The Tonsil Lymphocyte Landscape in Pediatric Tonsil Hyperplasia and Obstructive Sleep Apnea.

Tonsil hyperplasia is the most common cause of pediatric obstructive sleep apnea (OSA). Despite the growing knowledge in tissue immunology of tonsils, the immunopathology driving tonsil hyperplasia and OSA remains unknown. Here we used multi-parametric flow cytometry to analyze the composition and phenotype of tonsillar innate lymphoid cells (ILCs), T cells, and B cells from pediatric patients with OSA, who had previous polysomnography. Unbiased clustering analysis was used to delineate and compare lymphocyte heterogeneity between two patient groups: children with small tonsils and moderate OSA (n = 6) or large tonsils and very severe OSA (n = 13). We detected disturbed ILC and B cell proportions in patients with large tonsils, characterized by an increase in the frequency of naïve CD27-CD21hi B cells and a relative reduction of ILCs. The enrichment of naïve B cells was not commensurate with elevated Ki67 expression, suggesting defective differentiation and/or migration rather than cellular proliferation to be the causative mechanism. Finally, yet importantly, we provide the flow cytometry data to be used as a resource for additional translational studies aimed at investigating the immunological mechanisms of pediatric tonsil hyperplasia and OSA.

Diagnostic and Predictive Value of Immune-Related Genes in Crohn's Disease.

Abnormal immune cell infiltration is associated with the pathogenesis of Crohn's disease (CD). This study aimed to determine the diagnostic and predictive value of immune-related genes in CD. Seven Gene Expression Omnibus datasets that analyzed the gene expression in CD tissues were downloaded. Single-sample gene set enrichment analysis (ssGSEA) was used to estimate the infiltration of the immune cells in CD tissues. Immune-related genes were screened by overlapping the immune-related genes with differentially expressed genes (DEGs). The protein-protein interaction (PPI) network was used to identify key immune-related DEGs. Diagnostic value of CD and predictive value of anti-TNFα therapy were analyzed. Immunohistochemical (IHC) assay was used to verify gene expression in CD tissues. There were significant differences among CD tissues, paired CD tissues, and normal intestinal tissues regarding the infiltration of immune cells. AQP9, CD27, and HVCN1 were identified as the key genes of the three sub-clusters in the PPI network. AQP9, CD27, and HVCN1 had mild to moderate diagnostic value in CD, and the diagnostic value of AQP9 was better than that of CD27 and HVCN1. AQP9 expression was decreased in CD after patients underwent anti-TNFα therapy, but no obvious changes were observed in non-responders. AQP9 had a moderate predictive value in patients who had undergone treatment. IHC assay confirmed that the expression of AQP9, CD27, and HVCN1 in CD tissues was higher than that in normal intestinal tissues, and AQP9, CD27 was correlated with the activity of CD. Immune-related genes, AQP9, CD27, and HVCN1 may act as auxiliary diagnostic indicators for CD, and AQP9 could serve as a promising predictive indicator in patients who underwent anti-TNF therapy.

Virus-infected peripheral blood plasmablasts in a patient with severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne viral hemorrhagic disease with a high fatality rate. It is caused by the SFTS virus and is endemic in East Asian countries such as China, South Korea, and Japan. Previous studies have shown that plasmablasts appear transiently in peripheral blood during the acute phase of SFTS, but do not specify the characteristics of these plasmablasts. In this report, we describe the features of peripheral blood plasmablasts in a patient with SFTS. Immunohistochemical and immunofluorescence staining detected a small number of atypical lymphocytes expressing the SFTS virus antigen among peripheral leukocytes in a blood sample. The phenotype of the virus-infected cells was CD27+, CD38+, MUM1+, and CD138+, which is consistent with that of plasmablasts. This novel study demonstrates that plasmablasts in the peripheral blood of patients with SFTS are targets of the SFTS virus.

In Human Autoimmunity, a Substantial Component of the B Cell Repertoire Consists of Polyclonal, Barely Mutated IgG+ve B Cells.

