as patients with active disease. Conversely, TRAFD1 was more expressed in PBMC from treated CD subjects. Notably, TRAFD1 transcripts significantly increased in the patients analyzed longitudinally during the GFD, indicating a role in the downregulation of gluten-induced inflammatory pathways. Conclusion: Our study demonstrated that HLA-DQ2.5 and TRAFD1 molecules are two important mediators of anti-gluten immune response and inflammatory process.

Paracrine- and cell-contact-mediated immunomodulatory effects of human periodontal ligament-derived mesenchymal stromal cells on CD4+ T lymphocytes.

BACKGROUND: Mesenchymal stromal cells (MSCs) isolated from the periodontal ligament (hPDL-MSCs) have a high therapeutic potential, presumably due to their immunomodulatory properties. The interaction between hPDL-MSCs and immune cells is reciprocal and executed by diverse cytokine-triggered paracrine and direct cell-to-cell contact mechanisms. For the first time, this study aimed to directly compare the contribution of various mechanisms on this reciprocal interaction using different in vitro co-culture models at different inflammatory milieus. METHODS: Three co-culture models were used: indirect with 0.4 µm-pored insert, and direct with or without insert. After five days of co-culturing mitogen-activated CD4+ T lymphocytes with untreated, interleukin (IL)-1ß, or tumor necrosis factor (TNF)-α- treated hPDL-MSCs, the CD4+ T lymphocyte proliferation, viability, and cytokine secretion were investigated. The gene expression of soluble and membrane-bound immunomediators was investigated in the co-cultured hPDL-MSCs. RESULTS: Untreated hPDL-MSCs decreased the CD4+ T lymphocyte proliferation and viability more effectively in the direct co-culture models. The direct co-culture model without inserts showed a strikingly higher CD4+ T lymphocyte cell death rate. Adding IL-1ß to the co-culture models resulted in substantial CD4+ T lymphocyte response alterations, whereas adding TNF resulted in only moderate effects. The most changes in CD4+ T lymphocyte parameters upon the addition of IL-1ß or TNF-α in a direct co-culture model without insert were qualitatively different from those observed in two other models. Additionally, the co-culture models caused variability in the immunomediator gene expression in untreated and cytokine-triggered hPDL-MSCs. CONCLUSION: These results suggest that both paracrine and cell-to-cell contact mechanisms contribute to the reciprocal interaction between hPDL-MSCs and CD4+ T lymphocytes. The inflammatory environment affects each of these mechanisms, which depends on the type of cytokines used for the activation of MSCs' immunomodulatory activities. This fact should be considered by comparing the outcomes of the different models.

Tissue adaptation of CD4 T lymphocytes in homeostasis and cancer.

The immune system is traditionally classified as a defense system that can discriminate between self and non-self or dangerous and non-dangerous situations, unleashing a tolerogenic reaction or immune response. These activities are mainly coordinated by the interaction between innate and adaptive cells that act together to eliminate harmful stimuli and keep tissue healthy. However, healthy tissue is not always the end point of an immune response. Much evidence has been accumulated over the years, showing that the immune system has complex, diversified, and integrated functions that converge to maintaining tissue homeostasis, even in the absence of aggression, interacting with the tissue cells and allowing the functional maintenance of that tissue. One of the main cells known for their function in helping the immune response through the production of cytokines is CD4+ T lymphocytes. The cytokines produced by the different subtypes act not only on immune cells but also on tissue cells. Considering that tissues have specific mediators in their architecture, it is plausible that the presence and frequency of CD4+ T lymphocytes of specific subtypes (Th1, Th2, Th17, and others) maintain tissue homeostasis. In situations where homeostasis is disrupted, such as infections, allergies, inflammatory processes, and cancer, local CD4+ T lymphocytes respond to this disruption and, as in the healthy tissue, towards the equilibrium of tissue dynamics. CD4+ T lymphocytes can be manipulated by tumor cells to promote tumor development and metastasis, making them a prognostic factor in various types of cancer. Therefore, understanding the function of tissue-specific CD4+ T lymphocytes is essential in developing new strategies for treating tissue-specific diseases, as occurs in cancer. In this context, this article reviews the evidence for this hypothesis regarding the phenotypes and functions of CD4+ T lymphocytes and compares their contribution to maintaining tissue homeostasis in different organs in a steady state and during tumor progression.

