IgM Memory Research Articles

Acute Bone Marrow GvHD Is Associated With Delayed B Cell Neogenesis and Impaired Natural Antibody Response After Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GvHD) and severe infections are main complications limiting the success of allogeneic hematopoietic stem cell transplantion (alloHSCT). Delayed B cell reconstitution followed by B cell immune dysfunction considerably contributes to an increased risk for life-threatening infections. Several studies have shown that B cell regeneration is impaired in patients with systemic GvHD. Bone marrow (BM) suppression is often observed in parallel as GvHD symptoms appear suggesting the BM as a target of GvHD. Thus far, little is known about mechanisms of BM dysfunction during GvHD in alloHSCT patients. In this study, we investigated the reconstitution kinetics of peripheral blood B cell subsets in adult acute leukemic patients (n=52) before and within six months after alloHSCT by flow cytometry and correlated the data with RT-PCR quantified numbers of kappa-deleting-recombination-excision-circles (KREC), which are stable episomal plasmids generated during BM B cell development. Furthermore, we determined specific B cell antibody responses after in vitrostimulation with CpG, CD40L and T cell cytokines by EliSPOT analysis. To investigate BM as a direct target of allo-reactive T cells we performed histopathological stainings of BM biopsy samples obtained 3-4 weeks after alloHSCT. T cells were detected by specific anti-CD3 antibody staining and osteoblasts were morphologically evaluated. We observed in all patients a profound B cell immune deficiency already pre-transplant that proceeded within the first months post alloHSCT (mean B cells/ml blood±SEM: 11±3 pretransplant, 3±1 day14, 3±1 day28 post alloHSCT; 83±13 healthy control). Onset of B cell reconstitution is characterized by transitional B cell recovery representing the first B cell subset which emigrates from the BM. B cell reconstitution occurred either early (37% of patients) with a strong increase of transitional B cells between days 60-90 (mean transitional B cells/ml blood±SEM: Day 60, 36±10) or late (33% of patients) with delayed recovering transitional B cells (Day 180, 5±2). KREC copy numbers correlated highly positive and significantly with transitional B cell numbers (Spearman 0.94, p=0.017). Less correlation was obtained with naïve and CD27+ memory B cell recovery. Delayed onset of B cell reconstitution was significantly associated with both presence of systemic acute GvHD and full-intensity conditioning therapy (GvHD 71% vs non-GvHD 32%, Fisher´s exact p=0.044; full-intensity 41% vs reduced-intensity 5%, p=0.016). Supporting the hypothesis of bone marrow GvHD we could show a stronger infiltration of CD3+ T cells in the BM in late than in early recovering patients (≥5% T cell infiltration: 64% vs 17%, p=0.010). This increased T-cell infiltration was associated with reduced numbers of osteoblasts, known in mice to support B cell lymphopoiesis (no/few osteoblasts: 65% vs 17%, p=0.011). Impaired B cell lymphopoiesis further resulted in a delayed naïve and IgM memory B cell recovery compared to early recovering patients. No recovery of switched-memory B cells was seen for both patient groups within the analyzed time-period. Functionally, ex vivoactivation of patient B cells revealed higher numbers of IgM producing B cells specific for pneumococcal polysaccharide (PnP) at day 180 post alloHSCT in early than in late recovering patients. Polyclonal IgG producing B cells were significantly diminished in all patients. We conclude from these data, that early onset of B cell reconstitution is characterized by strong increase in regenerating transitional B cells within three months after alloHSCT. Herein, KREC appears as a suitable biomarker to monitor BM B cell output post-transplant. B cell regeneration is significantly delayed in patients showing increased occurrence of systemic acute GvHD and stronger T cell infiltration with loss of osteoblasts in the BM. Thus, delayed onset of B cell reconstitution might result from acute BM GvHD in which alloreactive T cells lead to an osteoblast niche destruction. Increased PnP specific IgM antibody responses are most likely result of higher numbers of early reconstituted transitional and IgM memory B cells but not naïve B cells that were shown not to produce IgM upon CpG stimulation (Capolunghi F et al. 2008). Thus, early B cell reconstitution might provide a first natural antibody immunity after alloHSCT, emphasizing the importance of a functional bone marrow niche. Disclosures: No relevant conflicts of interest to declare.

