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Introduction: Progressive Multifocal Leukoencephalopathy (PML) is a rare and deadly demyelinating disease caused by JC virus (JCV) replication in the central nervous system. PML occurs exclusively in patients with severe underlying immune deficiencies, including AIDS and hematological malignancies. PML has also emerged as a significant threat to patients on potent new immunosuppressive biologics, including natalizumab in multiple sclerosis. Methods: Here, we developed an IFN-γ release assay (IGRA) that mainly detects JCV-specific effector memory T cells and effectors T cells in the blood. Results: This assay was frequently positive in patients with active PML (with a positive JCV PCR in CSF) of various underlying immunosuppression causes (84% sensitivity). Only 3% of healthy donors had a positive response (97% specificity). The frequency of positivity also increased in multiple sclerosis patients according to the time on natalizumab (up to 36% in patients treated for more than 48 months, who are considered at a higher risk of PML). Discussion: The results show this assay's frequent or increased positivity in patients with PML or an increased risk of PML, respectively. The assay may help to stratify the risk of PML.

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The cross-talk between the innate and adaptive immune response represents the first defense weapon against the threat of pathogens. Substantial evidence has shown a relationship between immune phenotype lymphocytes and COVID-19 disease severity and/or implication in susceptibility to SARS-CoV-2 infection. Recently, belonging to ABO blood groups has been investigated as a correlation factor to COVID-19 disease. This pilot study investigated lymphocyte typing in a cohort of blood donors to understand the underlying mechanism in SARS-CoV-2 infection linked to the blood group. The study cohort consisted of 20-64-year-old subjects, without comorbidities, from both sexes, who were COVID-19 vaccinated with previous or no infection history. Whole blood samples, collected at A.O.R.N. Sant'Anna and San Sebastiano Hospital (Campania Region), were processed by multiparametric cytofluorimetric assay, to characterize CD4+ helper and CD8+ cytotoxic T cell CD3+ subpopulations. The CD45RA, CCR7, CD27, CD28, CD57 and PD-1 markers were investigated to delineate the peripheral T-cell maturation stages. Differences were detected in ABO blood types in CD3+, CD4+ gated on CD3+, CD8+ and CD8+ gated on CD3+ percentage. These results contribute to identifying a memory cell "identikit" profile in COVID-19 disease, thus leading to a useful tool in precision medicine.

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INTRODUCTION: Memory CD8+ T cells are essential for long-term immune protection in viral infections, including COVID-19. METHODS: This study examined the responses of CD8+ TEM, TEMRA, and TCM subsets from unvaccinated individuals who had recovered from mild and severe COVID-19 by flow cytometry. RESULTS AND DISCUSSION: The peptides triggered a higher frequency of CD8+ TCM cells in the recovered mild group. CD8+ TCM and TEM cells showed heterogeneity in CD137 expression between evaluated groups. In addition, a predominance of CD137 expression in naïve CD8+ T cells, TCM, and TEM was observed in the mild recovered group when stimulated with peptides. Furthermore, CD8+ TCM and TEM cell subsets from mild recovered volunteers had higher TNF-α expression. In contrast, the expression partner of IFN-γ, IL-10, and IL-17 indicated an antiviral signature by CD8+ TEMRA cells. These findings underscore the distinct functional capabilities of each memory T cell subset in individuals who have recovered from COVID-19 upon re-exposure to SARS-CoV-2 antigens.

