Selective emergence of antibody-secreting cells in the multiple sclerosis brain.

BACKGROUND: Although distinct brain-homing B cells have been identified in multiple sclerosis (MS), it is unknown how these further evolve to contribute to local pathology. We explored B-cell maturation in the central nervous system (CNS) of MS patients and determined their association with immunoglobulin (Ig) production, T-cell presence, and lesion formation. METHODS: Ex vivo flow cytometry was performed on post-mortem blood, cerebrospinal fluid (CSF), meninges and white matter from 28 MS and 10 control brain donors to characterize B cells and antibody-secreting cells (ASCs). MS brain tissue sections were analysed with immunostainings and microarrays. IgG index and CSF oligoclonal bands were measured with nephelometry, isoelectric focusing, and immunoblotting. Blood-derived B cells were cocultured under T follicular helper-like conditions to evaluate their ASC-differentiating capacity in vitro. FINDINGS: ASC versus B-cell ratios were increased in post-mortem CNS compartments of MS but not control donors. Local presence of ASCs associated with a mature CD45low phenotype, focal MS lesional activity, lesional Ig gene expression, and CSF IgG levels as well as clonality. In vitro B-cell maturation into ASCs did not differ between MS and control donors. Notably, lesional CD4+ memory T cells positively correlated with ASC presence, reflected by local interplay with T cells. INTERPRETATION: These findings provide evidence that local B cells at least in late-stage MS preferentially mature into ASCs, which are largely responsible for intrathecal and local Ig production. This is especially seen in active MS white matter lesions and likely depends on the interaction with CD4+ memory T cells. FUNDING: Stichting MS Research (19-1057 MS; 20-490f MS), National MS Fonds (OZ2018-003).

Dysregulated B cell differentiation towards antibody-secreting cells in neuromyelitis optica spectrum disorder.

BACKGROUND: Anti-aquaporin 4 (AQP4) antibody (AQP4-Ab) is involved in the pathogenesis of neuromyelitis optica spectrum disorder (NMOSD). However, the mechanism involved in AQP4-Ab production remains unclear. METHODS: We analyzed the immunophenotypes of patients with NMOSD and other neuroinflammatory diseases as well as healthy controls (HC) using flow cytometry. Transcriptome analysis of B cell subsets obtained from NMOSD patients and HCs was performed. The differentiation capacity of B cell subsets into antibody-secreting cells was analyzed. RESULTS: The frequencies of switched memory B (SMB) cells and plasmablasts were increased and that of naïve B cells was decreased in NMOSD patients compared with relapsing-remitting multiple sclerosis patients and HC. SMB cells from NMOSD patients had an enhanced potential to differentiate into antibody-secreting cells when cocultured with T peripheral helper cells. