Immune cell phenotype and functional defects in Netherton syndrome

Background

Netherton syndrome (NS) is a rare life-threatening syndrome caused by SPINK5 mutations leading to a skin barrier defect and a severe atopic diathesis. NS patients are prone to bacterial infections, but the understanding of the underlying immune deficiency is incomplete.

Results

We analyzed blood lymphocyte phenotypes and function in relation to clinical infections in 11 Finnish NS patients, aged 3 to 17?years, and healthy age-matched controls. The proportion of B cells (CD19⁺) and naïve B cells (CD27^?, IgD⁺) were high while memory B cells (CD27⁺) and switched memory B cells (CD27⁺IgM^?IgD^?), crucial for the secondary response to pathogens, was below or in the lowest quartile of the reference values in 8/11 (73%) and 9/11 (82%) patients, respectively. The proportion of activated non-differentiated B cells (CD21^low, CD38l^ow) was below or in the lowest quartile of the reference values in 10/11 (91%) patients. Despite normal T cell counts, the proportion of naïve CD4⁺ T cells was reduced significantly and the proportion of CD8⁺ T central memory significantly elevated. An increased proportion of CD57⁺ CD8⁺ T cells indicated increased differentiation potential of the T cells. The proportion of cytotoxic NK cells was elevated in NS patients in phenotypic analysis based on CD56DIM, CD16⁺ and CD27^? NK cells but in functional analysis, decreased expression of CD107a/b indicated impaired cytotoxicity.The T and NK cell phenotype seen in NS patients also significantly differed from that of age-matched atopic dermatitis (AD) patients, indicating a distinctive profile in NS. The frequency of skin infections correlated with the proportion of CD62L⁺ T cells, naïve CD4⁺ and CD27⁺ CD8⁺ T cells and with activated B cells. Clinically beneficial intravenous immunoglobulin therapy (IVIG) increased naïve T cells and terminal differentiated effector memory CD8⁺ cells and decreased the proportion of activated B cells and plasmablasts in three patients studied.

Conclusions

This study shows novel quantitative and functional aberrations in several lymphocyte subpopulations, which correlate with the frequency of infections in patients with Netherton syndrome. IVIG therapy normalized some dysbalancies and was clinically beneficial.

     

Dynamics of Memory B-Cell Populations in Blood,Lymph Nodes,and Bone Marrow during Antiretroviral Therapy and Envelope Boosting in Simian Immunodeficiency Virus SIVmac251-Infected Rhesus Macaques

Human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection causes B-cell dysregulation and the loss of memory B cells in peripheral blood mononuclear cells (PBMC). These effects are not completely reversed by antiretroviral treatment (ART). To further elucidate B-cell changes during chronic SIV infection and treatment, we investigated memory B-cell subpopulations and plasma cells/plasmablasts (PC/PB) in blood, bone marrow, and lymph nodes of rhesus macaques during ART and upon release from ART. Macaques previously immunized with SIV recombinants and the gp120 protein were included to assess the effects of prior vaccination. ART was administered for 11 weeks, with or without gp120 boosting at week 9. Naïve and resting, activated, and tissue-like memory B cells and PC/PB were evaluated by flow cytometry. Antibody-secreting cells (ASC) and serum antibody titers were assessed. No lasting changes in B-cell memory subpopulations occurred in bone marrow and lymph nodes, but significant decreases in numbers of activated memory B cells and increases in numbers of tissue-like memory B cells persisted in PBMC. Macaque PC/PB were found to be either CD27⁺ or CD27⁻ and therefore were defined as CD19⁺ CD38^hi CD138⁺. The numbers of these PC/PB were transiently increased in both PBMC and bone marrow following gp120 boosting of the unvaccinated and vaccinated macaque groups. Similarly, ASC numbers in PBMC and bone marrow of the two macaque groups also transiently increased following envelope boosting. Nevertheless, serum binding titers against SIVgp120 remained unchanged. Thus, even during chronic SIV infection, B cells respond to antigen, but long-term memory does not develop, perhaps due to germinal center destruction. Earlier and/or prolonged treatment to allow the generation of virus-specific long-term memory B cells should benefit ART/therapeutic vaccination regimens.     