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INTRODUCTION: Immune factors are important antecedents in the pathophysiology of necrotizing enterocolitis (NEC). However, studies on the peripheral blood lymphocyte subsets changes in NEC patients among different Bell stages and in patients requiring surgery are scarce. METHODS: 34 infants with NEC and 33 age-matched controls were included. Peripheral blood was collected within 48 h after NEC diagnosis. Peripheral blood B and T lymphocytes subsets were detected by 12-color flow cytometry. Cell ratios were calculated, and their relationship to disease severity and their roles as indicators for surgery were assessed. RESULTS: NEC patients showed elevated percentages of unSwB cells (CD27+IgD+ unswitched memory/activated B cells)/B cells, SwB cells (CD27+IgD-switched memory B cells)/B cells, CD8+ T (CD3+CD8+ T cells)/T cells, Tem (CD45RA-CCR7-effector memory T cells)/CD4+ T cells, Tem/CD8+ T cells and decreased Bn (CD27-IgD+ naïve B cells)/B cells, CD4+T (CD3+CD4+ T cells)/T cells, CD45RA+ CCR7+ naïve T cells (CD45RA+CCR7+ naïve T cells)/CD8+T cells. Compared to NEC patients at BELL stage I + II, patients at BELL stage III showed increased percentages of SwB cells/B cells, antibody secreting cell (ASC, CD3-CD20-CD27high CD38high ASCs)/B cells and Tem/CD4+ T cells, and decreased percentages of CD45RA+CCR7+ naïve T cells/CD4+ T cells. The Receiver Operating Characteristic Curve analysis showed that the sensitivity of ASC/B cells ratio (0.52%) is 86.67% and the specificity of Tem/CD4+T ratio (5.22%) is 100%, indicating that NEC patients required surgery. CONCLUSIONS: The severity of NEC exhibits codirectional changes with the maturation of B and T lymphocytes, especially CD4+ T cells. The increased ASC/B and Tem/CD4+ T cells could serve as potential indicators for NEC patients requiring surgery.

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BACKGROUND: The coronavirus pandemic has become the most critical global health threat of this century and the greatest challenge to the human population. The search for simple and quick diagnostic methods enabling the identification of patients infected with the SARS-CoV-2 virus may be a valuable method to track infection. OBJECTIVES: The aim of the study was the clinical and immunological characterization of patients by assessing the degrees of maturity of T lymphocytes from the 1st and 5th waves of coronavirus disease 2019 (COVID-19) in comparison to a healthy control group (HC). MATERIAL AND METHODS: We determined leukocyte and T lymphocyte subpopulations (recent thymic emigrant (RTE), naïve, effector, central memory and effector memory) in patients from the 1st COVID-19 wave (n = 23), the 5th COVID-19 wave (n = 38) and HC (n=20) using a panel of monoclonal antibodies using multiparameter flow cytometry. RESULTS: We observed a lower median proportion of lymphocytes and NK cells, and elevated percentage and number of neutrophils in patients from the 5th wave compared to the 1st. We found a reduced percentage of CD4+ effector memory cells in the 1st wave group compared to the 5th wave (14.1 vs 23.2, p < 0.05), and a higher percentage of RTE and naïve CD8+ cells in the 1st wave compared to the 5th wave (p < 0.05). The effector memory CD8+ cells were highest in the 5th wave compared to both 1st wave and HC patients (respectively, 35.1 vs 18.1 vs 19.3%, p < 0.05). The 5th wave group showed significantly more differences compared to HC. CONCLUSIONS: Our results showed a clear increase of effector cells with a simultaneous decrease in virgin T cells in the 5th COVID-19 infection. Monitoring lymphocyte subsets during infection allows assessment of the patient's immune status and of readiness of lymphocytes to respond to the immune response, and may be necessary to improve clinical outcomes.

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Cytomegalovirus (CMV) infection is the most critical infectious complication in recipients of hematopoietic cell transplantation (HCT) in the period between a therapeutic hematoablative treatment and the hematopoietic reconstitution of the immune system. Clinical investigation as well as the mouse model of experimental HCT have consistently shown that timely reconstitution of antiviral CD8 T cells is critical for preventing CMV disease in HCT recipients. Reconstitution of cells of the T-cell lineage generates naïve CD8 T cells with random specificities among which CMV-specific cells need to be primed by presentation of viral antigen for antigen-specific clonal expansion and generation of protective antiviral effector CD8 T cells. For CD8 T-cell priming two pathways are discussed: "direct antigen presentation" by infected professional antigen-presenting cells (pAPCs) and "antigen cross-presentation" by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Current view in CMV immunology favors the cross-priming hypothesis with the argument that viral immune evasion proteins, known to interfere with the MHC class-I pathway of direct antigen presentation by infected cells, would inhibit the CD8 T-cell response. While the mode of antigen presentation in the mouse model of CMV infection has been studied in the immunocompetent host under genetic or experimental conditions excluding either pathway of antigen presentation, we are not aware of any study addressing the medically relevant question of how newly generated naïve CD8 T cells become primed in the phase of lympho-hematopoietic reconstitution after HCT. Here we used the well-established mouse model of experimental HCT and infection with murine CMV (mCMV) and pursued the recently described approach of up- or down-modulating direct antigen presentation by using recombinant viruses lacking or overexpressing the central immune evasion protein m152 of mCMV, respectively. Our data reveal that the magnitude of the CD8 T-cell response directly reflects the level of direct antigen presentation.