B cells are critical for promoting autoimmunity and the success of B cell depletion therapy in rheumatoid arthritis (RA) confirms their importance in driving chronic inflammation. Whilst disease specific autoantibodies are useful diagnostically, our understanding of the pathogenic B cell repertoire remains unclear. Defining it would lead to novel insights and curative treatments. To address this, we have undertaken the largest study to date of over 150 RA patients, utilizing next generation sequencing (NGS) to analyze up to 200,000 BCR sequences per patient. The full-length antigen-binding variable region of the heavy chain (IgGHV) of the IgG B cell receptor (BCR) were sequenced. Surprisingly, RA patients do not express particular clonal expansions of B cells at diagnosis. Rather they express a polyclonal IgG repertoire with a significant increase in BCRs that have barely mutated away from the germline sequence. This pattern remains even after commencing disease modifying therapy. These hypomutated BCRs are expressed by TNF-alpha secreting IgG+veCD27-ve B cells, that are expanded in RA peripheral blood and enriched in the rheumatoid synovium. A similar B cell repertoire is expressed by patients with Sjögren's syndrome. A rate limiting step in the initiation of autoimmunity is the activation of B cells and this data reveals that a sizeable component of the human autoimmune B cell repertoire consists of polyclonal, hypomutated IgG+ve B cells, that may play a critical role in driving chronic inflammation.

CD27 antigen negative expression indicates poor prognosis in newly diagnosed multiple myeloma.

To investigate the role of CD27 in multiple myeloma(MM), bone marrow samples from 165 newly diagnosed MM were analysed by flow cytometry. CD27- group (n = 93) had higher level of plasma cell proportion (37.00% vs 22.50%, p < .05), ß2-MG (5.42 vs 3.20 mg/L, p < .05), calcium (2.45 vs 2.28 mmol/L, p < .05),higher percentage of ISS stage III (49.46% vs 22.22%, p < .05) and patients with ≥2 high-risk cytogenetics (24.73% vs 15.28%, p < .05) than CD27+ group (n = 72). After 4 cycles of chemotherapy, the overall response rate in CD27- group were lower than CD27+ group (56.67% vs 73.02%,p < .05). After a median follow-up of 18 months, progression-free survival was significantly shorter in CD27- group than in CD27+ group (22 vs 40 months, p < .05), so was overall survival (median OS not reached, p < .05). Gene sequencing showed more adverse mutations in CD27- group than CD27+ group.

The Dual Roles of Human γδ T Cells: Anti-Tumor or Tumor-Promoting.

γδ T cells are the unique T cell subgroup with their T cell receptors composed of γ chain and δ chain. Unlike αß T cells, γδ T cells are non-MHC-restricted in recognizing tumor antigens, and therefore defined as innate immune cells. Activated γδ T cells can promote the anti-tumor function of adaptive immune cells. They are considered as a bridge between adaptive immunity and innate immunity. However, several other studies have shown that γδ T cells can also promote tumor progression by inhibiting anti-tumor response. Therefore, γδ T cells may have both anti-tumor and tumor-promoting effects. In order to clarify this contradiction, in this review, we summarized the functions of the main subsets of human γδ T cells in how they exhibit their respective anti-tumor or pro-tumor effects in cancer. Then, we reviewed recent γδ T cell-based anti-tumor immunotherapy. Finally, we summarized the existing problems and prospect of this immunotherapy.

Measurement of sCD27 in the cerebrospinal fluid identifies patients with neuroinflammatory disease.

BACKGROUND: Laboratory tests to assist in the diagnosis and monitoring of neuroinflammatory diseases are scarce. The soluble form of the CD27 molecule (sCD27) is shed in high concentrations by activated T cells and can be detected in the cerebrospinal fluid. The aim of this study was to investigate whether CSF quantitation of sCD27 could discriminate between inflammatory and non-inflammatory neurological diseases. METHODS: The concentration of sCD27 was measured using a commercially available ELISA in 803 well-defined subjects from a study cohort comprised of 338 patients with neuroinflammatory disease, 338 with non-inflammatory neurological disease and 127 controls without neurological disease. RESULTS: The median value of cerebrospinal fluid sCD27 was 64 pg/mL (IQR 0-200) in controls, 58 pg/mL (IQR 0-130) in patients with non-inflammatory disease and 740 pg/mL (IQR 230-1800) in patients with inflammatory disease. The likelihood ratio of having an inflammatory disease was 10 (sensitivity 74% and specificity 93%) if the sCD27 concentration was >250 pg/mL. In patients with a known inflammatory condition, the likelihood ratio of having an infection was 10 (sensitivity 40% and specificity 96%) if the sCD27 concentration was >2500 pg/mL. CONCLUSIONS: The likelihood of having an inflammatory neurological condition is increased with elevated concentrations of sCD27 in cerebrospinal fluid. Rapid tests of sCD27 should be developed to assist clinicians in diagnosis of neuroinflammatory disease.