Frequency and phenotype of human immunodeficiency virus envelope-specific B cells from patients with broadly cross-neutralizing antibodies

Induction of broadly cross-reactive neutralizing antibodies (NAb) is an important goal for a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. Some HIV-infected patients make a NAb response that reacts with diverse strains of HIV-1, but most candidate vaccines have induced NAb only against a subset of highly sensitive isolates. To better understand the nature of broad NAb responses that arise during natural infection, we screened patients for sera able to neutralize diverse HIV strains and explored the frequency and phenotype of their peripheral Envelope-specific B cells. We screened 113 HIV-infected patients of various clinical statuses for the prevalence of broad NAb. Sera able to neutralize at least four of five viral isolates were found in over one-third of progressors and slow progressors, but much less frequently in aviremic long-term nonprogressors. Most Env-specific antibody-secreting B cells were CD27^hi CD38^hi plasmablasts, and the total plasmablast frequency was higher in HIV-infected patients than in uninfected donors. We found that 0.0031% of B cells and 0.047% of plasmablasts secreted Env-specific immunoglobulin G (IgG) in an enzyme-linked immunospot (ELISPOT) assay. We developed a novel staining protocol to label HIV-specific B cells with Env gp140 protein. A total of 0.09% of B cells were found to be Env-specific by this method, a frequency far higher than that indicated by ELISPOT assay. gp140-labeled B cells were predominantly CD27⁺ and surface IgG⁺. These data describe the breadth and titer of serum NAb and the frequency and phenotype of HIV-specific B cells in a cohort of patients with broad cross-neutralizing antibody responses that are potential goals for vaccines for HIV.     

Altered dendritic cell distribution in patients with common variable immunodeficiency

Recent data suggest a critical role for dendritic cells (DCs) in the generation of immunoglobulin-secreting plasma cells. In the work reported herein, we analyzed the frequency of peripheral blood plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) in a cohort of 44 adults with common variable immunodeficiency (CVID) classified according to their CD27 membrane expression status on B cells. A deep alteration in the distribution of DC subsets, especially of pDCs, in the peripheral blood of CVID patients was found. Patients with a reduced number of class-switched CD27⁺IgD^-IgM^- memory B cells and patients with granulomatous disease had a dramatic decrease in pDCs (P = 0.00005 and 0.0003 vs controls, respectively) and, to a lesser extent, of mDCs (P = 0.001 and 0.01 vs controls, respectively). In contrast, patients with normal numbers of switched memory B cells had a DC distribution pattern similar to that in controls. Taken together, our results raise the possibility that innate immunity contributes to pathogenesis in CVID.     

Heterogeneity of B cell clones: immunoglobulin-secreting subsets

Staphylococcus aureus Cowan I bacteria (SpA CoI) is known to be a polyclonal B-cell activator of human lymphocytes. In this study, we investigated which of the B-cell subsets SpA CoI could stimulate and induce immunoglobulin (Ig) production. B-Cell subsets were separated from peripheral blood and tonsil lymphocytes by rosette formation with E, EA_IgG, EAC, anti-Ig-conjugated ox erythrocytes (OE-anti-Ig), and protein A-conjugated OE (OE-Pro A), or on a bovine serum albumin (BSA) discontinuous density gradient. The cells responding to SpA CoI included E^?, C3 receptor-positive (C3R⁺), Fc receptor-negative (FcR^?), and surface Ig-positive (SIg⁺) B-cell subsets. These B-cell populations responded well to SpA CoI and produced significant amounts of IgG, IgM, and a lesser amount of IgA. Among SIg⁺ B cells, IgG, IgA, and IgM⁺ B-cell subsets responded to SpA CoI and produced large amounts of Ig belonging to each corresponding Ig class. IgD⁺ B cells failed to produce Ig of any class, except for minimal amounts of IgG and IgM. While both the protein A receptor-positive (Pro A · R⁺) and negative (Pro A · R^?) cells responded well to SpA CoI, Pro A · R⁺ B cells produced IgG mainly and Pro A · R^? B cells produced IgM. Fractionation of B cells on a BSA gradient revealed that comparatively small-sized and denser B-cell subsets responded well to SpA CoI and produced every class of Ig.     

Constitutively altered frequencies of circulating follicullar helper T cell counterparts and their subsets in rheumatoid arthritis

Introduction

Circulating CD4 T cells expressing CXCR5, ICOS and/or PD-1 are counterparts of follicular helper T cells (Tfh). There are three subpopulations of circulating Tfh (cTfh): CXCR5 + CXCR3 + CCR6- (Tfh-Th1), CXCR5 + CXCR3-CCR6- (Tfh-Th2) and CXCR5 + CXCR3-CCR6+ (Tfh-Th17). Our objective was to study the B cell helping capacity of cTfh subsets, and examine their frequency in Rheumatoid Arthritis (RA) patients, together with the frequency of circulating plasmablasts (CD19 + CD20-CD38^high).