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3' untranslated regions (3' UTRs) are critical elements of messenger RNAs, as they contain binding sites for RNA-binding proteins (RBPs) and microRNAs that affect various aspects of the RNA life cycle including transcript stability and cellular localization. In response to T cell receptor activation, T cells undergo massive expansion during the effector phase of the immune response and dynamically modify their 3' UTRs. Whether this serves to directly regulate the abundance of specific mRNAs or is a secondary effect of proliferation remains unclear. To study 3'-UTR dynamics in T helper cells, we investigated division-dependent alternative polyadenylation (APA). In addition, we generated 3' end UTR sequencing data from naive, activated, memory, and regulatory CD4+ T cells. 3'-UTR length changes were estimated using a nonnegative matrix factorization approach and were compared with those inferred from long-read PacBio sequencing. We found that APA events were transient and reverted after effector phase expansion. Using an orthogonal bulk RNA-seq data set, we did not find evidence of APA association with differential gene expression or transcript usage, indicating that APA has only a marginal effect on transcript abundance. 3'-UTR sequence analysis revealed conserved binding sites for T cell-relevant microRNAs and RBPs in the alternative 3' UTRs. These results indicate that poly(A) site usage could play an important role in the control of cell fate decisions and homeostasis.

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BACKGROUND: Memory T cells develop during an initial hypertensive episode, sensitizing mice to develop hypertension from further mild hypertensive challenges. We hypothesized that memory γδ T cells develop after a hypertensive challenge and sensitize mice to develop hypertension in response to a subsequent mild hypertensive challenge. METHODS: The first aim was to profile memory γδ T cells after a 14-day pressor dose angiotensin II (AngII) infusion (490 ng/kg/min, subcutaneously) in male mice. The second aim was to deplete γδ T cells during a second 14-day subpressor dose AngII challenge (140 ng/kg/min, subcutaneously) in mice pre-exposed to an initial pressor dose AngII challenge. The third aim was to transfer 2.5 × 105 live pre-activated or not γδ T cells from mice that had received a 14-day pressor dose AngII infusion or sham treatment, to naive recipient mice stimulated with a subpressor dose AngII infusion. RESULTS: Effector memory γδ T cells increased 5.2-fold in mesenteric vessels and perivascular adipose tissue, and 1.8-fold in mesenteric lymph nodes in pressor dose AngII-infused mice compared with sham-treated mice. Mice depleted of γδ T cells had 14 mm Hg lower systolic blood pressure (SBP) elevation than control mice from day 7 to 14 of subpressor dose AngII infusion. Adoptive transfer of γδ T cells from hypertensive mice induced an 18 mm Hg higher SBP elevation compared with a subpressor dose AngII infusion vs. γδ T cells transferred from sham-treated mice. CONCLUSIONS: Memory γδ T cells develop in response to hypertensive stimuli, and contribute to the pathogenesis of hypertension.

