M. Manuela Rosado

Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen.

Splenectomized and asplenic patients have a high incidence of infections by encapsulated bacteria and do not respond to polysaccharide vaccines. To understand whether the absence of the spleen is associated with a defined B cell defect, we analyzed B cell subsets in the peripheral blood. We found that a population of B cells known as immunoglobulin (Ig)M memory is lacking in patients without spleen. The absence of IgM memory B cells correlates with an impaired immune response to encapsulated bacteria not only in splenectomized patients, but also in individuals with an intact spleen. We show that the physiological and transient predisposition to pneumococcal infections of young children (0–2 yr) is associated with the lack of circulating IgM memory B cells and of serum antipolysaccharide IgM. We also demonstrate that IgM memory B cells are undetectable in a fraction of patients with common variable immunodeficiency, who have recurrent and invasive infections by encapsulated bacteria. IgM memory B cells, therefore, require the spleen for their generation and/or survival and are responsible for the protection against encapsulated bacteria.

Why do we need IgM memory B cells

Immunological memory is our reservoir of ready-to-use antibodies and memory B cells. Because of immunological memory a secondary infection will be very light or not occur at all. Antibodies and cells, generated in the germinal center in response to the first encounter with antigen, are highly specific, remain in the organism virtually forever and are mostly of IgG isotype. Long lived plasma cells homing to the bone marrow ensure the constant production of protective antibodies, whereas switched memory B cells proliferate and differentiate in response to secondary challenge. IgM memory B cells represent our first-line defense against infections. They are generated by a T-cell independent mechanism probably triggered by Toll-like receptor-9. They produce natural antibodies with anti-bacterial specificity and the spleen is indispensable for their maintenance. We will review the characteristics and functions of IgM memory B cells that explain their importance in the immediate protection from pathogens. IgM memory B cells, similar to mouse B-1a B cells, may be a remnant of a primitive immune system that developed in the spleen of cartilaginous fish and persisted throughout evolution notwithstanding the sophisticated tools of the adaptive immune system.

Switched memory B cells maintain specific memory independently of serum antibodies: the hepatitis B example.

The immunogenicity of a vaccine is conventionally measured through the level of serum Abs early after immunization, but to ensure protection specific Abs should be maintained long after primary vaccination. For hepatitis B, protective levels often decline over time, but breakthrough infections do not seem to occur. The aim of this study was to demonstrate whether, after hepatitis B vaccination, B‐cell memory persists even when serum Abs decline. We compared the frequency of anti‐hepatitis‐specific memory B cells that remain in the blood of 99 children five years after priming with Infanrix®‐hexa (GlaxoSmithKline) (n=34) or with Hexavac® (Sanofi Pasteur MSD) (n=65). These two vaccines differ in their ability to generate protective levels of IgG. Children with serum Abs under the protective level, <10u2009mIU/mL, received a booster dose of hepatitis B vaccine, and memory B cells and serum Abs were measured 2u2009wk later. We found that specific memory B cells had a similar frequency in all children independently of primary vaccine. Booster injection resulted in the increase of memory B cell frequencies (from 11.3 in 106 cells to 28.2 in 106 cells, p<0.01) and serum Abs (geometric mean concentration, GMC from 2.9 to 284u2009mIU/mL), demonstrating that circulating memory B cells effectively respond to Ag challenge even when specific Abs fall under the protective threshold.

Preserved antibody levels and loss of memory B cells against pneumococcus and tetanus after splenectomy: Tailoring better vaccination strategies

Splenectomized patients are exposed to an increased risk of septicemia caused by encapsulated bacteria. Defense against infection is ensured by preformed serum antibodies produced by long‐lived plasma cells and by memory B cells that secrete immunoglobulin in response to specific antigenic stimuli. Studying a group of asplenic individuals (57 adults and 21 children) without additional immunologic defects, we found that spleen removal does not alter serum anti‐pneumococcal polysaccharide (PnPS) IgG concentration, but reduces the number of PnPS‐specific memory B cells, of both IgM and IgG isotypes. The number of specific memory B cells was low in splenectomized adults and children that had received the PnPS vaccine either before or after splenectomy. Seven children were given the 13‐valent pneumococcal conjugated vaccine after splenectomy. In this group, the number of PnPS‐specific IgG memory B cells was similar to that of eusplenic children, suggesting that pneumococcal conjugated vaccine administered after splenectomy is able to restore the pool of anti‐PnPS IgG memory B cells. Our data further elucidate the crucial role of the spleen in the immunological response to infections caused by encapsulated bacteria and suggest that glycoconjugated vaccines may be the most suitable choice to generate IgG‐mediated protection in these patients.