SARS-Cov-2 spike induces intestinal barrier dysfunction through the interaction between CEACAM5 and Galectin-9.

Background: Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), as a typical tumor marker, has been found to exert immunomodulatory effects in many diseases. We previously reported the clinical and molecular evidences supporting that SARS-Cov-2 infected the gastrointestinal (GI) tract and found a reduction of CEACAM5 in COVID-19 patients' feces which associated with gut dysbiosis. Yet the role of CEACAM5 in GI infection is ill-defined. Methods: Mice models were established through intraperitoneally injecting with recombinant viral spike-Fc to mimic the intestinal inflammation. We collected duodenum, jejunum, ileum and colon samples after 6h, 2 days, 4 days and 7 days of spike-Fc or control-Fc injection to perform proteomic analysis. Blood was collected from healthy donors and peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation, then CD4+ T cells were isolated with magnetic beads and co-cultured with Caco-2 cells. Results: In addition to intestinal CEACAM5, the expression of tight junction and the percent of CD4+ T lymphocytes were significantly decreased in spike-Fc group compared to control (p < 0.05), accompanied with increased level of inflammatory factors. The KEGG analysis revealed differentially expressed proteins were mainly enriched in the coronavirus disease (COVID-19), tight junction, focal adhesion, adherens junction and PI3K-Akt signaling pathway. Protein-protein interaction (PPI) network analysis identified the interaction between CEACAM5 and Galectin-9 that was also verified by molecular docking and co-IP assay. We further confirmed a reduction of CEACAM5 in SARS-CoV-2 spike stimulated enterocytes could promote the expression of Galectin-9 protein in CD4+T cells. Then it gave rise to the increasing release of inflammatory factors and increased apoptosis of CD4+T cells by inhibition of PI3K/AKT/mTOR pathway. Ultimately intestinal barrier dysfunction happened. Conclusion: Our results indicated that CEACAM5 overexpression and Galectin-9 knockdown played a protective role in intestinal barrier injury upon spike-Fc stimulation. Collectively, our findings identified firstly that SARS-CoV-2 spike induced intestinal barrier dysfunction through the interaction between CEACAM5 and Galectin-9. The result provides potential therapeutic targets in intestinal barrier dysfunction for treating severe COVID patients.

Phenotypic and Functional Characterization of Memory CD4+ and CD8+ T Cells After Antigenic Stimulation.

The encounter of T cells with the antigen through the interaction of T cell receptors with peptides and major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) can generate effector response and memory T cells. Memory T cells developed following infections or vaccination may persist, leading to the generation of a specific immune response upon reexposure to the same pathogen through rapid clonal proliferation and activation of effector functions. T cell memory subsets can be identified based on the expression of several membrane markers such as CCR7, CD27, and CD45RA. Using fluorescent antibodies against these markers and a flow cytometer, it is possible to perform immunophenotyping via the analysis of cell surface expression of proteins by different subpopulations such as the subsets of naïve, effector, and memory T cells as well as via the analysis of functional markers that further characterize each sample. Intracellular cytokine staining allows for the evaluation of intracellular proteins expressed in T cells in response to antigenic stimulation. This chapter presents the phenotypic and functional characterization of memory T cells after antigenic stimulation, detailing the procedures for identifying intracellular and surface protein markers. Herein, we review and present a reproducible standardized protocol using antibodies for specific markers and applying flow cytometry.

[Functional analysis of virus-specific CD4(+)T cells and CD8(+)T cells in patients with liver injury caused by Epstein-Barr virus infection].