Methods

Peripheral blood was drawn from RA patients with active disease (RA-a, DAS28 >2.6) (n = 17), RA in remission (RA-r, DAS28 <2.6) (n = 17) and healthy controls (HC) (n = 34). cTfh and plasmablast frequencies were determined by flow cytometry. Cocultures of sorted CD4 + CXCR5+ T cell subpopulations were established with autologous CD19 + CD27- naïve B cells of HC, and concentrations of IgG, A and M were measured in supernatants.

Results

Isolated Tfh-Th2 and Tfh-Th17 but not Tfh-Th1 cells, induced naïve B cells to secrete IgG and IgA. The frequency of CXCR5+ cells gated for CD4+ T cells was not different among HC, RA-a and RA-r. In contrast, both RA-a and RA-r patients demonstrated an increased frequency of CD4 + CXCR5 + ICOS+ T cells and augmented (%Tfh-Th2 + %Tfh-Th17)/%Tfh-Th1 ratio as compared with HC. In addition, RA-a but not RA-r patients, showed an increased frequency of circulating plasmablasts.

Conclusion

Both RA-a and RA-r patients demonstrate an increased frequency of cTfh and overrepresentation of cTfh subsets bearing a B cell helper phenotype, suggesting that altered germinal center dynamics play a role in RA pathogenesis. In contrast, only RA-a patients show an increased proportion of circulating plasmablasts.     

Circulating CXCR5+CD4+ T Follicular-Like Helper Cell and Memory B Cell Responses to Human Papillomavirus Vaccines

Through the interaction of T follicular helper (Tfh) cells and B cells, efficacious vaccines can generate high-affinity, pathogen-neutralizing antibodies, and memory B cells. Using CXCR5, CXCR3, CCR6, CCR7, PD1, and ICOS as markers, Tfh-like cells can be identified in the circulation and be classified into three functionally distinct subsets that are PD1⁺ICOS⁺, PD1⁺ ICOS^-, or PD1^-ICOS^-. We used these markers to identify different subsets of CXCR5⁺CD4⁺ Tfh-like cells in response to highly immunogenic and efficacious vaccines for human papillomaviruses (HPV): Cervarix and Gardasil. In this small study, we used PBMC samples from 11 Gardasil recipients, and 8 Cervarix recipients from the Vaccine Research Center 902 Study to examine the induction of circulating Tfh-like cells and IgD^-CD38^HiCD27⁺ memory B cells by flow cytometry. PD1⁺ICOS⁺ CXCR3⁺CCR6^-CXCR5⁺CD4⁺ (Tfh1-like) cells were induced and peaked on Day (D) 7 post-first vaccination, but not as much on D7 post-third vaccination. We also observed a trend toward increase in PD1⁺ICOS⁺ CXCR3^-CCR6^-CXCR5⁺CD4⁺ (Tfh2-like) cells for both vaccines, and PD1⁺ICOS⁺ CXCR3^-CCR6⁺CXCR5⁺CD4⁺ (Tfh17-like) subset was induced by Cervarix post-first vaccination. There were also minimal changes in the other cellular subsets. In addition, Cervarix recipients had more memory B cells post-first vaccination than did Gardasil recipients at D14 and D30. We found frequencies of memory B cells at D30 correlated with anti-HPV16 and 18 antibody titers from D30, and the induction levels of memory B cells at D30 and PD1⁺ICOS⁺Tfh1-like cells at D7 post-first vaccination correlated for Cervarix. Our study showed that induction of circulating CXCR5⁺CD4⁺ Tfh-like subsets can be detected following immunization with HPV vaccines, and potentially be useful as a marker of immunogenicity of vaccines. However, further investigations should be extended to different cohorts with larger sample size to better understand the functions of these T cells, as well as their relationship with B cells and antibodies.     

The plasmablast-like leukocyte in the kidney of fugu (Takifugu rubripes)

In teleosts, the kidney is the major immune organ. From the kidney of fugu (Takifugu rubripes), we isolated a unique leukocyte population. This population shows properties similar to those of mammalian plasmablasts. First, adherent cells expressing IgM protein on their surface were obtained from the fugu kidney. Flow cytometry (FCM) showed that these cells were mainly composed of two cell populations: IgM⁺CD8α^? cells and IgM⁺CD8α⁺ cells. Further characterization of the IgM⁺CD8α^? population by RT-PCR demonstrated that the cells expressed secretory-type IgM as well as Bcl-6 and Blimp-1, developmental marker genes for the B cell lineage. Western blotting also showed that the cells secreted IgM protein. These results indicate that the IgM⁺CD8α^? cells are similar to cells at the plasmablast stage in mammals. This is the first report isolating plasmablast-like leukocytes in fish species. Our data also suggests that the teleosts kidney is a organ where B cells terminally differentiate into the plasma cells.     