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Human ageing is accompanied by poor responses to infection and decreased vaccine efficacy. While the causes of this can be attributed to defects in the immune system that increase with age, it is unknown whether mitochondrial dysfunction may also contribute to these phenomena. This study aims to assess mitochondrial dysfunction in CD4+ terminal effector memory T cells re-expressing CD45RA (TEMRA) cells and other CD4+ memory T cell subtypes, which are increased in number in the elderly population, with respect to how their metabolic responses to stimulation are altered compared to CD4+ naïve T cells. In this study, we show that CD4+ TEMRA cells exhibit altered mitochondrial dynamics compared to CD4+ naïve cells and CD4+ central and effector memory cells, with a 25% reduction in OPA1 expression. CD4+ TEMRA and memory cells show increased upregulation of Glucose transporter 1 following stimulation and higher levels of mitochondrial mass compared to CD4+ naïve T cells. Additionally, TEMRA cells exhibit a decrease in mitochondrial membrane potential compared to other CD4+ memory cell subsets by up to 50%. By comparing young to aged individuals, more significant mitochondria mass and lower membrane potential were observed in CD4+ TEMRA of young individuals. In conclusion, we suggest that CD4+ TEMRA cells may be impaired with respect to their metabolic response to stimulation, possibly contributing to impaired responses to infection and vaccination.

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BACKGROUND: Typical symptoms of uncomplicated dengue fever (DF) include headache, muscle pains, rash, cough, and vomiting. A proportion of cases progress to severe dengue hemorrhagic fever (DHF), associated with increased vascular permeability, thrombocytopenia, and hemorrhages. Progression to severe dengue is difficult to diagnose at the onset of fever, which complicates patient triage, posing a socio-economic burden on health systems. METHODS: To identify parameters associated with protection and susceptibility to DHF, we pursued a systems immunology approach integrating plasma chemokine profiling, high-dimensional mass cytometry and peripheral blood mononuclear cell (PBMC) transcriptomic analysis at the onset of fever in a prospective study conducted in Indonesia. RESULTS: After a secondary infection, progression to uncomplicated dengue featured transcriptional profiles associated with increased cell proliferation and metabolism, and an expansion of ICOS+CD4+ and CD8+ effector memory T cells. These responses were virtually absent in cases progressing to severe DHF, that instead mounted an innate-like response, characterised by inflammatory transcriptional profiles, high circulating levels of inflammatory chemokines and with high frequencies of CD4low non-classical monocytes predicting increased odds of severe disease. CONCLUSIONS: Our results suggests that effector memory T cell activation might play an important role ameliorating severe disease symptoms during a secondary dengue infection, and in the absence of that response, a strong innate inflammatory response is required to control viral replication. Our research also identified discrete cell populations predicting increased odds of severe disease, with potential diagnostic value.

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Introduction: The adaptive immune response mediated by T cells plays a vital role in the initiation and maintenance of blood pressure (BP) elevation. Memory T cells, which are antigen-specific T cells, can respond specifically to repeated hypertensive stimuli. Although the roles of memory T cells in animal models are well studied, their maintenance and functions in hypertensive patients are poorly understood. Method: Here, we focused on the circulating memory T cells of hypertensive patients. By using single-cell RNA sequencing technology, subsets of memory T cells were identified. Differentially expressed genes (DEGs) and functional pathways were explored for related biological functions in each population of memory T cells. Result and Discussion: Our study identified four subsets of memory T cells in the blood of hypertensive patients, with CD8 effector memory T (TEM) cells accounting for more cells and demonstrating more biological functions than CD4 TEM cells. CD8 TEM cells were further analyzed using single-cell RNA sequencing technology, and subpopulation 1 was demonstrated to contribute to BP elevation. The key marker genes CKS2, PLIN2, and CNBP were identified and validated by mass-spectrum flow cytometry. Our data suggest that CD8 TEM cells as well as the marker genes could be preventive targets for patients with hypertensive cardiovascular disease.

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This multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional (γδ-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naïve (TNaïve ), central memory (TCM ), effector memory (TEM ) and terminal effector (TTE ) T cells. Activated cattle T cells (TAV ) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-color, 10-parameter flow cytometry panel simultaneously identifies cattle TNaïve , TAV , TCM , TEM , TTE and γδ-TCR cells. This panel will improve our ability to examine T-cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimized for other bovid species as many of these reagents are likely to cross react.