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.

T Cell–Dependent IgM Memory B Cells Generated during Bacterial Infection Are Required for IgG Responses to Antigen Challenge

Immunological memory has long considered to be harbored in B cells that express high-affinity class-switched IgG. IgM-positive memory B cells can also be generated following immunization, although their physiological role has been unclear. In this study, we show that bacterial infection elicited a relatively large population of IgM memory B cells that were uniquely identified by their surface expression of CD11c, CD73, and programmed death-ligand 2. The cells lacked expression of cell surface markers typically expressed by germinal center B cells, were CD138 negative, and did not secrete Ab ex vivo. The population was also largely quiescent and accumulated somatic mutations. The IgM memory B cells were located in the region of the splenic marginal zone and were not detected in blood or other secondary lymphoid organs. Generation of the memory cells was CD4 T cell dependent and required IL-21R signaling. In vivo depletion of the IgM memory B cells abrogated the IgG recall responses to specific Ag challenge, demonstrating that the cell population was required for humoral memory, and underwent class-switch recombination following Ag encounter. Our findings demonstrate that T cell-dependent IgM memory B cells can be elicited at high frequency and can play an important role in maintaining long-term immunity during bacterial infection.

Tyrosine kinase inhibitors impair B-cell immune responses in CML through off-target inhibition of kinases important for cell signaling

AbstractTyrosine kinase inhibitors (TKIs) have significant off-target multikinase inhibitory effects. We aimed to study the impact of TKIs on the in vivo B-cell response to vaccination. Cellular and humoral responses to influenza and pneumococcal vaccines were evaluated in 51 chronic phase chronic myeloid leukemia (CML) patients on imatinib, or second-line dasatinib and nilotinib, and 24 controls. Following vaccination, CML patients on TKI had significant impairment of IgM humoral response to pneumococcus compared with controls (IgM titer 79.0 vs 200 U/mL, P = .0006), associated with significantly lower frequencies of peripheral blood IgM memory B cells. To elucidate whether CML itself or treatment with TKI was responsible for the impaired humoral response, we assessed memory B-cell subsets in paired samples collected before and after imatinib therapy. Treatment with imatinib was associated with significant reductions in IgM memory B cells. In vitro coincubation of B cells with plasma from CML patients on TKI or with imatinib, dasatinib, or nilotinib induced significant and dose-dependent inhibition of Bruton's tyrosine kinase and indirectly its downstream substrate, phospholipase-C-γ2, both important in B-cell signaling and survival. These data indicate that TKIs, through off-target inhibition of kinases important in B-cell signaling, reduce memory B-cell frequencies and induce significant impairment of B-cell responses in CML.

OP0138 B cell subsets phenotype in autoimmunity with immunodeficiency: Analysis of a large cohort of patients with apeced syndrome

BackgroundAutoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is a rare autosomal recessive syndrome due to mutations in AIRE gene, characterized by autoimmune endocrinopathies and mucocutaneous candidiasis. It is accompanied by generation...

Dependence of Immunoglobulin Class Switch Recombination in B Cells on Vesicular Release of ATP and CD73 Ectonucleotidase Activity

Immunoglobulin (Ig) isotype diversification by class switch recombination (CSR) is an essential process for mounting a protective humoral immune response. Ig CSR deficiencies in humans can result from an intrinsic B cell defect; however, most of these deficiencies are still molecularly undefined and diagnosed as common variable immunodeficiency (CVID). Here, we show that extracellular adenosine critically contributes to CSR in human naive and IgM memory B cells. In these cells, coordinate stimulation of B cell receptor and toll-like receptors results in the release of ATP stored in Ca(2+)-sensitive secretory vesicles. Plasma membrane ectonucleoside triphosphate diphosphohydrolase 1 CD39 and ecto-5'-nucleotidase CD73 hydrolyze ATP to adenosine, which induces CSR in B cells in an autonomous fashion. Notably, CVID patients with impaired class-switched antibody responses are selectively deficient in CD73 expression in B cells, suggesting that CD73-dependent adenosine generation contributes to the pathogenesis of this disease.

