The role of regulatory T-cells in the development of endometriosis.

Endometriosis is a benign disease of the female reproductive tract, characterized by the process of chronic inflammation and alterations in immune response. It is estimated to affect 2-19% of women in the general population and is commonly associated with symptoms of chronic pelvic pain and infertility. Regulatory T cells (Treg) are a subpopulation of T lymphocytes that are potent suppressors of inflammatory immune response, essential in preventing destructive immunity in all tissues. In endometriosis, several studies have investigated the possible role of Treg cells in the development of the disease. Most studies to date are heterogeneous in methodology and are based on a small number of cases, which means that it is impossible to define their exact role at present. Based on current knowledge, it seems that disturbed Treg homeostasis, leading to increased systemic and local inflammation within ectopic and eutopic endometrium, is present in women who eventually develop endometriosis. It is also evident that different subsets of human Treg cells have different roles in suppressing the immune response. Recent studies in patients with endometriosis have investigated naive/resting FOXP3lowCD45RA+ Treg cells, which upon T cell receptor stimulation, differentiate into activated/effector FOXP3highCD45RA- Treg cells, characterized by a strong immunosuppressive activity. In addition, critical factors controlling expression of Treg/effector genes, including reactive oxygen species and heme-responsive master transcription factor BACH2, were found to be upregulated in endometriotic lesions. As shown recently for cancer microenvironments, microbial inflammation may also contribute to the local composition of FOXP3+ subpopulations in endometriotic lesions. Furthermore, cytokines, such as IL-7, which control the homeostasis of Treg subsets through the tyrosine phosphorylation STAT5 signalling pathway, have also been shown to be dysregulated. To better understand the role of Treg in the development of endometriosis, future studies should use clear definitions of Tregs along with specific characterization of the non-Treg (FOXP3lowCD45RA-) fraction, which itself is a mixture of follicular Tregs and cells producing inflammatory cytokines.

New Flow Cytometric Methods for Monitoring STAT5 Signaling Reveal Responses to SARS-CoV-2 Antigen-Specific Stimulation in FOXP3+ Regulatory T Cells also in Patients with Advanced Chronic Lymphocytic Leukemia.

Increased frequency of CD4+CD25+ regulatory T-cells (Treg) has been associated with disease progression in chronic lymphocytic leukemia (CLL). Flow cytometric methods, which allow for the simultaneous analysis of their specific transcription factor Foxp3 and activated STAT proteins, together with proliferation can help to elucidate the signaling mechanisms driving Treg expansion and suppression of FOXP3- conventional CD4+T-cells (Tcon). Herein, we first report a novel approach in which STAT5 phosphorylation (pSTAT5) and proliferation (BrdU-FITC incorporation) could be analyzed specifically in FOXP3+ and FOXP3- responding cells after CD3/CD28 stimulation. The addition of magnetically purified CD4+CD25+ T-cells from healthy donors to cocultured autologous CD4+CD25- T-cells resulted in suppression of Tcon cell cycle progression accompanied by a decrease in pSTAT5. Next, a method using imaging flow cytometry is presented for the detection of cytokine-dependent pSTAT5 nuclear translocation in FOXP3-expressing cells. Finally, we discuss our experimental data obtained by combining Treg pSTAT5 analysis and antigen-specific stimulation with SARS-CoV-2 antigens. Applying these methods on samples from patients revealed Treg responses to antigen-specific stimulation and significantly higher basal pSTAT5 in CLL patients treated with immunochemotherapy. Thus, we speculate that through the use of this pharmacodynamic tool, the efficacy of immunosuppressive drugs and their possible off-target effects can be assessed.

Case report: Pneumocystis jirovecii pneumonia in a severe case of Aicardi-Goutières syndrome with an IFIH1 gain-of-function mutation mimicking combined immunodeficiency.

Aicardi-Goutières syndrome (AGS) is a genetically determined early-onset progressive encephalopathy caused by mutations leading to overexpression of type I interferon (IFN) and resulting in various clinical phenotypes. A gain-of-function (GOF) mutation in the IFIH1 gene is associated with robust production of type I IFN and activation of the Janus kinase (JAK) signal transducer and activator of the transcription (STAT) pathway, which can cause AGS type 7. We detail the clinical case of an infant who initially presented with Pneumocystis jirovecii pneumonia (PCP), had recurrent respiratory infections, and was later treated with a JAK inhibitor, baricitinib, because of a genetically confirmed GOF mutation in the IFIH1 gene. This spectrum of IFIH1 GOF mutations with overlapping features of hyperinflammation and severe opportunistic infection, which mimics combined immunodeficiency (CID), has not been described before. In this case, therapy with baricitinib effectively blocked IFN-α activation and reduced STAT1 signaling but had no effect on the progression of the neurological disease.