Objective: To analyze the functional differences between virus-specific CD4(+)T cells and CD8(+)T cells in patients infected with Epstein-Barr virus (EBV) who develop liver injury and those who do not. Methods: 45 cases of EBV infections were enrolled, including 28 cases developing liver injuries and 17 that did not. Mononuclear cells from peripheral blood were isolated. CD4(+)T cells and CD8(+)T cells were purified and cultured using recombinant EBV core antigen 2 (EBNA2) for 96 h with stimulation. The CCK-8 method was used to detect cell proliferation. Flow cytometry was used to detect the proportion of CD4(+)T cells and CD8(+)T cells. An enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of CD4(+)T cells secreting cytokines and CD8(+)T cells secreting molecular toxicity. Real-time quantitative PCR was used to detect the mRNA levels of transcription factors and molecular toxicity in CD4(+)T cell subsets. Flow cytometry was used to detect the immune checkpoints at molecular levels in CD8(+)T cells. The inter-group comparison was performed using a t-test or Mann-Whitney test. Results: There was no statistically significant difference (P > 0.05) in the proliferation proportion of peripheral blood mononuclear cells, CD4(+)T cells, and CD8(+)T cells after stimulation with recombinant EBNA2 between the EBV-infected non-liver injury group and the infected liver injury group (P > 0.05). There was no statistically significant difference in the proportion of CD4(+)T cells secreting related cytokines and the mRNA levels of transcription factors after stimulation with recombinant EBNA2 between the EBV-infected non-liver injury group and the infected liver injury group (P > 0.05).The levels of perforin secreted by CD8(+)T cells and granzyme B after stimulation with recombinant EBNA2 were higher in the EBV infection-induced liver injury group than those in the non-liver injury group [(75.51±23.33) pg/ml vs. (58.99±18.39) pg/ml, P = 0.017] [(117.8±44.55) pg/ml vs. (90.22±34.21) pg/ml, P = 0.034]. The mRNA levels of Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand in CD8(+)T cells in the liver injury group caused by EBV infection were approximately 1.5 and 1.2 times higher than those in the non-liver injury group, respectively, and the difference was statistically significant (P < 0.001), but there was no statistically significant difference in the proportional expression of programmed cell death-1 and cytotoxic T lymphocyte-associated antigen-4 in CD8(+)T cells between the EBV-infected non-liver injury group and infected liver injury group (P > 0.05) Conclusion: Patients with liver injury caused by EBV infection have strong virus-specific CD8(+) T cell toxic effects, which may mediate EBV-induced liver injury.

Prognostic impact of CD4+ and CD8+ tumor-infiltrating lymphocytes in patients with colorectal cancer.

OBJECTIVE: Tumor immune response has been suggested as an important indicator of cancer prognosis. This study was initiated to investigate the association between T lymphocytes and the prognosis of patients with colorectal cancer (CRC). METHODS: Included in this study were 129 CRC patients who received surgical treatment in Henan Provincial People's Hospital from January 2003 to January 2014. The level of CD4+ and CD8+ T lymphocytes in tissues was detected by immunohistochemistry (IHC). Survival analysis was conducted by the Kaplan-Meier method and Cox proportional hazards model. RESULTS: IHC staining showed that CD8+ T lymphocyte infiltration was high in 88 cases and low in 41 cases, while CD4+ T lymphocyte infiltration was high in 66 cases and low in 63 cases. The level of CD4+ and CD8+ T lymphocytes in CRC tissue was closely related to TNM stage and tumor invasion (p < 0.05). Follow-up analysis showed that both disease-free survival (DFS) and overall survival (OS) were better in patients with a high level of CD8+ and CD4 + CD8+ than those in patients with a low level (p < 0.05). Multivariate analysis showed that TNM stage, lymph node, CD8+ and CD4+ CD8+ were independent risk factors for DFS and OS (p < 0.05). CONCLUSION: High level of CD8+ and CD4+ CD8+ may prove to be a potential predictor of better prognosis of CRC patients.

CDK5R1, GSE1, HSPG2 and WDFY3 as indirect epigenetic-sensitive genes in atrial fibrillation.