Simultaneous Assessment of Rotavirus-Specific Memory B Cells and Serological Memory after B Cell Depletion Therapy with Rituximab

The mechanisms that contribute to the maintenance of serological memory are still unclear. Rotavirus (RV) memory B cells (mBc) are enriched in IgM⁺ and CD27^- subpopulations, which are associated with autoimmune diseases pathogenesis. In patients with autoimmune diseases treated with Rituximab (RTX), some autoantibodies (auto-Abs) decrease after treatment, but other auto-Abs and pathogen-specific IgG Abs remain unchanged. Thus, maintenance of autoimmune and pathogen-specific serological memory may depend on the type of antigen and/or Ab isotype evaluated. Antigen-specific mBc and antigen-specific Abs of different isotypes have not been simultaneously assessed in patients after RTX treatment. To study the relationship between mBc subpopulations and serological memory we characterized total, RV- and tetanus toxoid (TT)-specific mBc by flow cytometry in patients with autoimmune diseases before and after treatment with RTX. We also measured total, RV- and TT-Abs, and some auto-Abs by kinetic nephelometry, ELISA, and EliA tests, respectively. Minor differences were observed between the relative frequencies of RV-mBc in healthy controls and patients with autoimmune disease. After RTX treatment, naïve Bc and total, RV- and TT-specific mBc [IgM⁺, switched (IgA⁺/IgG⁺), IgM⁺ only, IgD⁺ only, and CD27^- (IgA⁺/IgG⁺/IgM⁺)] were significantly diminished. An important decrease in total plasma IgM and minor decreases in total IgG and IgA levels were also observed. IgM rheumatoid factor, IgG anti-CCP, and IgG anti-dsDNA were significantly diminished. In contrast, RV-IgA, RV-IgG and RV-IgG₁, and TT-IgG titers remained stable. In conclusion, in patients with autoimmunity, serological memory against RV and TT seem to be maintained by long-lived plasma cells, unaffected by RTX, and an important proportion of total IgM and serological memory against some auto-antigens seem to be maintained by short-lived plasma cells, dependent on mBc precursors depleted by RTX.     

A Lower Proportion of Regulatory B Cells in Patients with Henoch–Schoenlein Purpura Nephritis

Background

Henoch—Schoenlein purpura is the one of most common types of systemic vasculitis that involves impaired renal function and Henoch-Schoenlein purpura nephritis (HSPN). The diagnosis of this condition is largely based on immunohistologic detection of immunoglobulin A1-containing immune complex in the glomerular deposits of mesangium. Despite clinical advances, the etiopathogenesis of HSPN is still largely unknown.

Methods

In this study, we enrolled 25 newly diagnosed HSPN patients and 14 healthy controls. Then, fractions of B cell subtypes were determined in venous blood using flow cytometry. The serum interleukin (IL)-10 concentration was determined by enzyme-linked immunosorbent assay.

Results

Compared to those in healthy controls, the numbers of CD38⁺CD19⁺, CD86⁺CD19⁺, CD38⁺CD86⁺CD19⁺, and CD95⁺CD19⁺ B cells per microliter of blood were significantly higher in HSPN patients. In contrast, the numbers of CD5⁺CD19⁺, IL-10⁺CD19⁺, CD5⁺CD1d⁺CD19⁺, and IL-10⁺CD5⁺CD1d⁺CD19⁺ B cells per microliter of blood and the serum IL-10 concentration were significantly lower in HSPN patients. Following treatment, the numbers of CD38⁺CD19⁺ and CD86⁺CD19⁺ B cells per microliter of blood were significantly reduced in HSPN patients. However, the numbers of CD5⁺CD1d⁺CD19⁺, CD5⁺CD1d⁺IL-10⁺CD19⁺, and IL-10⁺CD19⁺ B cells per microliter of blood and the serum IL-10 concentration were significantly increased in HSPN patients following treatment. The estimated glomerular filtration rate (eGFR) was negatively correlated with the number of CD38⁺CD19⁺ B cells but positively correlated with the numbers of IL-10⁺CD19⁺, CD1d⁺CD5⁺CD19⁺, and IL-10⁺CD1d⁺CD5⁺CD19⁺B cells per microliter of blood and the serum IL-10 concentration. The 24-h urinary protein concentration was positively correlated with the number of CD38⁺CD19⁺B cells but negatively correlated with the numbers of IL-10⁺CD19⁺, CD1d⁺CD5⁺CD19⁺, and IL-10⁺CD1d⁺CD5⁺CD19⁺B cells per microliter of blood and the serum IL-10 concentration.