Assigning Cytomegalovirus Status in Children Awaiting Organ Transplant: Viral Shedding, CMV-Specific T Cells, and CD27-CD28-CD4+ T Cells.

Passive antibodies, maternal or transfusion-acquired, make serologic determination of pretransplant cytomegalovirus (CMV) status unreliable. We evaluated 3 assays unaffected by passive antibodies, in assignment of CMV infection status in children awaiting solid organ transplant and in controls: (1) CMV nucleic acid amplification testing (NAAT), (2) quantification of CMV-specific CD4+ T cells, and (3) quantification of CD27-CD28-CD4+ T cells. Our results highlight that CMV NAAT, from urine and oropharynx, is useful in confirming positive CMV status. Detection of CMV-specific CD4+ T cells was sensitive and specific in children >18 months but was less sensitive in children <12 months. CD27-CD28-CD4+ T cells are not likely useful in CMV risk stratification in children.

Dysfunction of CD19+CD24hiCD27+ B regulatory cells in patients with bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering skin disease characterized by the production of autoantibodies against the hemidesmosomal protein BP180. B regulatory cells (Bregs) are crucial in maintaining self-tolerance and suppressing autoantibody production. However, it is still unclear whether the dysfunctions of Bregs contributes to the autoantibody production in BP patients. In this study, we found that CD19+CD24hiCD27+ Bregs and IL-10+CD19+ Bregs were significantly increased in the peripheral blood samples of BP patients compared with that in healthy controls. Moreover, compared to Bregs from healthy individuals, we found that Bregs from BP patients fails to suppress the production of specific anti-BP180 autoantibody when co-cultured with patient-derived PBMCs. Additionally, Bregs from BP patients were defective in suppressing the CD4+ T cell proliferation and the cytokines expression (including IFN-γ, TNF-α and IL-4). Notably, we found that patient-derived Bregs produced high level of TNF-α and the TNF inhibitor etanercept could inhibit the autoantibody production in the culture system in vitro. Our results indicate that Bregs from BP patient appear phenotypically pro-inflammatory by their cytokine profile and are defective in immunosuppressive function, which suggest that Bregs play a pro-inflammatory role rather than a regulatory role in the pathogenesis of BP.

iNKT and memory B-cell alterations in HHV-8 multicentric Castleman disease.

Human herpesvirus 8 (HHV-8) is the causative agent of Kaposi sarcoma (KS) and multicentric Castleman disease (MCD), a life-threatening, virally induced B-cell lymphoproliferative disorder. HHV-8 is a B-lymphotropic γ-herpesvirus closely related to the Epstein-Barr virus (EBV). Invariant natural killer T (iNKT) cells are innate-like T cells that play a role in antiviral immunity, specifically in controlling viral replication in EBV-infected B cells. Decline of iNKT cells is associated with age or HIV infection, both situations associated with HHV-8-related diseases. We analyzed iNKT cells in both blood (n = 26) and spleen (n = 9) samples from 32 patients with HHV-8 MCD and compared them with patients with KS (n = 24) and healthy donors (n = 29). We determined that both circulating and splenic iNKT cell frequencies were markedly decreased in patients with HHV-8 MCD and were undetectable in 6 of them. Moreover, iNKT cells from patients with HHV-8 MCD displayed a proliferative defect after stimulation with α-galactosylceramide. These iNKT cell alterations were associated with an imbalance in B-cell subsets, including a significant decrease in memory B cells, particularly of marginal zone (MZ) B cells. Coculture experiments revealed that the decrease in iNKT cells contributed to the alterations in the B-cell subset distribution. These observations contribute to a better understanding of the complex interactions between HHV-8 and immune cells that cause HHV-8-related MCD.

Monitoring CD27+ memory B-cells in neuromyelitis optica spectrum disorders patients treated with rituximab: Results from a bicentric study.