BACKGROUND: Although mounting evidence supports that aberrant DNA methylation occurs in the hearts of patients with atrial fibrillation (AF), noninvasive epigenetic characterization of AF has not yet been defined. METHODS: We investigated DNA methylome changes in peripheral blood CD4+ T cells isolated from 10 patients with AF relative to 11 healthy subjects (HS) who were enrolled in the DIANA clinical trial (NCT04371809) via reduced-representation bisulfite sequencing (RRBS). RESULTS: An atrial-specific PPI network revealed 18 hub differentially methylated genes (DMGs), wherein ROC curve analysis revealed reasonable diagnostic performance of DNA methylation levels found within CDK5R1 (AUC = 0.76; p = 0.049), HSPG2 (AUC = 0.77; p = 0.038), WDFY3 (AUC = 0.78; p = 0.029), USP49 (AUC = 0.76; p = 0.049), GSE1 (AUC = 0.76; p = 0.049), AIFM1 (AUC = 0.76; p = 0.041), CDK5RAP2 (AUC = 0.81; p = 0.017), COL4A1 (AUC = 0.86; p < 0.001), SEPT8 (AUC = 0.90; p < 0.001), PFDN1 (AUC = 0.90; p < 0.01) and ACOT7 (AUC = 0.78; p = 0.032). Transcriptional profiling of the hub DMGs provided a significant overexpression of PSDM6 (p = 0.004), TFRC (p = 0.01), CDK5R1 (p < 0.001), HSPG2 (p = 0.01), WDFY3 (p < 0.001), USP49 (p = 0.004) and GSE1 (p = 0.021) in AF patients vs HS. CONCLUSIONS: CDK5R1, GSE1, HSPG2 and WDFY3 resulted the best discriminatory genes both at methylation and gene expression level. Our results provide several candidate diagnostic biomarkers with the potential to advance precision medicine in AF.

In HIV-Infected Immunological Non-Responders, Hepatitis C Virus Eradication Contributes to Incomplete Normalization of Systemic Inflammation Indexes, but Does Not Lead to Rapid CD4+ T-Cell Count Recovery.

In HIV-positive individuals taking antiretroviral therapy, coinfection with hepatitis C virus (HCV) increases systemic inflammation, which interferes with the CD4+ T-cells regeneration. This study evaluated the effect of HCV eradication on systemic inflammation and CD4+ T-cell regeneration in patients who gave poor response to antiretroviral therapy, the so-called "immunological non-responders" (INRs). HIV-infected patients who received a course of direct-acting antivirals for treating hepatitis C were examined. The control groups included HIV/HCV-coinfected INRs and relatively healthy volunteers. It was established for the first time that HCV eradication is not accompanied by a complete suppression of systemic inflammation, but improves the T-cell pool composition: in INRs, the blood CD4+/CD8+ T-lymphocyte ratio increases and approaches those of healthy individuals. Apparently, in INRs treated for hepatitis C, the immune system recovery takes time and may be incomplete.

Divergent CD4+ T-cell profiles are associated with anti-HLA alloimmunization status in platelet-transfused AML patients.

Introduction: Acute myeloid leukemia (AML) is one of the commonest hematologic disorders. Due to the high frequency of disease- or treatment-related thrombocytopenia, AML requires treatment with multiple platelet transfusions, which can trigger a humoral response directed against platelets. Some, but not all, AML patients develop an anti-HLA immune response after multiple transfusions. We therefore hypothesized that different immune activation profiles might be associated with anti-HLA alloimmunization status. Methods: We tested this hypothesis, by analyzing CD4+ T lymphocyte (TL) subsets and their immune control molecules in flow cytometry and single-cell multi-omics. Results: A comparison of immunological status between anti-HLA alloimmunized and non-alloimmunized AML patients identified differences in the phenotype and function of CD4+ TLs. CD4+ TLs from alloimmunized patients displayed features of immune activation, with higher levels of CD40 and OX40 than the cells of healthy donors. However, the most notable differences were observed in non-alloimmunized patients. These patients had lower levels of CD40 and OX40 than alloimmunized patients and higher levels of PD1. Moreover, the Treg compartment of non-alloimmunized patients was larger and more functional than that in alloimmunized patients. These results were supported by a multi-omics analysis of immune response molecules in conventional CD4+ TLs, Tfh circulating cells, and Tregs. Discussion: Our results thus reveal divergent CD4+ TL characteristics correlated with anti-HLA alloimmunization status in transfused AML patients. These differences, characterizing CD4+ TLs independently of any specific antigen, should be taken into account when considering the immune responses of patients to infections, vaccinations, or transplantations.

Selected biochemical parameters in the urine of HIV-infected patients in monitoring of kidney function.