Role of human IgM memory B cells in influenza vaccine response (P4401)

Abstract Influenza virus-specific memory B cells are critical to mount an accelerated antibody response against subsequent influenza infections. While multiple layers of humoral memory are involved in the protection against influenza, the unique role of IgM memory B cells is largely unknown. In this study, we demonstrated that human IgM memory B cells are similar to class-switched memory B cells in their basic effector functions: the ability to proliferate and to present antigens to T cells. However, ELISPOT analysis of peripheral blood mononuclear cells revealed that the frequency of influenza-specific IgM memory B cells is 10-fold higher than that of IgG memory B cells and doesn’t readily change by seasonal vaccination. Interestingly, IgM memory B cells exhibited broad cross-reactivity against contemporary seasonal as well as pandemic strains. Serum antibody analysis showed that although the level of influenza-specific IgM was approximately 20-fold less than that of IgG, the breadth of cross-reactivity of IgM-secreting cells is broader than that of IgG counterparts. Given that a recent study in mice showed that the majority of IgM memory B cells are preferentially recruited to the ensuing secondary response, we propose a model that upon vaccination, some IgM memory B cells are directly differentiated into highly cross-reactive IgM-secreting plasmablasts to confer the host early line of cross-protection, while the rest of IgM memory B cells are recruited to the secondary response.

Impact of PPV-23 vaccine in HIV patients under long term HAART (P4262)

Abstract HIV infection is characterized by profound changes in the B cell compartment, which includes, impaired antigen specific response and a significant loss of IgM memory B cells, amongst other defects. Despite HAART’s success in reducing the incidence of opportunistic infections, invasive pneumococcal disease in the HIV infected population continues to be high. PPV-23 vaccine is recommended as a prophylactic measure in the HAART treated population; however, its serological benefit is currently unclear. Although, HAART has been highly successful in reconstituting T cell function, the effect of HAART on antigen specific B cell reconstitution remains to be elucidated. We developed a novel method to identify pneumococcal polysaccharide responding B cells in circulation by conjugating vaccine serotype 14 and 23 to fluorescent molecules. Using this method, we investigated the polysaccharide responding B cells in HIV cohorts on long term HAART. Preliminary data suggests severe depletion of polysaccharide responding IgM memory B cells in HAART treated adults comparable to HAART naïve group; a striking contrast to HIV uninfected adults. This is in alignment with the diminished anti pneumococcal antibody response observed in the HAART treated group. HAART is thus unable to fully reconstitute the functional B cell subset required for protection against S.pneumoniae.

Abstract 480: Maintenance immunotherapy following carboplatin (CBDCA) - pemetrexed (PEM) chemotherapy in advanced adenocarcinoma (ADK) of the lung: A phase II study.

