Identifying Changes in Peripheral Lymphocyte Subpopulations in Adult Onset Type 1 Diabetes

T- and B-lymphocytes play an important role in the pathogenesis of type 1 diabetes (T1D), a chronic disease caused by the autoimmune destruction of the insulin-producing cells in the pancreatic islets. Flow cytometry allows their characterization in peripheral blood, letting to investigate changes in cellular subpopulations that can provide insights in T1D pathophysiology. With this purpose, CD4+ and CD8+ T cells (including naïve, central memory, effector memory and terminally differentiated effector (TEMRA), Th17 and Tregs) and B cells subsets (naïve, unswitched memory, switched memory and transitional B cells) were analysed in peripheral blood of adult T1D patients at disease onset and after ≥2 years using multiparametric flow cytometry. Here we report changes in the percentage of early and late effector memory CD4+ and CD8+ T cells as well as of naïve subsets, regulatory T cells and transitional B cells in peripheral blood of adult patients at onset of T1D when compared with HD. After 2 years follow-up these changes were maintained. Also, we found a decrease in percentage of Th17 and numbers of T cells with baseline. In order to identify potential biomarkers of disease, ROC curves were performed being late EM CD4 T cell subset the most promising candidate. In conclusion, the observed changes in the percentage and/or absolute number of lymphocyte subpopulations of adult T1D patients support the hypothesis that effector cells migrate to the pancreas and this autoimmune process perseveres along the disease. Moreover, multiparametric flow allows to identify those subsets with potential to be considered biomarkers of disease.

IRF4 Has a Unique Role in Early B Cell Development and Acts Prior to CD21 Expression to Control Marginal Zone B Cell Numbers

Strict control of B lymphocyte development is required for the ability to mount humoral immune responses to diverse foreign antigens while remaining self-tolerant. In the bone marrow, B lineage cells transit through several developmental stages in which they assemble a functional B cell receptor in a stepwise manner. The immunoglobulin heavy chain gene is rearranged at the pro-B stage. At the large pre-B stage, cells with a functional heavy chain expand in response to signals from IL-7 and the pre-BCR. Cells then cease proliferation at the small pre-B stage and rearrange the immunoglobulin light chain gene. The fully formed BCR is subsequently expressed on the surface of immature B cells and autoreactive cells are culled by central tolerance mechanisms. Once in the periphery, transitional B cells develop into mature B cell subsets such as marginal zone and follicular B cells. These developmental processes are controlled by transcription factor networks, central to which are IRF4 and IRF8. These were thought to act redundantly during B cell development in the bone marrow, with their functions diverging in the periphery where IRF4 limits the number of marginal zone B cells and is required for germinal center responses and plasma cell differentiation. Because of IRF4’s unique role in mature B cells, we hypothesized that it may also have functions earlier in B cell development that cannot be compensated for by IRF8. Indeed, we find that IRF4 has a unique role in upregulating the pre-B cell marker CD25, limiting IL-7 responsiveness, and promoting migration to CXCR4 such that IRF4-deficient mice have a partial block at the pre-B cell stage. We also find that IRF4 acts in early transitional B cells to restrict marginal zone B cell development, as deletion of IRF4 in mature B cells with CD21-cre impairs plasma cell differentiation but has no effect on marginal zone B cell numbers. These studies highlight IRF4 as the dominant IRF family member in early B lymphopoiesis.

