Simona Cascioli

CpG Drives Human Transitional B Cells to Terminal Differentiation and Production of Natural Antibodies

The receptor TLR9, recognizing unmethylated bacterial DNA (CpG), is expressed by B cells and plays a role in the maintenance of serological memory. Little is known about the response of B cells stimulated with CpG alone, without additional cytokines. In this study, we show for the first time the phenotypic modification, changes in gene expression, and functional events downstream to TLR9 stimulation in human B cell subsets. In addition, we demonstrate that upon CpG stimulation, IgM memory B cells differentiate into plasma cells producing IgM Abs directed against the capsular polysaccharides of Streptococcus pneumoniae. This novel finding proves that IgM memory is the B cell compartment responsible for the defense against encapsulated bacteria. We also show that cord blood transitional B cells, corresponding to new bone marrow emigrants, respond to CpG. Upon TLR9 engagement, they de novo express AID and Blimp-1, genes necessary for hypersomatic mutation, class-switch recombination, and plasma cell differentiation and produce Abs with anti-pneumococcal specificity. Transitional B cells, isolated from cord blood, have not been exposed to pneumococcus in vivo. In addition, it is known that Ag binding through the BCR causes apoptotic cell death at this stage of development. Therefore, the ability of transitional B cells to sense bacterial DNA through TLR9 represents a tool to rapidly build up the repertoire of natural Abs necessary for our first-line defense at birth.

Inhibition of B-Cell Proliferation and Antibody Production by Mesenchymal Stromal Cells Is Mediated by T Cells

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs), endowed with immunosuppressive and anti-inflammatory properties, represent a promising tool in immunoregulatory and regenerative cell therapy. Clarifying the interactions between MSCs and B-lymphocytes may be crucial for designing innovative MSC-based strategies in conditions in which B cells play a role, including systemic lupus erythematosus (SLE) and rejection of kidney transplantation. In this study, we show that, both in healthy subjects and in patients, in vitro B-cell proliferation, plasma-cell differentiation, and antibody production are inhibited by BM-derived MSCs when peripheral blood lymphocytes are stimulated with CpG, but not when sorted B cells are cultured with MSCs+CpG. Inhibition is restored in CpG+MSC cocultures when sorted T cells are added to sorted B cells, suggesting that this effect is mediated by T cells, with both CD4(+) and CD8(+) cells playing a role. Moreover, cell-cell contact between MSCs and T cells, but not between MSCs and B cells, is necessary to inhibit B-cell proliferation. Thus, the presence of functional T cells, as well as cell-cell contact between MSCs and T cells, are crucial for B-cell inhibition. This information can be relevant for implementing MSC-based therapeutic immune modulation in patients in whom T-cell function is impaired.

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, <10 mIU/mL, received a booster dose of hepatitis B vaccine, and memory B cells and serum Abs were measured 2 wk 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 284 mIU/mL), demonstrating that circulating memory B cells effectively respond to Ag challenge even when specific Abs fall under the protective threshold.

B Cell Reconstitution after Rituximab Treatment in Idiopathic Nephrotic Syndrome

The pathogenesis of nephrotic syndrome is unclear. However, the efficacy of rituximab, a B cell-depleting antibody, in nephrotic syndrome suggests a pathogenic role of B cells. In this retrospective study, we determined by flow cytometry levels of B and T cell subpopulations before and after rituximab infusion in 28 pediatric patients with frequently relapsing or steroid-dependent nephrotic syndrome. At baseline, patients had lower median percentages of transitional and mature B cells than age-matched healthy controls (P<0.001). Rituximab induced full depletion of B cells (<1% of lymphocytes). At 1 year, most patients exhibited complete total and mature B cell recovery, whereas memory B cell subsets remained significantly depleted. Total T cell concentration did not change with rituximab, whereas the CD4(+)/CD8(+) T cell ratio tended to increase. Fourteen patients relapsed within 24 months, with a median follow-up of 11.2 months (interquartile range, 8-17.7 months). We observed no difference at baseline between nonrelapsing and relapsing patients in several clinical parameters and cell subset concentrations. Reconstitution of all memory B cell subpopulations, number of immunosuppressive drugs, and dose of tacrolimus during the last 4 months of follow-up were predictive of relapse in univariate Cox regression analysis. However, only delayed reconstitution of switched memory B cells, independent of immunosuppressive treatment, was protective against relapse in multivariate (P<0.01) and receiver operator characteristic (P<0.01 for percentage of lymphocytes; P=0.02 for absolute count) analyses. Evaluation of switched memory B cell recovery after rituximab may be useful for predicting relapse in patients with nephrotic syndrome.