Abstract Background: Continuation maintenance therapy with PEM vs. placebo has been shown to improve progression-free survival (PFS) and overall survival (OS), in patients with advanced non squamous non small cell lung cancer, at the price of a substantial toxicity. Nevertheless, in these patients the observed lymphocytopenia induced by chemotherapy, is a negative independent prognostic factor. Bacterial extracts (OM-85) have been shown to enhance the activation of both IgM memory B lymphocytes (CD24+/CD27+ cells) and inteleukin-2 receptor-expressing lymphocytes (CD25+ cells) (Int J Immunopathol Pharmacol. 2006;19:551). In addition, we have demonstrated that 13-cis retinoic acid (RA) restored the chemotherapy-depressed immune function (Clin Cancer Res 2001;7:1251). With this rationale, we designed a phase II study of maintenance immunotherapy after first line CBDCA-PEM chemotherapy in order to maintain an achieved response, or at least stable disease, for as long as possible, with acceptable side effects. Methods: Histologically/cytologically confirmed patients with stage IIIB or IV non operable ADK of the lung with measurable disease, ECOG PS 0-2, age 18-75 years and adequate bone marrow, renal and liver function were eligible for the study. Treatment consisted of 6 courses of PEM 500 mg/m2 and CBDCA, AUC=5, on day 1 every 3 weeks. Patients with a clinical benefit were treated with OM-85, 7 mg/day, 10 days/month and RA 0.5 mg/kg, 5 days/week, 3 weeks/month, both given until progression. Results: From September 2007 to March 2012, 54 patients, with a median age of 65 years (range 33-75), 50% stage IIIB and 50% stage IV, were entered into the study. At a median follow up of 22 months, clinical benefit was observed in 41 patients (75%); Complete response 8 (14%), partial response 21 (39%), stable disease 12 (22%), progressive disease 13 (25%). One-year OS rate was 69%. After immunotherapy, a statistically significant (P<0.001) increase in lymphocyte number was observed in responders. Median PFS was 10.2 months, while median OS was 27.6 months. Four patients, rendered operable with treatment, underwent a radical surgical procedure. Grade 4 renal toxicity was observed in 1 patient, while grade 3-4 hematologic toxicity occurred in 9 patients (17%). Conclusion: This study shows that maintenance immunotherapy with the combination of OM-85 and RA, administered after chemotherapy in advanced ADK of the lung, is feasible and induces a statistically significant raise in lymphocyte number. In addition a favorable impact on PFS and OS was observed. Future randomized studies to investigate the role of maintenance immunotherapy strategies, are in program Citation Format: Francesco Recchia, Giampiero Candeloro, Alisia Cesta, Anna Di Blasio, Cornelia O.C. Recchia, Stefano Necozione, Giovambattista Desideri, Silvio Rea. Maintenance immunotherapy following carboplatin (CBDCA) - pemetrexed (PEM) chemotherapy in advanced adenocarcinoma (ADK) of the lung: A phase II study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 480. doi:10.1158/1538-7445.AM2013-480

Memory B cell compartment constitution and susceptibility to recurrent lower respiratory tract infections in young children

A proportion of children have recurrent LRTIs, mostly as a result of Spn, which persist after 2 years of age. Here, we investigate, by flow cytofluorometry, the constitution of the memory B cell compartment in 90 healthy children and 49 children with recurrent LRTIs to determine if an increased susceptibility to recurrent LRTIs results from a delayed or abnormal ontogeny with poor antibody-mediated protection. Total IgA, IgM, IgG, and IgG subclasses were measured by nephelometry, as well as antipneumococcal antibodies by ELISA. Pneumococcal vaccination status was obtained. We show that the memory B cells increase between birth and 2 years of age (1.6% vs. 21.1%, P<0.001) without further significant increase noted per additional years (3-4 years old: 23.3%; 4-5 years old: 22.2%, P>0.40) to reach adult-like values (31.8±11.8%, P=0.08). Proportions of switched and IgM memory B cells were similar in children and adults. Comparatively, LRTI children had no delay in the constitution of their memory B cell compartment (2-3 years old: 26.9%; 3-4 years old: 18.2%; 4-5 years old: 26.8%, P>0.05). Their switched and IgM memory B cells were similar among age categories, and the distribution was overall similar to that of healthy controls. LRTI children had normal total and pneumococcal serotype-specific antibody values but showed a rapid waning of antipneumococcal antibody levels after vaccination. In summary, our results show that the memory B cell compartment is already similarly constituted at 2 years of age in healthy and LRTI children and thus, cannot explain the increased susceptibility to bacterial pneumonia. However, the waning of antibodies might predispose children to recurrent infections in the absence of revaccination.

Follicular helper T cells may play an important role in the severity of primary Sjögren's syndrome

The aim of this study was to investigate the possible role of follicular helper T (TFH) cells in the pathogenesis of primary Sjögren's syndrome (pSS) by analyzing immune-competent cells and serological markers with special emphasis on clinical symptoms. We enrolled 50 pSS patients and 16 healthy individuals in the study. Patients had elevated ratio of peripheral TFH cells, however, when dividing patients into two groups defined by the presence of extraglandular manifestations (EGMs), only patients with EGMs differed from controls significantly. Moreover, TFH cell percentages correlated positively with both activated T cell and Tr1 cell values. On the contrary, TFH cell percentages showed negative correlation with both IgM and IgG memory B cell proportions. Elevated TFH percent\ages were observed in the anti-SSA/SSB positive patients, and also in patients with higher IL-12, IL-21 levels and focus score values. Increased TFH cell proportions seem to have an important role in disease development.