Immune Response to SARS-CoV-2 Vaccine in 2 Men

<b><i>Introduction:</i></b> In the trials of corona virus vaccines, detailed analyses of subsets of lymphocytes were not carried out. We present perhaps the most comprehensive immunological analysis of 29 subsets of B and T cells in 2 healthy subjects receiving 2 doses of the Pfizer SARS-CoV-2 (COVID-19) vaccine. <b><i>Methods:</i></b> Analyses were performed prior to vaccination, 3 weeks following the 1st dose, and 4 weeks following the 2nd dose. Total, naïve (T_N), and different memory and effector subsets (T_CM, T_EM, and T_EMRA) of CD4+ and CD8+ T cells; SARS-CoV-2 spike protein-specific tetramer+, and cytotoxic CD8+ T; subsets of T follicular cells (T_FH, T_FH1, T_FH2, T_FH1/T_FH17, and T_FH17); B-cell subsets (mature B cells, naive B cells, transitional B cells, marginal zone B cells, class-switched memory B cells, germinal center B cells, and CD21^low B cells), and plasmablasts; and regulatory lymphocytes (CD4+ Treg, CD8+ Treg, Breg, and T_FR cells) were evaluated with specific monoclonal antibodies by flow cytometry. <b><i>Results:</i></b> A lack of COVID-19 IgG antibodies after the 1st dose in one of 2 subjects was associated with increased regulatory lymphocytes and decreased plasmablasts. Seroconversion after the 2nd dose in this subject was associated with decreased T_FR cells and increased plasmablasts. In both subjects, CD4 T_EM and CD8 T_CM were markedly increased following the 2nd dose. T_FH1 and regulatory lymphocytes were increased (except Breg) following the 1st dose. A striking increase in SARS-CoV-2-specific CD8+ T cells was observed following the 2nd dose. <b><i>Conclusion:</i></b> Our data support the need for 2nd dose of vaccine to induce strong SARS-CoV-2 CD8 T-cell specific response and generation of memory subsets of CD4+ and CD8+ T cells. Regulatory lymphocytes appear to play a role in the magnitude of response.

B Lymphocyte Subsets in Children With Steroid-Sensitive Nephrotic Syndrome: A Longitudinal Study

Background: B-cell subsets may be involved in the pathogenesis of childhood steroid-sensitive nephrotic syndrome (SSNS). Horizontal control studies have shown that homeostasis of B-cell subsets changes at different stages of the SSNS. However, there is a lack of longitudinal studies that have investigated dynamic changes in B cell subpopulations.Methods: Blood samples were collected at the following time points from 15 children with SSNS treated at our hospital: before administration of steroid and after 3 days, 1 week, and 3, 6, 9, and 12 months. The proportions of circulating total B cells (CD19+), transitional B cells (CD19+CD24highCD38high), mature B cells (CD19+CD24lowCD38intermediate), and memory B cells (CD19+CD24highCD38−) were monitored by flow cytometry.Results: The proportion of CD19+ B cells before steroid administration was significantly higher than that observed at any other time point or in the healthy control (HC) group (p &lt; 0.001). However, this proportion was significantly lower than that in the HC group at 12 months (p = 0.031). Transitional B cells before (%BL 9.5 ± 4.4) and 3 days after steroid administration (%BL 10.6 ± 5.1) were significantly higher than at any other time point or in the HC group (p &lt; 0.001). Although these cells declined after the 3rd day the percentage was still significantly lower than that of the HC group at 12 months (p = 0.029). Memory B cells increased gradually after steroid administration and decreased to the normal range after 9 months.Conclusions: B cell subpopulations show dynamic changes in children with SSNS, suggesting that they are involved in the pathogenesis of the disorder. Further studies are required to determine whether this change can guide individualized treatment.

Marginating transitional B cells modulate neutrophils in the lung during inflammation and pneumonia

Pulmonary innate immunity is required for host defense; however, excessive neutrophil inflammation can cause life-threatening acute lung injury. B lymphocytes can be regulatory, yet little is known about peripheral transitional IgM+ B cells in terms of regulatory properties. Using single-cell RNA sequencing, we discovered eight IgM+ B cell subsets with unique gene regulatory networks in the lung circulation dominated by transitional type 1 B and type 2 B (T2B) cells. Lung intravital confocal microscopy revealed that T2B cells marginate in the pulmonary capillaries via CD49e and require CXCL13 and CXCR5. During lung inflammation, marginated T2B cells dampened excessive neutrophil vascular inflammation via the specialized proresolving molecule lipoxin A4 (LXA4). Exogenous CXCL13 dampened excessive neutrophilic inflammation by increasing marginated B cells, and LXA4 recapitulated neutrophil regulation in B cell–deficient mice during inflammation and fungal pneumonia. Thus, the lung microvasculature is enriched in multiple IgM+ B cell subsets with marginating capillary T2B cells that dampen neutrophil responses.