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BACKGROUND: A Wilms' tumor 1 (WT1) oral vaccine, Bifidobacterium longum (B. longum) 420, in which the bacterium is used as a vector for WT1 protein, triggers immune responses through cellular immunity consisting of cytotoxic T lymphocytes (CTLs) and other immunocompetent cells (e.g., helper T cells). We developed a novel, oral, helper epitope-containing WT1 protein vaccine (B. longum 2656) to examine whether or not B. longum 420/2656 combination further accelerates the CD4+ T cell help-enhanced antitumor activity in a model of murine leukemia. METHODS: C1498-murine WT1-a genetically-engineered, murine leukemia cell line to express murine WT1-was used as tumor cell. Female C57BL/6 J mice were allocated to the B. longum 420, 2656, and 420/2656 combination groups. The day of subcutaneous inoculation of tumor cells was considered as day 0, and successful engraftment was verified on day 7. The oral administration of the vaccine by gavage was initiated on day 8. Tumor volume, the frequency and phenotypes of WT1-specific CTLs in CD8+ T cells in peripheral blood (PB) and tumor-infiltrating lymphocytes (TILs), as well as the proportion of interferon-gamma (INF-γ)-producing CD3+CD4+ T cells pulsed with WT135-52 peptide in splenocytes and TILs were determined. RESULTS: Tumor volume was significantly smaller (p < 0.01) in the B. longum 420/2656 combination group than in the B. longum 420 group on day 24. WT1-specific CTL frequency in CD8+ T cells in PB was significantly greater in the B. longum 420/2656 combination group than in the B. longum 420 group at weeks 4 (p < 0.05) and 6 (p < 0.01). The proportion of WT1-specific, effector memory CTLs in PB increased significantly in the B. longum 420/2656 combination group than in the B. longum 420 group at weeks 4 and 6 (p < 0.05 each). WT1-specific CTL frequency in intratumoral CD8+ T cells and the proportion of IFN-γ-producing CD3+CD4+ T cells in intratumoral CD4+ T cells increased significantly (p < 0.05 each) in the B. longum 420/2656 combination group than in the 420 group. CONCLUSIONS: B. longum 420/2656 combination further accelerated antitumor activity that relies on WT1-specific CTLs in the tumor compared with B. longum 420.

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Tuberculosis caused by Mycobacterium tuberculosis (M.tb) is one of the main causes of human death in the world. Bacillus Calmette-Guérin (BCG) provides limited protection in adolescents and adults. To explore the factors reducing efficacy of BCG vaccine, we assess the impacts of interleukin (IL)-10 and alarmins S100A8/A9 on T-cell memory. We found that BCG-induced IL-10 inhibited production of S100A8/A9 in human peripheral blood mononuclear cells (PBMCs) and murine splenocytes. S100A9 deficiency inhibited IFN-γ production by CD4+ T cells in the early phase of BCG immunization and hindered the development of effector memory T helper type 1 (Th1) cells, while IL-10 deficiency promoted Th1 memory and blocking IL-10 signaling enhanced Th1 protective recall response against M.tb. IL-10 inhibited the binding of transcription factor CCAAT enhancer binding protein beta to S100a8/a9 promoter leading to S100A8/A9 reduction. S100A8/A9 heterodimer enhanced the IFN-γ production via receptor for advanced glycation end products signaling in CD4+ T cells. Our results demonstrate a hurdle to development of Th1 memory after BCG immunization and clarify the mechanism of the regulation of Th1 memory by IL-10 and S100A8/A9.