BACKGROUND: Rituximab (RTX) is increasingly used in the treatment of neuromyelitis optica spectrum disorder (NMO-SD). Administration regimen is not consensual as there is no reliable biomarker of RTX efficacy. In most cases, after induction, RTX is administered systematically every 6months. OBJECTIVE: To assess efficacy and safety of a maintenance regimen based on CD19+ CD27+ memory B-cell (mBc) detection. METHODS: We conducted a study in two French centers, including patients with NMO-SD who received an induction therapy with RTX. We compared the number of administered infusions, relapses and EDSS depending on two maintenance schemes (S1: administration of 1g RTX infusion every 6months or S2: a scheme based on regular mBc detection. 1g RTX was administered if mBc was >0.05%) RESULTS: 40 patients were included (mean age: 40.2years, F/M sex ratio: 5/1). Aquaporin-4 antibodies were positive in 75% patients. Under S1 regimen, all patients received 2 infusions per year, whereas under S2, they received 1.62 infusion per year. The mean interval between infusions under S2 was 7.4months, without decrease of clinical efficacy. CONCLUSION: In our study, mBc-based administration of RTX allowed personalizing treatment administration and in several cases to lower the cumulative dose without loss of efficacy.

Impaired Upregulation of the Costimulatory Molecules, CD27 and CD28, on CD4+ T Cells from HIV Patients Receiving ART Is Associated with Poor Proliferative Responses.

HIV patients beginning antiretroviral therapy (ART) with advanced immunodeficiency often retain low CD4+ T cell counts despite virological control. We examined proliferative responses and upregulation of costimulatory molecules, following anti-CD3 stimulation, in HIV patients with persistent CD4+ T cell deficiency on ART. Aviremic HIV patients with nadir CD4+ T cell counts <100 cells/µL and who had received ART for a median time of 7 (range 1-11) years were categorized into those achieving low (<350 cells/µL; n = 13) or normal (>500 cells/µL; n = 20) CD4+ T cell counts. Ten healthy controls were also recruited. CD4+ T cell proliferation (Ki67) and upregulation of costimulatory molecules (CD27 and CD28) after anti-CD3 stimulation were assessed by flow cytometry. Results were related to proportions of CD4+ T cells expressing markers of T cell senescence (CD57), activation (HLA-DR), and apoptotic potential (Fas). Expression of CD27 and/or CD28 on uncultured CD4+ T cells was similar in patients with normal CD4+ T cell counts and healthy controls, but lower in patients with low CD4+ T cell counts. Proportions of CD4+ T cells expressing CD27 and/or CD28 correlated inversely with CD4+ T cell expression of CD57, HLA-DR, and Fas. After anti-CD3 stimulation, induction of CD27hiCD28hi expression was independent of CD4+ T cell counts, but lower in HIV patients than in healthy controls. Induction of CD27hiCD28hi expression correlated with induction of Ki67 expression in total, naïve, and CD31+ naïve CD4+ T cells from patients. In HIV patients responding to ART, impaired induction of CD27 and CD28 on CD4+ T cells after stimulation with anti-CD3 is associated with poor proliferative responses as well as greater CD4+ T cell activation and immunosenescence.

Interaction between tumour-infiltrating B cells and T cells controls the progression of hepatocellular carcinoma.

OBJECTIVE: The nature of the tumour-infiltrating leucocytes (TILs) is known to impact clinical outcome in carcinomas, including hepatocellular carcinoma (HCC). However, the role of tumour-infiltrating B cells (TIBs) remains controversial. Here, we investigate the impact of TIBs and their interaction with T cells on HCC patient prognosis. DESIGN: Tissue samples were obtained from 112 patients with HCC from Singapore, Hong Kong and Zurich and analysed using immunohistochemistry and immunofluorescence. RNA expression of CD19, CD8A, IFNG was analysed using quantitative PCR. The phenotype of freshly isolated TILs was analysed using flow cytometry. A mouse model depleted of mature B cells was used for functional study. RESULTS: Tumour-infiltrating T cells and B cells were observed in close contact with each other and their densities are correlated with superior survival in patients with HCC. Furthermore, the density of TIBs was correlated with an enhanced expression of granzyme B and IFN-γ, as well as with reduced tumour viability defined by low expression of Ki-67, and an enhanced expression of activated caspase-3 on tumour cells. CD27 and CD40 costimulatory molecules and TILs expressing activation marker CD38 in the tumour were also correlated with patient survival. Mice depleted of mature B cells and transplanted with murine hepatoma cells showed reduced tumour control and decreased local T cell activation, further indicating the important role of B cells. CONCLUSIONS: The close proximity of tumour-infiltrating T cells and B cells indicates a functional interaction between them that is linked to an enhanced local immune activation and contributes to better prognosis for patients with HCC.