Altered Homeostasis of Regulatory T Lymphocytes and Differential Regulation of STAT1/STAT5 in CD4+ T Lymphocytes in Childhood-onset Systemic Lupus Erythematosus.

OBJECTIVE: Childhood-onset systemic lupus erythematosus (cSLE) is usually a more severe and aggressive disease than adult-onset SLE (aSLE), but cellular and subcellular reasons for these differences are not well understood. The present study analyzed Th subsets, STAT1/STAT5 signaling response, and cytokine profiles of cSLE. METHODS: FOXP3+ regulatory (Treg) and effector Th subsets, expression and phosphorylation of STAT1/STAT5 in Th, and cytokine profiles were measured in the peripheral blood of patients with cSLE and healthy controls (HC), using flow cytometry and immunoassay on a biochip. RESULTS: Significant correlation between expression of the activation marker HLA-DR and decreased Th counts, an increase in the percentage of FOXP3+ Th, and a decrease in the activated Treg (aTreg) subset among them were found in cSLE. In contrast to our previous findings in aSLE, no significant differences in percentages and a significant decrease in the numbers of the naive-resting Treg (rTreg) subset compared to HC were found. The percentages of CD25- cells, possibly reflecting interleukin 2 depletion, were significantly increased in cSLE aTreg, but not in the rTreg subset. Consistent with the results of our previous studies in aSLE, increased expression of STAT1, along with significant correlation between decreased Th counts and their increased basal phosphorylation of STAT5, were also found in cSLE. CONCLUSION: Our results suggest that the key difference in Treg homeostasis between cSLE and aSLE is in the rTreg subset. However, perturbed aTreg homeostasis, increased levels of STAT1 protein, and homeostatic STAT5 signaling appear to be intrinsic characteristics of the disease, present in cSLE and aSLE alike.

STAT signaling as a marker of SLE disease severity and implications for clinical therapy.

The Janus kinase/signal transduction and activator of transcription (JAK-STAT) signaling pathway is implicated in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE). While small-molecule JAK inhibitors (Jakinibs) are currently under investigation for SLE, results of recent studies suggest, that the efficacy of drugs such as methotrexate (MTX) may also be due to their ability to suppress phosphorylation of STAT proteins. A previously identified STAT5 phosphorylation (pSTAT5) and STAT1 protein expression ¼signature« in circulating CD4+ T cells of patients with SLE was associated with perturbed homeostasis between conventional (Tcon) and activated regulatory (aTreg) subset and with time-adjusted cumulative disease activity during follow-up. Initial observations in SLE patient cohort were validated with additional markers of disease severity and patients were stratified according to medication status. Preliminary results show that lower CD4+ T-cell counts in patients with SLE are associated with higher pSTAT5 levels and Tcon homeostatic proliferation, which was previously found to drive lymphopenia associated autoimmunity. Relapsing disease was better predicted by pSTAT5 levels than CD4 counts. Further, significant correlation was found between mean pSTAT5 levels during follow- up and the markers of disease severity. As patients with SLE, also patients with rheumatoid arthritis (RA) not receiving methotrexate, had significantly higher increase in CD4+ T-cell pSTAT5 levels compared to patients not receiving this specific therapy. However, the difference in pSTAT5 between Tcon and aTreg was independent of treatment with MTX and significantly increased only in patients with SLE. CD4 depletion, driving homeostatic proliferation of Tcon subset, is therefore associated with higher pSTAT5 levels, which confer worse prognosis in patients with SLE. While treatment with MTX may decrease overall pSTAT5 levels in CD4+ T-cells also from patients with RA, increased pSTAT5 levels in Tcon relative to aTreg subset are specific for SLE.

Can flow cytometry reinvent the sentinel lymph node concept in gastric cancer patients?