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Introduction: Tobacco smoking generates airway inflammation in chronic obstructive pulmonary disease (COPD), and its involvement in the development of lung cancer is still among the leading causes of early death. Therefore, we aimed to have a better understanding of the disbalance in immunoregulation in chronic inflammatory conditions in smoker subjects with stable COPD (stCOPD), exacerbating COPD (exCOPD), or non-small cell lung cancer (NSCLC). Methods: Smoker controls without chronic illness were recruited as controls. Through extensive mapping of single cells, surface receptor quantification was achieved by single-cell mass cytometry (CyTOF) with 29 antibodies. The CyTOF characterized 14 main immune subsets such as CD4+, CD8+, CD4+/CD8+, CD4-/CD8-, and γ/δ T cells and other subsets such as CD4+ or CD8+ NKT cells, NK cells, B cells, plasmablasts, monocytes, CD11cdim, mDCs, and pDCs. The CD4+ central memory (CM) T cells (CD4+/CD45RA-/CD45RO+/CD197+) and CD4+ effector memory (EM) T cells (CD4+/CD45RA-/CD45RO+/CD197-) were FACS-sorted for RNA-Seq analysis. Plasma samples were assayed by Luminex MAGPIX® for the quantitative measurement of 17 soluble immuno-oncology mediators (BTLA, CD28, CD80, CD27, CD40, CD86, CTLA-4, GITR, GITRL, HVEM, ICOS, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, TLR-2) in the four studied groups. Results: Our focus was on T-cell-dependent differences in COPD and NSCLC, where peripheral CD4+ central memory and CD4+ effector memory cells showed a significant reduction in exCOPD and CD4+ CM showed elevation in NSCLC. The transcriptome analysis delineated a perfect correlation of differentially expressed genes between exacerbating COPD and NSCLC-derived peripheral CD4+ CM or CD4+ EM cells. The measurement of 17 immuno-oncology soluble mediators revealed a disease-associated phenotype in the peripheral blood of stCOPD, exCOPD, and NSCLC patients. Discussion: The applied single-cell mass cytometry, the whole transcriptome profiling of peripheral CD4+ memory cells, and the quantification of 17 plasma mediators provided complex data that may contribute to the understanding of the disbalance in immune homeostasis generated or sustained by tobacco smoking in COPD and NSCLC.

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Background: Human cytomegalovirus (HCMV) can reactivate in the mammary gland during lactation and is shed into breast milk of nearly every HCMV-IgG-seropositive mother of a preterm infant. Dynamics of breast milk leukocytes during lactation, as well as blood leukocytes and the comparison between both in the context of HCMV reactivation is not well understood. Methods: Here, we present the BlooMil study that aimed at comparing changes of immune cells in blood and breast milk from HCMV-seropositive- vs -seronegative mothers, collected at four time ranges up to two months post-partum. Viral load was monitored by qPCR and nested PCR. Multiparameter flow cytometry was used to identify leukocyte subsets. Results: CD3+ T cell frequencies were found to increase rapidly in HCMV-seropositive mothers' milk, while they remained unchanged in matched blood samples, and in both blood and breast milk of HCMV-seronegatives. The activation marker HLA-DR was more strongly expressed on CD4+ and CD8+ T cells in all breast milk samples than matched blood samples, but HCMV-seropositive mothers displayed a significant increase of HLA-DR+ CD4+ and HLA-DR+ CD8+ T cells during lactation. The CD4+/CD8+ T cell ratio was lower in breast milk of HCMV-seropositive mothers than in the blood. HCMV-specific CD8+ T cell frequencies (recognizing pp65 or IE1) were elevated in breast milk relative to blood, which might be due to clonal expansion of these cells during local HCMV reactivation. Breast milk contained very low frequencies of naïve T cells with no significant differences depending on serostatus. Conclusion: Taken together, we conclude that the distribution of breast milk leukocyte populations is different from blood leukocytes and may contribute to the decrease of breast milk viral load in the late phase of HCMV reactivation in the mammary gland.