Conclusion

Our results suggest that CD38⁺CD19⁺ and CD1d⁺CD5⁺CD19⁺ B cells (Bregs) contribute to the pathogenesis of HSPN.     

Impairment of Pneumococcal Antigen Specific Isotype-Switched Igg Memory B-Cell Immunity in HIV Infected Malawian Adults

Pneumococcal disease is associated with a particularly high morbidity and mortality amongst adults in HIV endemic countries. Our previous findings implicating a B-cell defect in HIV-infected children from the same population led us to comprehensively characterize B-cell subsets in minimally symptomatic HIV-infected Malawian adults and investigate the isotype-switched IgG memory B-cell immune response to the pneumococcus. We show that similar to vertically acquired HIV-infected Malawian children, horizontally acquired HIV infection in these adults is associated with IgM memory B-cell (CD19⁺ CD27⁺ IgM⁺ IgD⁺) depletion, B-cell activation and impairment of specific IgG B-cell memory to a range of pneumococcal proteins. Our data suggest that HIV infection affects both T-cell independent and T-cell dependent B-cell maturation, potentially leading to impairment of humoral responses to extracellular pathogens such as the pneumococcus, and thus leaving this population susceptible to invasive disease.     

Epstein-Barr Virus Infection of Na?ve B Cells In Vitro Frequently Selects Clones with Mutated Immunoglobulin Genotypes: Implications for Virus Biology

Epstein-Barr virus (EBV), a lymphomagenic human herpesvirus, colonises the host through polyclonal B cell-growth-transforming infections yet establishes persistence only in IgD⁺ CD27⁺ non-switched memory (NSM) and IgD⁻ CD27⁺ switched memory (SM) B cells, not in IgD⁺ CD27⁻ naïve (N) cells. How this selectivity is achieved remains poorly understood. Here we show that purified N, NSM and SM cell preparations are equally transformable in vitro to lymphoblastoid cells lines (LCLs) that, despite upregulating the activation-induced cytidine deaminase (AID) enzyme necessary for Ig isotype switching and Ig gene hypermutation, still retain the surface Ig phenotype of their parental cells. However, both N- and NSM-derived lines remain inducible to Ig isotype switching by surrogate T cell signals. More importantly, IgH gene analysis of N cell infections revealed two features quite distinct from parallel mitogen-activated cultures. Firstly, following 4 weeks of EBV-driven polyclonal proliferation, individual clonotypes then become increasingly dominant; secondly, in around 35% cases these clonotypes carry Ig gene mutations which both resemble AID products and, when analysed in prospectively-harvested cultures, appear to have arisen by sequence diversification in vitro. Thus EBV infection per se can drive at least some naïve B cells to acquire Ig memory genotypes; furthermore, such cells are often favoured during an LCL''s evolution to monoclonality. Extrapolating to viral infections in vivo, these findings could help to explain how EBV-infected cells become restricted to memory B cell subsets and why EBV-driven lymphoproliferative lesions, in primary infection and/or immunocompromised settings, so frequently involve clones with memory genotypes.     

Characterization of hematopoietic stem cell subsets from patients with multiple myeloma after mobilization with plerixafor

Background aimsPrevious studies have demonstrated that the combination of granulocyte–colony-stimulating factor (G-CSF) + plerixafor is more efficient in mobilizing CD34⁺ hematopoietic stem cells (HSC) into the peripheral blood than G-CSF alone. In this study we analyzed the impact of adding plerixafor to G-CSF upon the mobilization of different HSC subsets.MethodsWe characterized the immunophenotype of HSC subsets isolated from the peripheral blood of eight patients with multiple myeloma (MM) before and after treatment with plerixafor. All patients were supposed to collect stem cells prior to high-dose chemotherapy and consecutive autologous stem cell transplantation, and therefore received front-line mobilization with 4 days of G-CSF followed by a single dose of plerixafor. Samples of peripheral blood were analyzed comparatively by flow cytometry directly before and 12 h after administration of plerixafor.ResultsThe number of aldehyde dehydrogenase (ALDH)^bright and CD34⁺ cells was significantly higher after plerixafor treatment (1.2–5.0 and 1.5–6.0 times; both P < 0.01) and an enrichment of the very primitive CD34⁺ CD38^? and ALDH^bright CD34⁺ CD38^? HSC subsets was detectable. Additionally, two distinct ALDH⁺ subsets could be clearly distinguished. The small ALDH^high subset showed a higher number of CD34⁺ CD38^? cells in contrast to the total ALDH^bright subpopulation and probably represented a very primitive subpopulation of HSC.ConclusionsA combined staining of ALDH, CD34 and CD38 might represent a powerful tool for the identification of a very rare and primitive hematopoietic stem cell subset. The addition of plerixafor mobilized not only more CD34⁺ cells but was also able to increase the proportion of more primitive stem cell subsets.     