Evaluation of the B Cell Compartment in CVID and Respi Patients

S D A Y 819 Evaluation of the B Cell Compartment in CVID and Respi Patients Tracy Hwangpo, MD, PhD, Ewa Szymanksa, Cunren Liu, Marsha Brand, Elizabeth Brown, Harry Schroeder; UAB, UAB, AL. RATIONALE: Recent studies suggest that clinical symptoms in CVID can correlate with a reduction in the memory B cell compartment. We are characterizing various B cell populations of patients without disease, with CVID, and with REcurrent Sino-Pulmonary Infections and normal serum immunoglobulins (RESPI) as an effort to find distinguishing features among them. METHODS: Whole blood was collected from over 140 study subjects. Total lymphocytes, marginal zone-like B cells, IgM+ memory B cells, class-switched B cells, transitional B cells, immature B cells, and na€ive B cells were isolated and evaluated by flow cytometry. Immunoglobulins and HLA haplotyewere also examined. Unpaired t-test was used to analyze the means of these cells. RESULTS:We found that by disease phenotype, RESPI patients appear to have lower IgG levels compared to control patients. All B cell subsets were decreased in CVID patients, particularly classed-switchedmemory B cells. There is a gradient for marginal zone-like B cells, with CVID having the lowest numbers compared to RESPI and controls. IgM+ memory B cells were found to be lower in RESPI patients compared to controls. We also found patients with the B4/B88 haplotype tended to have lower B cell subsets. Analysis is ongoing for transitional, naive, and immature B cells. CONCLUSIONS: We postulate that the transition fromRESPI patients to CVID patientsmay involve the development of defects in B cell maturation signaling or memory B cell generation. We identified the distinctive features of lower IgM memory B cells in RESPI patients compared to controls. Characterizing the B cell compartment may elucidate their importance in disease identification and progression.

Basic and clinical immunology – 3036. Investigation of peripheral follicular helper T-cells in primary Sjögren's syndrome

Background The exact mechanisms behind the immune disturbances in primary Sjogrens syndrome (pSS) are still not known in details, but evidence suggests that abnormal T- and B-cell activation plays a critical role in the disease develop- ment. Recent studies emphasized the important role of follicular helper T (TFH) cells, which contribute to B-cell proliferation and differentiation, as well as antibody pro- duction. The aim of this study was to investigate the possi- ble role of TFH cells in the pathogenesis of pSS, by analyzing a wide spectrum of immune-competent cells and serological markers with a special emphasis on clinical symptoms of the disease. Methods Results Patients with pSS showed elevated ratio of peripheral TFH cells. However, when we divided patients into two groups defined by the presence or absence of EGMs, only patients with EGMs had significant differences, while values of patients without EGMs were similar to healthy controls. We found a significant positive correlation between acti- vated Ta nd TFH cell percentages as well as between the Tr1 and TFH cells. Significant negative correlations were observed between IgM memory B and TFH cell values and between IgG memory B and TFH cell proportions. Elevated TFH percentages were observed in the anti-SSA positive patients, compared to autoantibody negative patients and healthy controls. Moreover, the percentages of TFH cells were significantly elevated in patients with higher IL-12 and IL-21 levels. Conclusions Our data suggest that percentages of peripheral TFH cells are increased in pSS, especially in patients suffering from a systemic, more pronounced course of disease (pSS with EGMs). Moreover, the elevated TFH cell percentages cor- relates with activation of immune system, proportions of memory B cells and titers of autoantibodies, which implies that TFH cells may play an important role in the disease development.