Peripheral regulatory T cells and serum transforming growth factor‐β: Relationship with clinical response to infliximab in Crohn's disease

Background: CD4+Foxp3+ regulatory T cells (Treg) inhibit T‐cell proliferation in vitro and are effective in suppressing colitis in mouse models. Tumor necrosis factor (TNF)‐&agr;, which is centrally involved in Crohns disease (CD) pathogenesis, also impairs Treg function. Here we investigated the influence of anti‐TNF therapy on Treg frequency and function in CD. Methods: Twenty CD patients were treated with infliximab administered at weeks 0, 2, and 6. Blood was collected immediately before the first infusion and after 10 weeks. Treg frequency was quantified by flow cytometry. Treg function was measured using a standard coculture assay. Serum levels of transforming growth factor (TGF)‐&bgr;1 and interleukin (IL)‐10 were measured by enzyme‐linked immunosorbent assay (ELISA). Results: Pretreatment Treg frequency and serum TGF‐&bgr;1 levels were significantly higher in nonresponder than responder patients. Clinical improvement in 12 CD patients was associated with a significant increase of Treg frequency after 10 weeks. Treg were functionally active before and after treatment with infliximab, both in responder and nonresponder CD patients. In responder patients the restoration of Treg pool was accompanied by a parallel significant increase of serum TGF‐&bgr;1 and IL‐10. No significant change in the elevated Treg or serum TGF‐&bgr;1 was seen in nonresponder patients. Conclusions: This study suggests that there may be a relationship between numbers of Treg in the blood, serum TGF‐&bgr;1, and response to infliximab; however, further prospective studies are needed. (Inflamm Bowel Dis 2010)

Plasma Cells in the Mucosa of Patients with Inflammatory Bowel Disease Produce Granzyme B and Possess Cytotoxic Activities

In both Crohn’s disease (CD) and ulcerative colitis (UC), the gut is massively infiltrated with B cells and plasma cells, but the role of these cell types in the pathogenesis of gut tissue damage remains largely unknown. Human B cells express granzyme B (GrB) when cultured with IL-21, a cytokine overproduced in CD and UC mucosa. We therefore examined whether mucosal B cells express GrB and have cytotoxic activity in inflammatory bowel disease (IBD). GrB-expressing CD19+ and IgA+ cells were seen in the normal intestinal mucosa, but they were significantly more frequent in both CD and UC. In contrast, only a minority of CD19+ and IgA+ cells expressed perforin with no difference between IBD and controls. GrB-producing CD19+ cells expressed CD27 and were CD38high and CD20 negative. CD19+ B cells from IBD patients induced HCT-116 cell death. IL-21 enhanced GrB expression in control CD19+ B cells and increased their cytotoxic activity. These data indicate that IBD-related inflammation is marked by mucosal accumulation of cytotoxic, GrB-expressing CD19+ and IgA+ cells, suggesting a role for these cells in IBD-associated epithelial damage.

Splenic function and IgM-memory B cells in Crohn's disease patients treated with infliximab

Background: Under experimental chronic inflammation, tumor necrosis factor (TNF)‐&agr; plays a role in damaging spleen marginal zone. This latter has a crucial function in mounting B cell‐dependent immune responses against infections by encapsulated bacteria. In Crohns disease (CD), a chronic inflammatory disorder where TNF‐&agr; is centrally involved, impaired splenic function may increase the susceptibility to bacterial infections. On this basis, we aimed to investigate the influence of anti‐TNF therapy on splenic function in CD patients. Methods: Peripheral blood samples were obtained from 15 CD patients before and after treatment with infliximab administered at weeks 0, 2, and 6 at a dose of 5 mg/kg. Counting of erythrocytes with membrane abnormalities (pitted red cells) was used as an indicator of splenic function. Multicolor flow cytometry was performed to analyze circulating B cells. Results: A substantial clinical improvement in 10 of the 15 CD patients was associated with a significant reduction of pitted red cells (from median 6.0% to 3.6%; P < 0.01) after 10 weeks of treatment. In responder patients the improvement of splenic function was accompanied by a parallel increase of circulating IgM‐memory B cells (from median 6.9% to 13.3%; P < 0.005). Splenic function was not ameliorated in nonresponder patients. Conclusions: Splenic function improved in CD patients who responded to infliximab and was accompanied by a concomitant restoration of the IgM‐memory B cell pool responsible for the protection against encapsulated bacteria. Restoration of splenic function after infliximab treatment is intriguing and requires further investigation.