BACKGROUND: The focused sentinel lymph node (SLN) concept we proposed previously relied on real time-quantitative polymerase chain reaction (RT-qPCR) to detect tumor cells, which is too elaborate for intraoperative use. Therefore, we evaluated flow cytometry for intraoperative detection of tumor cells in SLNs. METHODS: Sixty-five consecutive gastric cancer patients were included. SLN analysis was carried out for a single SLN from each patient, using the molecular methods of RT-qPCR (first 30 patients) and flow cytometry (final 35 patients). All LNs underwent hematoxylin and eosin staining and immunohistochemical examination. RESULTS: Extraction of the SLN from a high-risk station was an important determinant for accurate prediction of LN metastases. For RT-qPCR, the sensitivity and specificity of detection were 72.7% and 81.8%, respectively, and for flow cytometry, 36.8% and 100%, respectively. When only high-risk SLNs were analyzed and specimens with <10% viability of leukocytes were excluded, the sensitivity and specificity of flow cytometry were 60% and 100%, respectively. Multivariate analysis identified significant predictors for LN metastases as the molecular method of SLN analysis (P = 0.021; 95% confidence interval [CI]: 1.304-24.284) and lymphovascular invasion (P = 0.002; 95% CI: 2.142-28.555). In subgroup analysis of high-risk SLNs, only RT-qPCR was a significant predictor for LN metastases (P = 0.016; 95% CI: 1.581-91.084). CONCLUSIONS: Flow cytometry of high-risk SLNs, excluding specimens with low cell viability is a rapid, cost-effective, widely obtainable, and highly specific method for SLN metastases detection although it lacks the necessary sensitivity. Therefore, it cannot be recommended as a stand-alone method for the detection of LN metastases during operations.

STAT5 phosphorylation in CD4 T cells from patients with SLE is related to changes in their subsets and follow-up disease severity.

Activation of the STAT5 signaling pathway up-regulates antiapoptotic protein Bcl2 and drives proliferation of autoreactive conventional CD4 T cells (Tcons). In systemic lupus erythematosus (SLE), an increased T cell Bcl2 content and perturbed homeostasis of CD45RA-FOXP3hi activated regulatory T cells (aTregs) were described. We assessed Tcon/Treg subsets and phosphorylation of STAT5 (pSTAT5) in blood T cells from patients with SLE by using conventional and imaging flow cytometry. Forty-one patients with SLE, 33 healthy controls, and 29 patients with rheumatoid arthritis were included. Long-term monitoring was performed in 39 patients with SLE, which were followed longitudinally for up to 1000 d. Significantly increased Bcl2 protein content in T cells from patients with SLE was associated with IL-7-dependent STAT5 activation, expressed as increased basal levels and nuclear localization of pSTAT5. pSTAT5 levels were significantly increased in the FOXP3 low-expressing CD4+ T cell subsets but not in the aTreg subset, which was significantly decreased in patients with SLE. In contrast to aTreg, SLE Tcon displayed significantly increased pSTAT5 and Bcl2 levels. Moreover, the percentage of Tcon-expressing proliferation marker Ki-67 was significantly increased in patients with SLE and was positively correlated with CD4 T cell pSTAT5 levels. Finally, a subgroup of patients characterized by an increased Tcon-pSTAT5/aTreg-pSTAT5 ratio experienced a more aggressive-relapsing disease course and displayed higher time-adjusted cumulative CD4 T cell pSTAT5 levels during follow-up, which were positively correlated with time-adjusted cumulative disease activity. Our results indicate that imbalanced STAT5 phosphorylation, which is related to Bcl2 and Ki-67 expression, may confer survival and proliferative advantage to Tcon over aTreg and could represent a possible marker of SLE disease severity.

Increased Levels of STAT1 Protein in Blood CD4 T Cells from Systemic Lupus Erythematosus Patients Are Associated with Perturbed Homeostasis of Activated CD45RA-FOXP3hi Regulatory Subset and Follow-Up Disease Severity.

In murine systemic lupus erythematosus (SLE), aberrant regulation of interferon (IFN)-alpha-STAT1 signaling and perturbed homeostasis of CD4+FOXP3+ regulatory T cells (Tregs) were described. In the present study, STAT1 signaling and circulating Treg subsets were assessed by flow cytometry in 39 SLE patients and their potential association with disease course was examined during long-term follow-up. Levels of STAT1 protein as measured by median fluorescence intensity (MFI) were significantly increased in SLE CD4 T cells when compared with rheumatoid arthritis patients and healthy controls and were positively correlated with disease activity. The highest STAT1 MFI was found in CD45RA-FOXP3hi-activated Treg (aTreg) subset, which demonstrated the highest STAT1 phosphorylation responses among SLE CD4 T cells and significant decrease in proliferation marker Ki-67 expression after IFN-alpha stimulation. Percentage of Ki-67+ aTregs was significantly decreased in SLE patients and was negatively correlated with CD4 T cell STAT1 MFI. A subgroup of SLE patients characterized by lower aTreg counts experienced more severe relapsing disease course during 1,000 days of follow-up. Mean CD4 T cell STAT1 MFI in follow-up samples from SLE patients was negatively correlated with mean of follow-up aTreg counts. Our findings indicate that augmented STAT1 signaling may be involved in perturbed aTreg homeostasis, which could represent a possible marker of SLE disease severity.