Splenic hypofunction in patients with an incidental finding of small-sized spleen at abdominal ultrasound

Splenic hypofunction is an acquired condition, ranging from a reversible mild hyposplenism to severe splenic atrophy, and may be accompanied by a reduction in spleen size. It is potentially associated with a number of different diseases, including hematological, immunological, gastroenterological and iatrogenic disorders. Functional hyposplenism, as well as asplenia, has been shown to predispose to thromboembolic events, autoimmunity and infectious complications, the latter of which are the most widely recognized risk of these states [1]. In particular, a significantly higher relative risk of pneumococcal sepsis has been demonstrated in patients affected by splenic hypofunction or atrophy [2] due to reduced levels of circulating IgM memory B cells, a unique B cell population that needs the spleen for their survival and generation [3]. Nevertheless, the relationship between splenic function and size in hyposplenic individuals is unknown. Since a small-sized spleen can be easily detected in the course of an abdominal ultrasound examination, we aimed at evaluating splenic function through pitted erythrocyte counting in patients with an incidental finding of atrophic spleen at abdominal ultrasound. Between October 2009 and January 2011, 4,585 patients were referred to the Ultrasound Unit of our Department of Internal Medicine to undergo an abdominal ultrasound evaluation. The most frequent indications were abdominal pain (49 %), malignancy (37 %), liver and biliary disorders (10 %), bowel disorders (7 %), and nephrolithiasis (5 %). All images were obtained by an expert sonographer, using an Esaote model MyLab 70 XVG, with a 1–8 MHz convex probe and an ATL model HDI 3500, a 5.2 MHz convex probe (Philips Medical Systems, Bothel, Washington). The length of the spleen, defined as the maximum distance between the dome and the tip of the spleen, was measured on a longitudinal section with the patient in the supine or right lateral decubitus position and on deep inspiration. Patients, who were diagnosed as having a small spleen: defined as a spleen length B8 cm in men and B7.5 cm in women [4], underwent peripheral blood collection for the evaluation of spleen function. Splenic function was assessed by counting the number of pitted red cells—that is, erythrocytes bearing membrane abnormalities visible under interference phase microscopy as a so-called ‘pit’. Briefly, one drop of fresh venous blood was taken and mixed with 0.5 ml 3 % buffered glutaraldehyde solution, pH 7.4, in a plastic tube. One thousand erythrocytes were examined in a wet preparation (magnification, 1,0009) with a directinterference phase microscope Leica DMLB (Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) equipped with Nomarsky optics by the same experienced observer unaware of the study. The percentage of pitted erythrocytes was calculated and taken as a measure of splenic function (upper limit of normal = 4 %) [5]. A peripheral blood sample was also collected from 52 splenectomized patients, who were used as positive controls. Data were analyzed in the GraphPad Prism statistical PC program (GraphPad Software, San Diego, CA) using the paired t test and the Mann–Whitney U test. Correlations were studied by Spearman’s rank correlation test. A level of p \ 0.05 was considered statistically significant. A small spleen was found in 203 out of 4,585 patients. Only 128 (mean age 61.5 years; 90 females and 38 males) A. Di Sabatino (&) G. Carnevale Maffe L. Brunetti M. Guerci G. R. Corazza First Department of Medicine, Fondazione IRCCS Policlinico San Matteo, University of Pavia, Piazzale Golgi 19, 27100 Pavia, Italy e-mail: a.disabatino@smatteo.pv.it

PD-1 is a novel regulator of human B-cell activation

The outcome of the adaptive immune response is determined by the integration of both positive and negative signals, respectively, induced upon the triggering of co-signaling receptors. One of them, programmed cell death 1 (PDCD1/PD-1) has largely been shown to be involved in the negative regulation of T-cell activation. However, PD-1 is also expressed on human B cells, and its role(s) in the process of human B-cell activation remains uncertain thus far. In this study, we describe the expression of PD-1 on the major human B-cells subsets isolated from peripheral blood and lymph nodes. We showed that PD-1 was expressed on naive B cells, was differentially expressed on peripheral IgM memory as compared with memory B cells and was lost on germinal center B cells. Expression of PD-1 ligands (PD-Ls) was induced by TLR9 activation. Finally, we showed that PD-1 was recruited to the B-cell receptor upon triggering. We determined that during TLR9 activation, blockade of PD-1/PD-Ls pathways indeed increased B-cell activation, proliferation and the production of inflammatory cytokines. Altogether, our results show, that, as reported in T cells, PD-1/PD-Ls complexes acted as inhibitors of the B-cell activation cascade and highlight the importance of devising future therapies able to modulate lymphocyte activation through the targeting of the PD-1/PD-Ls pathways.