Altered B cell homeostasis and toll-like receptor 9-driven response in type 1 diabetes carriers of the C1858T PTPN22 allelic variant: implications in the disease pathogenesis.

Type 1 diabetes is an autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Among the genetic variants associated with type 1 diabetes, the C1858T (Lyp) polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene alters the function of T cells but also of B cells in innate and adaptive immunity. The Lyp variant was shown to diminish interferon production and responses upon Toll-like receptor stimulation in macrophages and dendritic cells, possibly leading to uncontrolled infections as triggers of the diabetogenic process. The aim of this study was to unravel the yet uncharacterized effects that the variant could exert on the immune and autoimmune responses, particularly regarding the B cell phenotype, in the peripheral blood lymphocytes of diabetic patients and healthy controls in basal conditions and after unmethylated bacterial DNA CpG stimulation. The presence of the Lyp variant resulted in a significant increase in the percentage of transitional B cells in C/T carriers patients and controls compared to C/C patients and controls, in C/T carrier patients compared to C/C controls and in C/T carrier patients compared to C/C patients. A significant reduction in the memory B cells was also observed in the presence of the risk variant. After four days of CpG stimulation, there was a significant increase in the abundance of IgM+ memory B cells in C/T carrier diabetics than in C/C subjects and in the groups of C/T carrier individuals than in C/C individuals. IgM− memory B cells tended to differentiate more precociously into plasma cells than IgM+ memory B cells in heterozygous C/T subjects compared to the C/C subjects. The increased Toll-like receptor response that led to expanded T cell-independent IgM+ memory B cells should be further investigated to determine the putative contribution of innate immune responses in the disease pathogenesis.

Microbiota and chronic inflammatory arthritis: an interwoven link

BackgroundOnly recently, the scientific community gained insights on the importance of the intestinal resident flora for the host’s health and disease. Gut microbiota in fact plays a crucial role in modulating innate and acquired immune responses and thus interferes with the fragile balance inflammation versus tolerance.Main bodyCorrelations between gut bacteria composition and the severity of inflammation have been studied in inflammatory bowel diseases. More recently similar alterations in the gut microbiota have been reported in patients with spondyloarthritis, whereas in rheumatoid arthritis an accumulating body of evidence evokes a pathogenic role for the altered oral microbiota in disease development and course. In the context of dysbiosis it is also important to remember that different environmental factors like stress, smoke and dietary components can induce strong bacterial changes and consequent exposure of the intestinal epithelium to a variety of different metabolites, many of which have an unknown function. In this perspective, and in complex disorders like autoimmune diseases, not only the genetic makeup, sex and immunologic context of the individual but also the structure of his microbial community should be taken into account.ConclusionsHere we provide a review of the role of the microbiota in the onset, severity and progression of chronic inflammatory arthritis as well as its impact on the therapeutic management of these patients. Furthermore we point-out the complex interwoven link between gut-joint-brain and immune system by reviewing the most recent data on the literature on the importance of environmental factors such as diet, smoke and stress.

Repeated vaccinations do not improve specific immune defenses against Hepatitis B in non-responder health care workers.

Hepatitis B is a major infectious occupational hazard for health care workers and can be prevented with a safe and effective vaccine. The serum titer of anti-HBsAg antibodies is the most commonly used correlate of protection and post-vaccination anti-HBsAg concentrations of ≥ 10 mIU/ml are considered protective. Subjects with post-vaccination anti-HBsAg titers of <10 mIU/ml 1-6 months post-vaccination, who tested negative for HBsAg and anti-HBc, are defined as non-responders. The question of whether non-responders should be repeatedly vaccinated is still open. The aim of the study was to (i) evaluate the distribution of lymphocyte subpopulations and the percentage of HBsAg-specific memory B cells in responders and non-responders (ii) assess whether non-responders can be induced to produce antibodies after administration of a booster dose of vaccine (iii) determine whether booster vaccination increases the number of specific memory B cells in non-responders. Combining flow-cytometry, ELISPOT and serology we tested the integrity and function of the immune system in 24 health care workers, confirmed to be non-responders after at least three vaccine injections. We compared the results with those obtained in 21 responders working in the same institution. We found that the great majority of the non-responders had a functional immune system and a preserved ability to respond to other conventional antigens. Our most important findings are that the frequency of HBsAg-specific memory B cells is comparable in non-responders and controls and that booster immunization does not lead either to antibody production or memory B cell increase in non-responders.