Introduction: For years, there has been an increase in the number of cases of chronic kidney disease (CKD) in human immunodeficiency virus (HIV)-infected patients. Renal dysfunction can be caused by direct effects of HIV on the kidneys but also of applied combined antiretroviral therapy (cART). Therefore there is a need of renal function diagnosis to monitor the development of kidney disturbances. In this study the urinary levels of selected low molecular weight proteins (LMWP) in HIV-infected patients were measured and related to current CD4+ T lymphocyte (LT CD4+) count, the glomerular filtration rate (eGFR) value and the applied cART. Material and methods: The levels of 5 LMWP - kidney injury molecule-1 (KIM-1), neutrophil gelatinase associated lipocalin (NGAL), glutathione S-transferase α (GST-α) and π (GST-π) isoenzymes and neopterin (NPT) - in urine were measured in HIV-infected patients and healthy controls by enzyme-linked immunosorbent assay. Results: Taking into account the current LT CD4+ count, KIM-1, NGAL and GST-α showed statistically significant differences between groups with the CD4+ count < 500 and ≥ 500 cells (< 0.001). Depending on the eGFR, apart from KIM-1 and NGAL, NPT showed statistically significant differences in the investigated groups with normal and lower eGFR values (< 0.001). In terms of applied cART, the best parameters in the assessment of kidney damage were NGAL, GST-π and NPT (< 0.001). Conclusions: This research shows that the analyzed LMWP parameters are useful in the assessment of kidney damage in HIV patients during cART, especially NPT, NGAL and GST-π. However, future studies should be conducted on larger groups.

Clinical implications of aberrant PD-1 expression for acute leukemia prognosis.

BACKGROUND: Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are the most common types of leukemia in adults with an overall poor prognosis. PD-1 alone or combined with other immune checkpoint blockade is a promising research direction for the treatment of acute leukemia (AL) patients. However, clinical Implications of aberrant PD-1 expression in peripheral CD4+ and CD8+ T lymphocytes of AML and ALL patients in assessing the prognosis of diseases, remains inconclusive. METHODS: In the present study, we used flow cytometry to evaluate PD-1 expression on the surface of CD4+ and CD8+ T lymphocytes in the peripheral circulation of AML and ALL patients and its clinical significance. A total of 53 AML patients, 44 ALL patients and 28 healthy controls were enrolled in this study and peripheral blood specimens were detected by flow cytometry. RESULTS: Our results indicated that percentages of CD4+ PD1+ and CD8+ PD1+ T lymphocytes in newly diagnosed and non-remission groups were significantly higher than healthy control both in AML and ALL patients. The high level of CD4+ PD1+ and CD8+ PD1+ T lymphocytes were respectively poor prognostic indicators of AML patients and ALL patients but had no significant correlation with most common clinical risks. CONCLUSIONS: Our findings show that aberrant PD-1 expression correlates with the prognosis of AL patient and may thus serve as poor prognostic indicators. Immunotherapy using PD-1 inhibitors may be a promising strategy for AML and ALL patients with peripheral circulating CD4+ PD1+ and CD8+ PD1+ T lymphocytes positively expressed, respectively.

Regulatory Rheumatoid Factor is Specific to PD-1 and Uses PD-1 Pathway to Control CD4 T Lymphocytes.

BACKGROUND: We have earlier discovered a new factor of autoimmunity downregulation, called regulatory rheumatoid factor (regRF). Being anti-idiotypic antibodies, regRF restricts the expansion of CD4+ T lymphocytes to the idiotype of which it is specific, according to the negative feedback principle. It has been shown that only activated CD4+ T lymphocytes are the target of regRF. However, it is still not clear the way regRF distinguishes activated cells from naive ones. RegRF molecules, apart from individual paratopes specific to unique sequences of B- and T-cell receptors, have a shared paratope. We assume that regRF by means of a shared paratope recognizes one of the surface activation molecules of CD4+ T lymphocytes and initiates the cell death. Programmed death-1 (PD-1) has been tested as a potential receptor of the shared regRF paratope and transmitter of the negative regRF signal into activated CD4+ T lymphocytes. METHODS: The specificity of the shared regRF paratope to PD-1 was determined by ELISA. T cell activation was performed with immobilized anti-CD3ε antibodies. Flow cytometry was used to study the effect of regRF on PD-1+CD4+ lymphocytes. RESULTS: We found that regRF binds to PD-1. IgG Fc fragments carrying epitopes specific to the shared paratope of regRF compete with PD-1 for binding to regRF. It follows that regRF recognizes specifically PD-1 by means of a shared paratope. RegRF-containing serum reduced the number of PD-1+CD4+ lymphocytes in proportion to their increase resulting from the action of anti-CD3ε antibodies. CONCLUSION: RegRF uses PD-1 pathway to control activated CD4+ T lymphocytes.