An age‐related numerical and functional deficit in CD19+CD24hiCD38hi B cells is associated with an increase in systemic autoimmunity

Autoimmunity increases with aging indicative of reduced immune tolerance, but the mechanisms involved are poorly defined. In recent years, subsets of B cells with immunoregulatory properties have been identified in murine models of autoimmune disorders, and these cells downregulate immune responses via secretion of IL10. In humans, immature transitional B cells with a CD19⁺CD24^hiCD38^hi phenotype have been reported to regulate immune responses via IL10 production. We found the frequency and numbers of CD19⁺CD24^hiCD38^hi cells were reduced in the PBMC pool with age. IL10 expression and secretion following activation via either CD40, or Toll‐like receptors was also impaired in CD19⁺CD24^hiCD38^hi B cells from healthy older donors. When investigating the mechanisms involved, we found that CD19⁺CD24^hiCD38^hi B‐cell function was compromised by age‐related effects on both T cells and B cells: specifically, CD40 ligand expression was lower in CD4 T cells from older donors following CD3 stimulation, and signalling through CD40 was impaired in CD19⁺CD24^hiCD38^hi B cells from elders as evidenced by reduced phosphorylation (Y705) and activation of STAT3. However, there was no age‐associated change in expression of costimulatory molecules CD80 and CD86 on CD19⁺CD24^hiCD38^hi cells, suggesting IL10‐dependent immune suppression is impaired, but contact‐dependent suppressive capacity is intact with age. Finally, we found a negative correlation between CD19⁺CD24^hiCD38^hi B‐cell IL10 production and autoantibody (Rheumatoid factor) levels in older adults. We therefore propose that an age‐related decline in CD19⁺CD24^hiCD38^hi B cell number and function may contribute towards the increased autoimmunity and reduced immune tolerance seen with aging.     

Peripheral regulatory cells immunophenotyping in Primary Sj?gren's Syndrome: a cross-sectional study

Introduction

IL-10--producing B cells, Foxp3-expressing T cells (Tregs) and the IDO-expressing dendritic cells (pDC) are able to modulate inflammatory processes, to induce immunological tolerance and, in turn, to inhibit the pathogenesis of autoimmune disease.The aim of the study was to characterize and to enumerate peripheral IL-10--producing B cells, Tregs and pDCregs in primary Sjögren''s Syndrome (pSS) patients in regard of their clinical and serologic activity.

Methods

Fifty pSS patients and 25 healthy individuals were included in the study. CD19⁺--expressing peripheral B lymphocytes were purified by positive selection. CD19⁺/CD24^hi/CD38^hi/IL-10--producing B cells, CD4⁺/CD25^hi/Foxp3⁺ and CD8⁺/CD28^-/Foxp3⁺ Tregs, as well as CCR6⁺/CD123⁺/IDO⁺ DCs, were quantitated by flow cytometry.

Results

Immature/transitional circulating IgA⁺ IL-10--producing B cells had higher levels in pSS patients versus control group, whereas CD19⁺/CD38^hi/IgG⁺/IL-10⁺ cells had lower percentage versus control. Indeed CD19⁺/CD24^hi/CD38^hi/CD5⁺/IL-10⁺, CD19⁺/CD24^hi/CD38^hi/CD10⁺/IL-10⁺, CD19⁺/CD24^hi/CD38^hi/CD20⁺/IL-10⁺, CD19⁺/CD24^hi/CD38^hi/CD27^-/IL-10⁺, and CD19⁺/CD24^hi/CD38^hi/CXCR7⁺/IL-10⁺ cells had higher frequency in clinical inactive pSS patients when compared with control group. Remarkably, only percentages of CD19⁺/CD24^hi/CD38^hi/CD10⁺/IL-10⁺ and CD19⁺/CD24^hi/CD38^hi/CD27^-/IL-10⁺ subsets were increased in pSS serologic inactive versus control group (P < 0.05). The percentage of IDO-expressing pDC cells was higher in pSS patients regardless of their clinical or serologic activity. There were no statistically significant differences in the percentage of CD4⁺/CD25^hi/Foxp3⁺ Tregs between patient groups versus controls. Nonetheless, a decrease in the frequency of CD8⁺/CD28^-/Foxp3⁺ Tregs was found in inactive pSS patients versus controls (P < 0.05).

Conclusions

The findings of this exploratory study show that clinical inactive pSS patients have an increased frequency of IL-10--producing B cells and IDO-expressing pDC cells.     