Immunodeficiency in Vici syndrome: a heterogeneous phenotype.

Vici syndrome is a rare congenital multisystem disorder characterized by agenesis of the corpus callosum, hypotonia, developmental delay, hypopigmentation, cataract, cardiomyopathy, and immunological abnormalities. Recurrent infections, mainly affecting the respiratory tract, have been reported in the majority of cases, representing an important risk factor for morbidity and mortality. The immunological phenotype of patients is extremely variable, ranging from a combined immunodeficiency to nearly normal immunity. We report on a new patient with Vici syndrome, in whom we have extensively investigated immunological features. Despite a mild impairment of the cellular compartment, a defect of humoral immunity was found, requiring treatment with intravenous immunoglobulin. A wider knowledge of immune system abnormalities of Vici syndrome will help to plan strategies for treatment and prevention of infections, such as immunoglobulin replacement and antimicrobial prophylaxis, resulting in improved survival rates.

Abatacept (cytotoxic T lymphocyte antigen 4-immunoglobulin) improves B cell function and regulatory T cell inhibitory capacity in rheumatoid arthritis patients non-responding to anti-tumour necrosis factor-α agents

The use of biological agents combined with methotrexate (MTX) in rheumatoid arthritis (RA) patients has strongly improved disease outcome. In this study, the effects of abatacept on the size and function of circulating B and T cells in RA patients not responding to anti‐tumour necrosis factor (TNF)‐α have been analysed, with the aim of identifying immunological parameters helpful to choosing suitable tailored therapies. We analysed the frequency of peripheral B and T cell subsets, B cell function and T regulatory cell (Treg) inhibitory function in 20 moderate/severe RA patients, according to the European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) criteria, primary non‐responders to one TNF‐α blocking agent, who received abatacept + MTX. Patients were studied before and 6 months after therapy. We found that abatacept therapy significantly reduced disease activity score on 44 joints (DAS)/erythrocyte sedimentation rate (ESR) values without causing severe side effects. The size of the circulating B and T cell compartments in RA patients was not significantly different from healthy donors, but B cell proliferation and plasma cell differentiation was impaired before therapy and restored by abatacept. While Treg cell frequency was normal, its inhibitory function was absent before therapy and was partially recovered 6 months after abatacept. B and Treg cell function is impaired in RA patients not responding to the first anti‐TNF‐α agent. Abatacept therapy was able to rescue immune function and led to an effective and safe clinical outcome, suggesting that RA patients, in whom anti‐TNF‐α failed, are immunologically prone to benefit from an agent targeting a different pathway.

Human B-cell memory is shaped by age- and tissue-specific T-independent and GC-dependent events

Switched and IgM memory B cells execute different and noninterchangeable functions. We studied memory B cells in children of different ages, in peripheral blood and spleen and compared them with those of children born asplenic or unable to build germinal centers. We show that, whereas switched memory B cells are mostly generated in the germinal centers at all ages, IgM memory B cells can be distinct in three types with different developmental history. Innate IgM memory B cells, the largest pool in infants, are generated in the spleen by a germinal center‐independent mechanism. With age, if the spleen is present and germinal centers are functional, innate IgM memory B cells are remodelled and accumulate somatic mutations. The third type of IgM memory B cell is a by‐product of the germinal center reaction. Our data suggest that the B‐cell memory developmental program is implemented during the first 5–6 years of life.