Endotoxemia contributes to CD27+ memory B-cell apoptosis via enhanced sensitivity to Fas ligation in patients with Cirrhosis.

Peripheral CD27+ memory B-cells become quantitatively reduced and dysfunctional in patients with cirrhosis through poorly characterized mechanisms. We hypothesized that the disappearance of CD27+ memory B-cells results from enhanced sensitivity to apoptosis caused by exposure to gut microbial translocation products. Using isolated naïve and memory B-cells from patients with cirrhosis and age-matched controls, ex vivo and activation-induced sensitivity to Fas-mediated apoptosis was assessed under relevant experimental conditions. We observed differential expression of CD95(Fas) in CD27+ B-cells from cirrhotic patients that was inversely correlated with peripheral CD27+ B-cell frequency. While memory B-cells from cirrhotic patients were resistant to Fas-mediated apoptosis ex vivo, Toll-like receptor 4(TLR4)-ligation restored Fas-sensitivity. Sensitivity to Fas-mediated apoptosis could be transferred to healthy donor memory B-cells by co-culturing these cells with plasma from cirrhotic patients, a sensitivity partially mediated by Fas and TLR4 signaling, and partially rescued via B-cell receptor crosslinking. We conclude that peripheral CD27+ memory B-cells in cirrhosis exhibit increased sensitivity to Fas-induced apoptosis in an activation-dependent manner to which endotoxin contributes, associated with reduced frequency of circulating memory B-cells. Destruction of this critical cell subset may contribute to the cirrhotic immunodeficiency state and heightened risk of systemic infections in advanced liver disease.

Bone marrow B lymphocytes in multiple myeloma and MGUS: Focus on distribution of naïve cells and memory subsets.

BACKGROUND/AIMS: Multiple myeloma (MM) is caused by proliferation of clonal plasma cells (cPCs) in bone marrow (BM), associated with numerical and functional defects in immune subsets. An impairment of B cell compartment is involved in onset/progression of the disease. METHODS: By flow cytometry, we studied distribution of naïve/transitional (IgD(+)CD27(-)), memory unswitched (IgD(+)CD27(+)), memory switched (IgD(-)CD27(+)) and double negative (DN) (IgD(-)CD27(-)) B lymphocytes in BM of control subjects, and responding and relapsing patients. RESULTS: We observed an increased percentage of IgD(+)CD27(+) B cells in healthy controls vs responding patients (p<0.05). Treated non complete responders exhibited an expanded DN compartment vs stringent complete responders (p=0.011); in turn IgD(+)CD27(-) subpopulation was larger in stringent complete responders vs other responding patients (p=0.006). None of the studied B cell subsets showed clonal restriction. Correlation analysis revealed negative correlations between naïve/transitional and DN B cells in all groups, except in newly diagnosed subjects. CONCLUSIONS: This may be considered a feasible start point to explore the importance of B cells in the immunosuppressive MM BM microenvironment, correlating these findings with immunosenescence and therapy related increased risk of infection. Moreover, we propose a possible role of naïve/transitional and DN B cells as predictive markers in treated patients.

Use of several immunological markers to model the probability of active tuberculosis.

Blood-based biomarkers tests are attractive alternative for diagnosing tuberculosis to assays depending on mycobacteria detection. Given several immunological markers we used logistic regression to model the probability of active tuberculosis in a cohort of patients with active or latent tuberculosis, showing an increased accuracy in distinguishing active from latent tuberculosis.

Altered phenotypic and functional characteristics of CD3+CD56+ NKT-like cells in human gastric cancer.