From impending toxic megacolon to multiple organ failure in severe ulcerative colitis

We thank Hokama et al. [1] for their appreciation of our review on ulcerative colitis [2]. The case they describe of a patient affected by ulcerative colitis, which was complicated by impending toxic megacolon, gives us the opportunity to focus attention on a problem that has been somewhat neglected in the literature, i.e. the possible evolution of severe ulcerative colitis into toxic megacolon, and finally multiple organ failure. Apart from a few sporadic case reports, the only two studies in the literature with adequate large numbers of patients are both from the same group: a retrospective study by Caprilli et al. [3] and a prospective one by Latella et al. [4], on, respectively, 180 and 45 patients with severe ulcerative colitis. If we compare the findings from the two different studies, we observe that the prevalence of complications, surgery, mortality and multiple organ failure is quite comparable (Table 1). Impending toxic megacolon represents the most frequent complication, followed by toxic megacolon, hemorrhage and perforation. The patients who more frequently develop multiple organ failure are those at the onset of the disease, with extensive disease (pancolitis), and in most cases the multiple organ failure is preceded by impending toxic megacolon or toxic megacolon [3, 4]. Of note, multiple organ failure is burdened with a high mortality rate, ranging from 72 [3] to 100 % [4], and may occur even after colectomy in 27–50 % of cases (late multiple organ failure.) The development of pneumococcal sepsis and subsequent late multiple organ failure seems to be more common in ulcerative colitis patients with extensive disease, and complicated by splenic hypofunction or atrophy. The defect of circulating IgM memory B cell pool—a unique B cell population needing the spleen for their survival and responsible for the defence against infections by encapsulated bacteria—which affects almost half of the patients with ulcerative colitis may provide a pathophysiological explanation for this finding [5]. In conclusion, multiple organ failure is an important cause of mortality in severe ulcerative colitis, and the radiological finding of impending toxic megacolon may make it possible to identify a subset of patients with severe ulcerative colitis at risk for toxic megacolon and subsequent multiple organ failure [3, 4]. However, both these studies were conducted in the pre-biologic era, and due to the revolutionary impact of the monoclonal anti-TNF-a antibody infliximab in the treatment of inflammatory bowel diseases, it would be interesting to ascertain whether the prevalence of toxic megacolon and following multiple organ failure has changed in severe ulcerative colitis during the last decade. A. Di Sabatino (&) P. Giuffrida G. R. Corazza Clinica Medica I, Centro per lo Studio e la Cura delle Malattie Infiammatorie Croniche Intestinali, Fondazione IRCCS Policlinico S. Matteo, Universita di Pavia, Piazzale Golgi 5, 27100 Pavia, Italy e-mail: a.disabatino@smatteo.pv.it

Reduced CD19 expression and decreased memory B cell numbers in transient hypogammaglobulinemia of infancy

We aimed to evaluate the role of the CD19 complex in the pathogenesis of transient hypogammaglobulinemia of infancy (THI) and to better characterize the subsets of memory B cells. The study population consisted of 22 male and 14 female patients with a mean age at presentation of 20±9.9months. The CD19 complex and B cell subsets were evaluated by flow cytometry. While the CD19 median fluorescence index (MFI) in patients with THI was significantly lower than controls (122.9±66.7 in patients; 184.2±39 in controls, p<0.01), expression of CD21 and CD81 was increased (94.4±3, 96.8±2.5% in patients; 91±3.9; 94.7±3.5% in controls, p<0.01 vs. p<0.05, respectively). The expressions of switched memory B cells and IgM memory B cells were found to be reduced in THI. Considering that the CD19 complex regulates the events following antigen stimulation, the change in CD19 complex detected in THI may be related to insufficiency of antibody production.