B‐cell activation with CD40L or CpG measures the function of B‐cell subsets and identifies specific defects in immunodeficient patients

Around 65% of primary immunodeficiencies are antibody deficiencies. Functional tests are useful tools to study B‐cell functions in vitro. However, no accepted guidelines for performing and evaluating functional tests have been issued yet. Here, we report our experience on the study of B‐cell functions in infancy and throughout childhood. We show that T‐independent stimulation with CpG measures proliferation and differentiation potential of memory B cells. Switched memory B cells respond better than IgM memory B cells. On the other hand, CD40L, a T‐dependent stimulus, does not induce plasma cell differentiation, but causes proliferation of naïve and memory B cells. During childhood, the production of plasmablasts in response to CpG increases with age mirroring the development of memory B cells. The response to CD40L does not change with age. In patients with selective IgA deficiency (SIgAD), we observed that switched memory B cells are reduced due to the absence of IgA memory B cells. In agreement, IgA plasma cells are not generated in response to CpG. Unexpectedly, B cells from SIgAD patients show a reduced proliferative response to CD40L. Our results demonstrate that functional tests are an important tool to assess the functions of the humoral immune system.

The possible implication of the S250C variant of the autoimmune regulator protein in a patient with autoimmunity and immunodeficiency: in silico analysis suggests a molecular pathogenic mechanism for the variant.

Autoimmunity can develop from an often undetermined interplay of genetic and environmental factors. Rare forms of autoimmune conditions may also result from single gene mutations as for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, an autosomal recessive disease associated with mutated forms of the autoimmune regulator gene. It was proposed that genetic variability in the autoimmune regulator locus, in particular heterozygous loss-of-function mutations, might favor the development of organ-specific autoimmunity by affecting the presentation of self-antigens in the thymus. Indeed, heterozygous mutations of the autoimmune regulator gene were reported in patients with organ-specific autoimmunity. Also, in primary immunodeficiencies, a breakdown in central/peripheral tolerance frequently produces association with autoimmunity. The causative link may involve a common genetic background and several gene defects have been identified as putative culprits. We report a unique patient, a 14 year old male from Lazio region, affected by common variable immunodeficiency associated with autoimmune manifestations (alopecia, onychodystrophy) and heterozygote for the S250C variant located in the SAND domain of the autoimmune regulator gene protein. To our knowledge this is the first report of the S250C variant in a patient bearing this unusual combination of autoimmunity and immunodeficiency. To obtain insights into the possible molecular effects of the S250C variant, we have carried out an in silico analysis of the SAND domain structure of the autoimmune regulator protein. In particular, homology modeling has allowed us to observe that the cysteine introduced by the S250C variant is surrounded by cationic residues, and by means of molecular dynamics simulations together with pKa calculations, we have shown that these residues remain stably proximal to cysteine-250 lowering its pKa and thus conferring high chemical reactivity to the mutated residue. We propose that the enhanced reactivity of cysteine-250, which is likely to impair the protein function but probably insufficient to produce alone a phenotype as a heterozygous S250C variant due to compensation mechanisms, might become manifest when combined with other genetic/environmental factors. These results can provide the rationale for the patients unusual phenotype, shedding new light into the pathogenesis of the clinical association of autoimmunity and immunodeficiency.

Rituximab in a childhood-onset idiopathic refractory chronic inflammatory demyelinating polyneuropathy.

Childhood-onset chronic inflammatory demyelinating polyneuropathy (CIDP) are generally responsive to conventional immunosuppressant treatments. However about 20% of patients may be refractory to several treatments and the disease has poor outcome. Rituximab is becoming a promising treatment in selected adult cases of severe CIDPs. We report the effectiveness of Rituximab in a refractory childhood-onset CIDP and we suggest this treatment as an effective choice in unresponsive childhood CIDP.