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BACKGROUND: SARS-CoV-2 pre-existing T-cell immune reactivity can be present in some people. A general perturbation of the main peripheral lymphocyte subsets has been described in severe COVID-19 patients, but very few studies assessed the general memory T-cell homeostasis in the acute phase of COVID-19. Here, we performed a general analysis of the main memory T cell populations in the peripheral blood of patients admitted to the hospital for a confirmed or probable COVID-19 diagnosis. METHODS: In this cross-sectional study, adult patients (aged ≥ 18 years) needing hospital admission for respiratory disease due to confirmed or probable COVID-19, were recruited before starting the therapeutic protocol for this disease. In addition to the assessment of the general lymphocyte subpopulations in the early phase of COVID-19, central memory T cells (Tmcentr cells: CD45RO+CCR7+) and effector memory T cells (Tmeff cells: CD45RO+CCR7-) were assessed by multi-color flow cytometry, in comparison to a control group. RESULTS: During the study period, 148 study participants were recruited. Among them, 58 patients turned out positive for SARS-CoV-2 PCR (including both patients with interstitial pneumonia [PCR+Pn+] and without this complication [PCR+Pn-]), whereas the remaining 90 patients resulted to be SARS-CoV-2 PCR negative, even though all were affected with interstitial pneumonia [PCR-Pn+]. Additionally, 28 control patients without any ongoing respiratory disease were recruited. A clear unbalance in the T memory compartment emerged from this analysis on the whole pool of T cells (CD3+ cells), showing a significant increase in Tmcentr cells and, conversely, a significant decrease in Tmeff cells in both pneumonia groups (PCR+Pn+ and PCR-Pn+) compared to the controls; PCR+Pn- group showed trends comprised between patients with pneumonia (from one side) and the control group (from the other side). This perturbation inside the memory T cell compartment was also observed in the individual analysis of the four main T cell subpopulations, based upon the differential expression of CD4 and/or CD8 markers. CONCLUSION: Overall, we observed both absolute and relative increases of Tmcentr cells and decrease of Tmeff cells in patients affected with interstitial pneumonia (regardless of the positive or negative results of SARS-CoV-2 PCR), compared to controls. These results need confirmation from additional research, in order to consider this finding as a potential biological marker of interstitial lung involvement in patients affected with viral respiratory infections.

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Upon antigen recognition by the T cell receptor (TCR), a complex signaling network orchestrated by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs) regulates the transmission of the extracellular signal to the nucleus. The role of the PTPs Src-homology 2 (SH2) domain-containing phosphatase 1 (SHP1, Ptpn6) and Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2, Ptpn11) have been studied in various cell types including T cells. Whereas SHP1 acts as an essential negative regulator of the proximal steps in T cell signalling, the role of SHP2 in T cell activation is still a matter of debate. Here, we analyzed the role of the constitutively active SHP2-D61Y-mutant in T cell activation using knock-in mice expressing the mutant form Ptpn11D61Y in T cells. We observed reduced numbers of CD8+ and increased numbers of CD4+ T cells in the bone marrow and spleen of young and aged SHP2-D61Y-mutant mice as well as in Influenza A Virus (IAV)-infected mice compared to controls. In addition, we found elevated frequencies of effector memory CD8+ T cells and an upregulation of the programmed cell death protein 1 (PD-1)-receptor on both CD4+ and CD8+ T cells. Functional analysis of SHP2-D61Y-mutated T cells revealed an induction of late apoptosis/necrosis, a reduced proliferation and altered signaling upon TCR stimulation. However, the ability of D61Y-mutant mice to clear viral infection was not affected. In conclusion, our data indicate an important regulatory role of SHP2 in T cell function, where the effect is determined by the kinetics of SHP2 phosphatase activity and differs in the presence of the permanently active and the temporally regulated phosphatase. Due to interaction of SHP2 with the PD-1-receptor targeting the protein-tyrosine phosphatase might be a valuable tool to enhance T cell activities in immunotherapy.

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Lymphocytes play a leading role in regulation of the immune system in lung cancer patients. The recognition of T cells profile may help in prediction of effectiveness of anticancer immunotherapy. The aim of the study was to determine the dominant subpopulation of CD4+ and CD8+ lymphocytes in metastatic and non-metastatic lymph nodes (LNs) of lung cancer patients. LNs aspirates were obtained during EBUS/TBNA procedure and cells were analyzed by flow cytometry. We showed a higher percentage of CD4+ and CD8+ effector memory T cells in the metastatic than in the non-metastatic LNs (28.6 vs. 15.3% and 28.6 vs. 14.0%, p< 0.05). The proportion of CD45RO+ T regulatory cells (CD45RO+ Tregs) was higher in the metastatic LNs than in the non-metastatic ones (65.6 vs. 31%, p< 0.05). We reported the significant differences in T cell subsets depending on the lung cancer metastatic process. We observed that the effector memory T cells were predominant subpopulations in metastatic LNs. Lymphocyte profile in LNs is easy to evaluate by flow cytometry of EBUS/TBNA samples and may reflect the immune status in lung cancer.

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