PDL241, a novel humanized monoclonal antibody,reveals CD319 as a therapeutic target for rheumatoid arthritis

Introduction

Targeting the CD20 antigen has been a successful therapeutic intervention in the treatment of rheumatoid arthritis (RA). However, in some patients with an inadequate response to anti-CD20 therapy, a persistence of CD20^- plasmablasts is noted. The strong expression of CD319 on CD20^- plasmablast and plasma cell populations in RA synovium led to the investigation of the potential of CD319 as a therapeutic target.

Methods

PDL241, a novel humanized IgG₁ monoclonal antibody (mAb) to CD319, was generated and examined for its ability to inhibit immunoglobulin production from plasmablasts and plasma cells generated from peripheral blood mononuclear cells (PBMC) in the presence and absence of RA synovial fibroblasts (RA-SF). The in vivo activity of PDL241 was determined in a human PBMC transfer into NOD scid IL-2 gamma chain knockout (NSG) mouse model. Finally, the ability of PDL241 to ameliorate experimental arthritis was evaluated in a collagen-induced arthritis (CIA) model in rhesus monkeys.

Results

PDL241 bound to plasmablasts and plasma cells but not naïve B cells. Consistent with the binding profile, PDL241 inhibited the production of IgM from in vitro PBMC cultures by the depletion of CD319⁺ plasmablasts and plasma cells but not B cells. The activity of PDL241 was dependent on an intact Fc portion of the IgG₁ and mediated predominantly by natural killer cells. Inhibition of IgM production was also observed in the human PBMC transfer to NSG mouse model. Treatment of rhesus monkeys in a CIA model with PDL241 led to a significant inhibition of anti-collagen IgG and IgM antibodies. A beneficial effect on joint related parameters, including bone remodeling, histopathology, and joint swelling was also observed.

Conclusions

The activity of PDL241 in both in vitro and in vivo models highlights the potential of CD319 as a therapeutic target in RA.     

Pediatric Helicobacter pylori infection and circulating T-lymphocyte activation and differentiation

Background: In this study, H. pylori‐infected and noninfected children with gastritis were compared to a control group with respect to circulating CD4⁺ and CD8⁺ T lymphocytes expressing activation and differentiation markers. Additionally, the lymphocyte phenotypes of children with gastritis were correlated with the gastric inflammation scores. Materials and Methods: H. pylori infection status was assessed based on [¹³C]urea breath test, rapid urease test, and histology. Analysis of the lymphocyte surface molecule expression was carried out by triple‐color flow cytometry. Results: The group of H. pylori‐infected children showed an elevated proportion of peripheral B cells with CD19^low, along with a twofold increase in the percentage of memory (CD45RO⁺) CD4⁺ and CD8⁺ T‐cell subsets (p < .05). Moreover, a positive correlation between the age and the percentage of these subsets was seen (r = .38, p = .04 and r = .56, p < .01, respectively). Children with gastritis but without infection had a slightly increased percentage of CD8⁺ T cells and CD56⁺ NK cells, CD3^high T cells and CD45RO^high CD4⁺ T‐cell subsets (p < .05). Both H. pylori‐infected and noninfected children with gastritis were characterized by an increased percentage of memory/effector CD4⁺ T cells, the presence of NK cells with CD56^high, memory T‐cell subset with CD4^high, and naive, memory, memory/effector, and effector T‐cell subsets with CD8^high (p < .05). Gastric inflammation scores correlated positively with the percentage of CD4⁺ T lymphocytes in H. pylori‐infected children (r = .42, p = .03). In noninfected children, gastric inflammation scores correlated positively with the percentage of B cells (r = .45, p = .04). Conclusion: In H. pylori‐negative children, gastritis was associated with an increased percentage of activated NK and T cells, and intermediate‐differentiated peripheral blood CD4⁺ T cells, which was more pronounced in H. pylori‐positive children who also showed an increased B‐cell response. However, increased inflammation was only associated with the elevation of CD4⁺ T‐cell percentage in H. pylori‐positive children as well as B‐cell percentage in H. pylori‐negative children with gastritis.     