Impaired Regulation by IL-35 in Systemic Sclerosis.

This study investigated the role of IL-35 in systemic sclerosis (SSc) patients, focusing on CD4+ T cell response and immunomodulatory cytokine production. By comparing the cytokine levels in healthy donors (HD) and SSc patients using ELISAs, we found a significantly lower plasma IL-35 concentration in the SSc patients (52.1 ± 5.6 vs. 143 ± 11.1, p < 0.001). Notably, the IL-35 levels showed a negative correlation with TGF-ß (p < 0.001) and IL-17 (p = 0.04). Assessing the IL-35R expression across cell types in the SSc patients and HDs via flow cytometry, we found higher levels on monocytes (40.7 + 5.7 vs. 20.3 ± 1.9, p < 0.001) and lower levels on CD8+ T cells (61.8 ± 9.2 vs. 83.4 ± 0.8, p < 0.05) in the SSc patients. The addition of recombinant IL-35 to stimulated peripheral blood mononuclear cells reduced the IL-17+CD4+ T cell percentage (9.0 ± 1.5 vs. 4.8 ± 0.7, p < 0.05) and increased the IL-35+CD4+ T percentage (4.1 ± 2.3 vs. 10.2 ± 0.8, p < 0.001). In a Treg:Tresponder cell Sco-culture assay with HD and SSc samples, rIL35 decreased the cell proliferation and levels of IL-17A (178.2 ± 30.5 pg/mL vs. 37.4 ± 6.4 pg/mL, p < 0.001) and TGF-ß (4194 ± 777 pg/mL vs. 2413 ± 608 pg/mL, p < 0.01). Furthermore, we observed a positive correlation between the modified Rodnan skin score (mRSS) and TGF-ß (p < 0.001), while there was a negative correlation between mRSS and IL-35 (p = 0.004). Interestingly, higher levels of plasmatic IL-35 were detected in individuals with limited disease compared to those with diffuse disease (60.1 ± 8.0 vs. 832.3 ± 4.1, p < 0.05). These findings suggest that IL-35 exhibits anti-inflammatory properties in SSc and it may serve as a marker for disease severity and a therapeutic target.

Alarming rise in HIV cases in Pakistan: Challenges and future recommendations at hand.

Background: Human immunodeficiency virus (HIV) is a retrovirus that suppresses the immune system by reducing the CD4+ T lymphocytes level. It has become a global challenge with fast prevalence ratio. Like other developing countries, Pakistan is also struggling for overcoming this viral disease since very first reported case in 1987. Aim: To update the society on the alarming rise in HIV cases in Pakistan: challenges and future recommendations at hand. Materials and Methods: The review paper utilized different search engines such pubmed central, scopus, web of science, google scholar etc. to conduct this review paper. Results: Lack of awareness, low literacy rate, practice of unhygienic equipment in healthcare departments, unstable economy, and unsafe sexual practices are the major factors behind the increasing rate of AIDS in Pakistan. Conclusion: By regulating healthcare practices and policies, promoting psychological counseling to HIV positive patients, educating the society and minimizing commercial sex practices, Pakistan can overcome this viral disease.

Genome-wide DNA methylation profiling of CD4+ T lymphocytes identifies differentially methylated loci associated with adult primary refractory immune thrombocytopenia.