The impact of hypoxia on B cells in COVID-19

Prominent early features of COVID-19 include severe, often clinically silent, hypoxia and a pronounced reduction in B cells, the latter important in defence against SARS-CoV-2. This brought to mind the phenotype of mice with VHL-deficient B cells, in which Hypoxia-Inducible Factors are constitutively active, suggesting hypoxia might drive B cell abnormalities in COVID-19. We demonstrated the breadth of early and persistent defects in B cell subsets in moderate/severe COVID-19, including reduced marginal zone-like, memory and transitional B cells, changes we also observed in B cell VHL-deficient mice. This was corroborated by hypoxia-related transcriptional changes in COVID-19 patients, and by similar B cell abnormalities in mice kept in hypoxic conditions, including reduced marginal zone and germinal center B cells. Thus hypoxia might contribute to B cell pathology in COVID-19, and in other hypoxic states. Through this mechanism it may impact on COVID-19 outcome, and be remediable through early oxygen therapy.

Immune Cells Profiling in ANCA-Associated Vasculitis Patients—Relation to Disease Activity

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are a group of necrotizing multiorgan autoimmune vasculitides that predominantly affect small blood vessels and are associated with the presence of ANCAs. The aim was to assess regulatory and effector cell populations accompanied by the suPAR biomarker level and link the so-defined immune state to the AAV disease activity. The research involved a multicomponent description of an immune state encompassing a range of B and T cell subsets such as transitional/regulatory B cells (CD19+CD24++CD38++), naïve B cells (CD19+CD24INTCD38INT), Th17 cells, T regulatory cells (CD4+CD25+FoxP3+) and cytotoxic CD4+CD28− cells by flow cytometry. The suPAR plasma level was measured by ELISA. The results indicate that AAV is associated with an increased suPAR plasma level and immune fingerprint characterized by an expansion of Th17 cells and T cells lacking the costimulatory molecule CD28, accompanied by a decrease of regulatory populations (Tregs and transitional B cells) and NK cells. Decreased numbers of regulatory T cells and transitional B cells were shown to be linked to activation of the AAV disease while the increased suPAR plasma level—to AAV-related deterioration of kidney function. The observed immune fingerprint might be a reflection of peripheral tolerance failure responsible for development and progression of ANCA-associated vasculitides.

Immunological features that determine the strength of antibody responses to BNT162b2 mRNA vaccine against SARS-CoV-2

We analyzed peripheral blood mononuclear cells (PBMCs) of each 20 individuals with a high anti-SARS-CoV-2 antibody titer and a low antibody titer out of 1,774 healthcare workers who received BNT162b2 mRNA vaccine. A higher antibody titer was associated with the frequencies of naive and transitional B cells before vaccination. In addition, fold changes in the frequency of activated CD8+ T cells upon vaccination were correlated with the antibody titers.

CD27-CD38lowCD21low B-Cells Are Increased in Axial Spondyloarthritis

B-cells have received little attention in axial spondyloarthritis (axSpA) and for this reason their role in pathogenesis remains unclear. However, there are indications that B-cells may be involved in the disease process. Our objective was to obtain insights into the composition of the peripheral B-cell compartment of axSpA patients compared to healthy donors (HD) and patients with primary Sjögren’s syndrome (pSS), a typical B-cell-associated autoimmune disease. Special emphasis was given to CD27-negative B-cells expressing low levels of CD21 (CD21low B-cells), since this subset is implicated in autoimmune diseases with strong involvement of B-cells. Transitional B-cells (CD38hi) were excluded from the analysis of the CD27-CD21low B-cell compartment. This study included 45 axSpA patients, 20 pSS patients and 30 HDs. Intriguingly, compared to HDs the frequency of CD27-CD38lowCD21low B-cells was significantly elevated in both axSpA and pSS patients (P&lt;0.0001 for both comparisons). The frequency of CD27-CD38lowCD21low B-cells expressing the activation-induced immune markers T-bet and CD11c was decreased in axSpA patients compared to HDs. A higher proportion of CD27-CD38lowCD21low B-cells expressed the chemokine receptor CXCR3 in axSpA compared to HDs, suggestive for active involvement of these cells in an inflammatory process. The frequency of CD27-CD38lowCD21low B-cells in axSpA patients correlated positively with age and erythrocyte sedimentation rate. Furthermore, axSpA patients with extra-skeletal manifestations (ESM) showed increased frequencies of CD27-CD38lowCD21low B-cells compared to patients without ESM. In conclusion, our findings are suggestive of active B-cell involvement in the pathogenesis of axSpA, against prevailing dogma.