CD3+CD56+ natural killer T (NKT)-like cells are a group of CD3+ T cells sharing characteristics of NK and T cells and constitute a major component of host anti-tumor immune response in human cancer. However, the nature, function and clinical relevance of CD3+CD56+ NKT-like cells in human gastric cancer (GC) remain unclear. In this study, we showed that the frequencies of CD3+CD56+NKT-like cells in GC tumors were significantly decreased and low levels of tumor-infiltrating CD3+CD56+ NKT-like cells were positively correlated with poor survival and disease progression. Most CD3+CD56+NKT-like cells in GC tumors were CD45RA-CD27+/- central/effector-memory cells with decreased activity and lower expression levels of CD69, NKG2D and DNAM-1 than those in non-tumor tissues. We further observed that tumor-infiltrating CD3+CD56+ NKT-like cells had impaired effector function as shown by decreased IFN-γ, TNF-α, granzyme B and Ki-67 expression. Moreover, in vitro studies showed that soluble factors released from GC tumors could induce the functional impairment of CD3+CD56+ NKT-like cells. Collectively, our data indicate that decreased tumor-infiltrating CD3+CD56+ NKT-like cells with impaired effector function are associated with tumor progression and poor survival of GC patients, which may contribute to immune escape of GC.

Comparative phenotypical analysis of B cells in fresh and cryopreserved mononuclear cells from blood and tissue of rhesus macaques.

Cryopreservation of peripheral blood mononuclear cells (PBMCs) is common for large clinical trials for which phenotypical characterization of lymphocytes is retrospectively performed in specialized core laboratories. It is therefore essential to assess the comparability between fresh and frozen samples. No side-by-side comparison of B and plasma cells of rhesus macaques (RM), which serve as useful models for several human diseases has been conducted until now. Hence, we performed an extensive comparative analysis between fresh and thawed mononuclear cells (MNCs) from blood and various tissues of healthy RM to analyze for the possible effects of cryopreservation on phenotype and functionality. Our data demonstrate that -80°C cryopreservation induces profound changes compared to fresh ex vivo-derived material. Percentages of B cells were stable in PBMCs, but were increased in all organs analyzed. The expression of CD27, a marker for differentiation between naïve and memory B cells, was massively reduced in PBMCs and MNC from organs with the most severe changes observed in cells from bone marrow (BM). Additionally, similar low percentages of CD27(+) memory B cells were detected in PBMCs and BM samples stored in liquid nitrogen. Therefore, cryopreservation is not suitable for the phenotypical and functional characterization of B cells. Further optimization of cryoconservation protocols monitoring the surface expression of CD27, which was identified as a marker for the quality of cryopreserved material of RM, will be essential.

Supranormal thymic output up to 2 decades after HIV-1 infection.

OBJECTIVES: AIDS is caused by CD4 T-cell depletion. Although combination antiretroviral therapy can restore blood T-cell numbers, the clonal diversity of the reconstituting cells, critical for immunocompetence, is not well defined. METHODS: We performed an extensive analysis of parameters of thymic function in perinatally HIV-1-infected (n = 39) and control (n = 28) participants ranging from 13 to 23 years of age. CD4 T cells including naive (CD27 CD45RA) and recent thymic emigrant (RTE) (CD31/CD45RA) cells, were quantified by flow cytometry. Deep sequencing was used to examine T-cell receptor (TCR) sequence diversity in sorted RTE CD4 T cells. RESULTS: Infected participants had reduced CD4 T-cell levels with predominant depletion of the memory subset and preservation of naive cells. RTE CD4 T-cell levels were normal in most infected individuals, and enhanced thymopoiesis was indicated by higher proportions of CD4 T cells containing TCR recombination excision circles. Memory CD4 T-cell depletion was highly associated with CD8 T-cell activation in HIV-1-infected persons and plasma interlekin-7 levels were correlated with naive CD4 T cells, suggesting activation-driven loss and compensatory enhancement of thymopoiesis. Deep sequencing of CD4 T-cell receptor sequences in well compensated infected persons demonstrated supranormal diversity, providing additional evidence of enhanced thymic output. CONCLUSION: Despite up to two decades of infection, many individuals have remarkable thymic reserve to compensate for ongoing CD4 T-cell loss, although there is ongoing viral replication and immune activation despite combination antiretroviral therapy. The longer term sustainability of this physiology remains to be determined.