The Role of STAT Signaling Pathways in the Pathogenesis of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder with a broad spectrum of clinical presentations and association with multiple immunological abnormalities. Recent research of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway-revealed aberrant STAT signaling in inflammatory conditions and autoimmune diseases including SLE. STAT proteins are major components in interferon (IFN)-dependent gene expression and are responsible for signal transduction of over 50 cytokines, hormones, and growth factors regulating key cellular processes such as survival, proliferation, and differentiation. This review summarizes the present evidence from experimental animal models and patients with SLE for the involvement of STAT pathways in the pathogenesis of SLE underlining the role of different members of the STAT family. Genome-wide association studies provided evidence that variations in STAT4 gene are linked to the development of SLE in humans. First integration with genome-wide epigenomics data suggests that control of CD4+ T cell differentiation in which STATs play a major role may be an important component of the genetic contribution to disease susceptibility. Increased transcript and total protein STAT1 levels were described both in SLE T and B cells suggestive of the priming mechanisms that augment STAT1 signaling responses to IFN. STAT3 has a crucial role in Th17 differentiation, T follicular helper, and B cells, and STAT3 inhibition could represent a possible future therapeutic target in SLE. STAT5B appears to act as a critical modulator of human Treg development and function. While the imbalance between phosphorylated STAT5 and STAT3 in human SLE T cells was implicated in dysregulated IL-10 expression, Treg-specific deletion of STAT3 in mouse model even enhanced Th17-mediated inflammation. Finally, we present also a comprehensive analysis of studies investigating STAT signaling responses in conventional and regulatory subsets of SLE T and B cells and possible implications of STAT inhibition for clinical therapy.

Age-Related Differences in Percentages of Regulatory and Effector T Lymphocytes and Their Subsets in Healthy Individuals and Characteristic STAT1/STAT5 Signalling Response in Helper T Lymphocytes.

The dynamic process of the development of the immune system can in itself result in age-related immune malfunctions. In this study, we analysed lymphocyte subsets in the peripheral blood of 60 healthy donors, divided into groups of children, adolescents, and adults, focusing on effector (Teff) and regulatory (Treg) T lymphocytes and STAT1/STAT5 signalling response in helper T lymphocytes (Th) in adults, using flow cytometry. Our results demonstrate a decrease in the percentage of total Tregs and an increase in the percentage of total Teffs with age and a consequential immense increase in the Teff/Treg ratio. The increase of Teffs was most apparent in Th1, Th1Th17, and Th17CD161- subsets. Significant Th lymphocyte STAT1 expression differences were observed between children and adolescents, which were associated with the decrease in activated Tregs. Higher expression of STAT1 was found in FoxP3hi than in FoxP3low Th lymphocytes, while significant IL-2 induced STAT5 phosphorylation differences were found among the subsets of Th lymphocytes in adults. Our study demonstrates age-related changes in circulating Teff and Treg, as well as significant differences in STAT5/STAT1 signalling among FoxP3+ Th lymphocytes, providing new advances in the understanding of immunosenescence.

A co-culture model of the developing small intestine offers new insight in the early immunomodulation of enterocytes and macrophages by Lactobacillus spp. through STAT1 and NF-kB p65 translocation.

The early establishment of a complete microbiome has been shown to play an integral part in the development and maintenance of an intact intestine and its immune system, although much remains unknown about the specific mechanisms of immune modulation in newborns. In our study we show in a co-culture model of the undeveloped small intestine that members of Lactobacillus spp. influence STAT1 and NF-kB p65 nuclear translocation in both intestinal epithelial cells as well as underlying macrophages. Moreover, by using imaging flow cytometry we were able to monitor each individual cell and create a framework of the percentage of cells in which translocation occurred in challenged versus control cell populations. We also observed a significant difference in baseline translocation in intestinal cells when cultured alone versus those in a co-culture model, underpinning the importance of 3D models over monolayer set-ups in epithelial in vitro research. In conclusion, our work offers new insights into the potential routes by which the commensal microbiome primes the early immune system to fight pathogens, and shows how strain-specific these mechanisms really are.