Chronic Hepatitis C Virus Infection Breaks Tolerance and Drives Polyclonal Expansion of Autoreactive B Cells

Chronic Hepatitis C virus (HCV) infection has been linked with B cell lymphoproliferative disorders and several autoimmune-related diseases. The mechanisms of how chronic viral infection affects B cell development and predisposes the patients to autoimmune manifestations are poorly understood. In this study, we established an experimental system to probe the B cell responses and characterize the antibodies from chronic-HCV-infected individuals. We identified an unusual polyclonal expansion of the IgM memory B cell subset in some patients. This B cell subset is known to be tightly regulated, and autoreactive cells are eliminated by tolerance mechanisms. Genetic analysis of the immunoglobulin (Ig) heavy chain variable gene (V(H)) sequences of the expanded cell population showed that the levels of somatic hypermutation (SHM) correlate with the extent of cell expansion in the patients and that the V(H) genes exhibit signs of antigen-mediated selection. Functional analysis of the cloned B cell receptors demonstrated autoreactivity in some of the expanded IgM memory B cells in the patients which is not found in healthy donors. In summary, this study demonstrated that, in some patients, chronic HCV infection disrupts the tolerance mechanism that normally deletes autoreactive B cells, therefore increasing the risk of developing autoimmune antibodies. Long-term follow-up of this expanded B cell subset within the infected individuals will help determine whether these cells are predictors of more-serious clinical manifestations.

Single cell network profiling (SCNP) assay allows for the identification of heterogeneity in the activation of multiple signaling pathways in distinct human B cell subsets (174.12)

Abstract The specific functions of phenotypically defined B cell subsets in the regulation of protective immune responses and the pathogenesis of immune-mediated diseases is incompletely understood. SCNP is a multi-parametric flow cytometry based approach that simultaneously measures intracellular signaling in multiple cell subsets in heterogeneous tissues. In this study, SCNP was applied to examine 34 signaling nodes (paired modulator/intracellular readout) in B cell subsets (CD27-IgM+IgD+ naïve B cells, total CD27+ memory B cells, CD27+IgM-IgD- switched memory B cells, CD27+IgM+IgD+ IgM memory B cells) in peripheral blood mononuclear cells from 6 healthy donors. Signaling responses in B cell subsets were masked when the total B cell population was analyzed. Specifically, a higher responsiveness was observed in the memory B cell subset relative to the naïve; 14 signaling nodes showed statistically significant differences between these subsets (p&amp;lt;.05, paired Wilcoxon test) of which 12 displayed stronger activation in response to CD40L, SDF1α, PMA, IFNα, IFNγ, IL10 and IL27 modulation in the memory B cells. 11 signaling nodes differed significantly in IgM memory vs. switched memory B cells (p&amp;lt;.05, paired Wilcoxon test); 7/11 displayed stronger activation in response to CpG, R848, thapsigargin, IFNγ and IL10 modulation in IgM memory B cells. An increased understanding of B cell subset signaling may allow for the development of more effective B cell targeted therapies.

IgM memory B cells generated during bacterial infection are required for secondary IgG responses to antigenic challenge (43.2)

Abstract Immunological memory was long considered to be harbored in B cells that express high affinity class-switched IgG. More recently, IgM memory B cells have been identified following immunization with nominal antigens and adjuvants. Based on the expression of CD11c, we have identified a large population of such cells using a natural model of infection by the bacterium Ehrlichia muris. These CD11c+ IgM memory cells exhibit phenotypic characteristics of memory B cells, including expression of CD73, CD11b, and PD-L2. In addition, these CD11c+ IgM memory cells lack expression of CD138, are largely quiescent, and have accumulated somatic mutations. Although these cells did not proliferate or secrete antibody ex vivo, they produced antigen-specific IgM upon in vitro stimulation with mitogens. Furthermore, in vivo depletion abrogated the IgG recall response to specific antigenic challenge. Our findings are consistent with previous data demonstrating that IgM memory cells undergo class switch recombination and affinity maturation following re-encounter with cognate antigen. We propose that these CD11c+ IgM memory B cells are responsible for the IgG produced following secondary challenge in ehrlichial infection, and likely function to provide greater flexibility to variant antigenic challenges.