The Elevated Secreted Immunoglobulin D Enhanced the Activation of Peripheral Blood Mononuclear Cells in Rheumatoid Arthritis

Immunoglobulin D (IgD) is a surface immunoglobulin that is expressed as either membrane IgD (mIgD) or secreted IgD (sIgD). Researchers have shown that sIgD is often elevated in patients with autoimmune diseases. The possible roles of sIgD on the function of peripheral blood mononuclear cells (PBMCs) in rheumatoid arthritis (RA) are still unclear. In this study, we compared the expression of sIgD, mIgD and IgD receptor (IgDR) in RA patients and healthy controls, and investigated the effect of sIgD on the function of PBMCs. We found that the levels of sIgD, mIgD and IgDR were significantly higher in RA patients compared with healthy controls. The concentrations of sIgD were positively correlated with soluble receptor activator of nuclear factor-κB ligand (sRANKL), rheumatoid factor (RF) and C-reactive protein (CRP) in RA patients. Strikingly, IgD could enhance the proliferation of PBMCs and induce IL-1α, IL-1β, TNF-α, IL-6 and IL-10 production from PBMCs. Moreover, the percentage of activated T cell subsets (CD4⁺CD69⁺, CD4⁺CD154⁺) and activated B cell subsets (CD19⁺CD23⁺, CD19⁺CD21⁺, CD19⁺IgD⁺ and CD19^-CD138⁺) were increased by IgD. The percentage of unactivated T cell subset (CD4⁺CD62L⁺) and immature B cell subset (CD19⁺IgM⁺IgD^-) were decreased by IgD in PBMCs. Furthermore, the expressions of IgDR on T and B cells were significantly increased by treatment with IgD. Our results demonstrate that IgD enhanced the activation of PBMCs, which may contribute to RA pathogenesis. Therefore, IgD could be a potential novel immunotherapeutic target for the management of RA.     

Plasmablasts as Migratory IgG-Producing Cells in the Pathogenesis of Neuromyelitis Optica

Neuromyelitis optica (NMO) is an inflammatory disease characterized by recurrent attacks of optic neuritis and myelitis. It is generally accepted that autoantibodies against aquaporin 4 water channel protein play a pathogenic role in neuromyelitis optica. We have recently reported that plasmablasts are increased in the peripheral blood of this autoimmune disease, and are capable of producing autoantibodies against aquaporin 4. Here, we demonstrate that CD138⁺HLA-DR⁺ plasmablasts, a subset of IgG-producing cells, are increased in the peripheral blood and are enriched among the cerebrospinal fluid (CSF) lymphocytes during the relapse of neuromyelitis optica. Notably, these CD138⁺HLA-DR⁺ plasmablasts overexpress CXCR3, whose ligands are present in the cerebrospinal fluid during the relapse of neuromyelitis optica. These results led us to speculate that plasmablasts producing anti-aquaporin 4 autoantibodies might traffic toward the central nervous system (CNS). Furthermore, we performed single-cell sorting of plasmablasts from peripheral blood and CSF samples from NMO and sequenced the complementarity-determining regions (CDRs) of the IgG heavy chain expressed by the sorted plasmablast clones. There were high frequencies of mutations in the CDRs compared with framework regions, indicating that these plasmablast clones would represent a post-germinal center B-cell lineage. Consistent with the preceding results, the plasmablast clones from the peripheral blood shared the same CDR sequences with the clones from the CSF. These results indicate that IgG-producing plasmablasts, which are guided by helper T-cells, may migrate from the peripheral blood preferentially to the CSF. Since migratory plasmablasts could be involved in the inflammatory pathology of NMO, the B-cell subset and their migration might be an attractive therapeutic target.     

High levels of memory B cells are associated with response to a first tumor necrosis factor inhibitor in patients with rheumatoid arthritis in a longitudinal prospective study

Introduction

Tumor necrosis factor inhibitor (TNFi) therapy is effective for rheumatoid arthritis (RA). Some researchers have suggested that TNFi therapy affects B-cell homeostasis. We studied the effect of TNFi therapy on the distribution of peripheral B-cell subsets to elucidate B-cell–related biomarkers to predict the TNFi response.

Methods

Peripheral B cells were analyzed for expression of CD19, CD27, CD38 and immunoglobulin D in 31 healthy donors and 96 RA patients, including 21 patients who were followed 3 months after TNFi initiation.

Results

Treatment with steroids significantly altered the distribution of B-cell subsets. After we adjusted for age, sex and steroid dose, we found that patients with RA had B-cell subset proportions similar to controls. B-cell subset distributions did not differ upon use of TNFi at baseline or before or after TNFi introduction. TNFi responders (according to European League Against Rheumatism criteria) at 3 months had significantly higher proportions of CD27⁺ memory B cells at baseline, and ≥26% CD27⁺ cells at inclusion was associated with a relative risk of 4.9 (1.3 to 18.6) for response to TNFi treatment. CD27⁺ cells produced three times more TNFα than did TNFi-naïve B cells and were correlated with interferon γ produced from CD4⁺ cells in patients without TNFi treatment.

Conclusions

In patients with RA, high levels of baseline memory B cells were associated with response to TNFi, which may be related to TNFα-dependent activation of the T helper type 1 cell pathway.