Hepatosplenic gamma-delta T-cell lymphoma in a female patient after delivery.

Hepatosplenic γδ T-cell lymphoma (HSTCL) is a very rare peripheral T-cell lymphoma characterized by extranodal infiltration of mature malignant post-thymic T-lymphocytes into sinusoids of the liver and spleen without lymphadenopathy and significant cytopenias. The aetiology of the disease is unknown. We describe the case of a female patient in whom HSTCL developed after delivery and who was previously without disease. Flow cytometry and liver puncture are essential for diagnosing HSTCL, especially in patients with unexplained pancytopenia and hepatosplenomegaly. Since phenotypic results can easily be misinterpreted as non-malignant, the examiner should have enough experience to recognize clonal changes of T-lymphocytes. Namely, in contrast to B-lymphocytes, T-lymphocytes do not have an efficient indicator of clonality and are recognized by flow cytometry based only on aberrant expression of commonly present antigens of T-cell and NK-cell subsets. At present, there is no known cure for HSTCL with a maximum survival up to 2 years.

Sperm morphological abnormalities as indicators of DNA fragmentation and fertilization in ICSI.

PURPOSE: To determine the relationship between sperm morphological abnormalities, DNA fragmentation and fertilization rate in intracytoplasmic sperm injection (ICSI). METHODS: Sperm samples from 20 ICSI cycles were analysed. Morphology was assessed according to strict criteria, and DNA fragmentation was measured by terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end labelling (TUNEL) using flow cytometry. RESULTS: A negative correlation was found between the percentage of spermatozoa with elongated heads and fertilization rate. There was a significant difference in the amount of morphological abnormalities between sperm samples with low and high degree of DNA fragmentation. The percentages of amorphous heads and overall head abnormalities were significantly higher in sperm samples with elevated degree of DNA fragmentation. No correlation was found between sperm DNA fragmentation and fertilization rate. CONCLUSIONS: Head abnormalities, especially amorphous heads, are related to elevated degree of DNA fragmentation. Elongated heads, when detected as predominant abnormal form in sperm samples, may affect fertilization in ICSI.

Dideoxy fluoro-ketopyranosyl nucleosides as potent antiviral agents: synthesis and biological evaluation of 2,3- and 3,4-dideoxy-3-fluoro-4- and -2-keto-beta-d-glucopyranosyl derivatives of N(4)-benzoyl cytosine.

The synthesis of the dideoxy fluoro ketopyranonucleoside analogues, 1-(2,3-dideoxy-3-fluoro-6-O-trityl-beta-d-glycero-hexopyranosyl-4-ulose)-N(4)-benzoyl cytosine (7a), 1-(3,4-dideoxy-3-fluoro-6-O-trityl-beta-d-glycero-hexopyranosyl-2-ulose)-N(4)-benzoyl cytosine (13a) and their detritylated analogues 8a and 14a, respectively, is described. Condensation of peracetylated 3-deoxy-3-fluoro-D-glucopyranose (1) with silylated N(4)-benzoyl cytosine, followed by selective deprotection and isopropylidenation afforded compound 2. Routine deoxygenation at position 2', followed by a deprotection-selective reprotection sequence afforded the partially tritylated dideoxy nucleoside of cytosine 6, which upon oxidation of the free hydroxyl group at the 4'-position, furnished the desired tritylated 2,3-dideoxy-3-fluoro ketonucleoside 7a in equilibrium with its hydrated form 7b. Compound 2 was the starting material for the synthesis of the dideoxy fluoro ketopyranonucleoside 13a. Similarly, several subsequent protection and deprotection steps as well as routine deoxygenation at position 4', followed by oxidation of the free hydroxyl group at the 2'-position of the partially tritylated dideoxy nucleoside 12, yielded the desired carbonyl compound 13a in equilibrium with its hydrated form 13b. Finally, trityl removal from 7a/b and 13a/b provided the unprotected 2,3-dideoxy-3-fluoro-4-keto and 3,4-dideoxy-3-fluoro-2-ketopyranonucleoside analogues 8a and 14a, in equilibrium with their gem-diol forms 8b and 14b. None of the compounds showed inhibitory activity against a wide variety of DNA and RNA viruses at subtoxic concentrations, except 7a/b that was highly efficient against rotavirus infection. Nucleoside 7a/b also exhibited cytostatic activity against cells of various cancers. BrdU-cell cycle analysis revealed that the mechanism of cytostatic activity may be related to a delay in G1/S phase and initiation of programmed cell death.