BACKGROUND: DNA methylation played a crucial role in the pathogenesis of immune thrombocytopenia (ITP). However, genome-wide DNA methylation analysis has not been applied thus far. The present study aimed to provide the first DNA methylation profiling for ITP. METHODS: Peripheral blood CD4+ T lymphocytes samples were collected from 4 primary refractory ITP cases and 4 age-matched healthy controls, and DNA methylome profiling was performed using Infinium MethylationEPIC BeadChip. Differentially methylated CpG sites were further validated in another independent cohort of 10 ITP patients and 10 healthy controls using qRT-PCR. RESULTS: The DNA methylome profiling identified a total of 260 differentially methylated CpG sites mapping to 72 hypermethylated and 64 hypomethylated genes. These genes were mainly enriched in the actin nucleation of the Arp2/3 complex, vesicle transport, histone H3-K36 demethylation, Th1 and Th2 cell differentiation, and Notch signaling pathway according to the GO and KEGG databases. The mRNA expression of CASP9, C1orf109, and AMD1 were significantly different. CONCLUSIONS: Given the altered DNA methylation profiling of ITP, our study provides new insights into its genetic mechanism and suggests candidate biomarkers for the diagnosis and treatment of ITP.

The cholinergic anti-inflammatory pathway inhibits inflammation without lymphocyte relay.

The magnitude of innate inflammatory immune responses is dependent on interactions between peripheral neural and immune cells. In particular, a cholinergic anti-inflammatory pathway (CAP) has been identified in the spleen whereby noradrenaline (NA) released by splenic nerves binds to ß2-adrenergic receptors (ß2-AR) on CD4+ T cells which, in turn, release acetylcholine (ACh). The binding of ACh to α7 acetylcholine receptors (α7-AChR) expressed by splenic macrophages inhibits the production of inflammatory cytokines, including tumor necrosis factor (TNF). However, the role of ACh-secreting CD4+ T-cells in the CAP is still controversial and largely based on the absence of this anti-inflammatory pathway in mice lacking T-cells (nude, FoxN1-/-). Using four conscious, non-lymphopenic transgenic mouse models, we found that, rather than acting on CD4+ T-cells, NA released by splenic nerve terminals acts directly onto ß2-AR on splenic myeloid cells to exert this anti-inflammatory effect. We also show that, while larger doses of LPS are needed to trigger CAP in nude mouse strain compared to other strains, TNF production can be inhibited in these animals lacking CD4+ T-cell by stimulating either the vagus or the splenic nerve. We demonstrate that CD4+ T-cells are dispensable for the CAP after antibody-mediated CD4+ T-cell depletion in wild type mice. Furthermore, we found that NA-mediated inhibition of in vitro LPS-induced TNF secretion by human or porcine splenocytes does not require α7-AChR signaling. Altogether our data demonstrate that activation of the CAP by stimulation of vagus or splenic nerves in mice is mainly mediated by direct binding of NA to ß2-AR on splenic macrophages, and suggest that the same mechanism is at play in larger species.

Targeting L-Selectin Lymphocytes to Deliver Immunosuppressive Drug in Lymph Nodes for Durable Multiple Sclerosis Treatment.

Inflammation induced by autoreactive CD4+ T lymphocytes is a major factor in the pathogenesis of multiple sclerosis (MS). Immunosuppressive drugs, such as FTY720, are subsequently developed to prevent the migration of CD4+ T lymphocytes to the central nervous system (CNS). However, these immunosuppressive drugs have limited accumulation in lymph nodes (LNs), resulting in poor efficacy. Here, this work develops a nanoplatform for delivering immunosuppressive drugs to LNs for durable MS treatment. Human CD47 peptide and L-selectin targeting aptamer are modified on the nanoparticles encapsulated with FTY720 (clnFTY) for self-passivation and the targeting of L-selectin on lymphocytes, a homing receptor for T-cells entering LNs. Using this natural process, clnFTY nanoparticles efficiently deliver FTY720 to LNs and delay disease progression in experimental autoimmune encephalomyelitis (EAE) mice following a single dose treatment over a 42-day observational period. Considering the daily dosing requirement of FTY720, this strategy greatly improves its therapeutic efficiency. The ability of clnFTY nanoparticles to target lymphocytes, reduce sphingosine-1-phosphate receptor 1 (S1PR1) expression, and suppress inflammatory cytokines release are demonstrated in clinical blood samples from MS patients. Taken together, this study demonstrates that targeted LNs delivery may greatly extend the treatment cycle of immunosuppressive drugs for durable MS treatment.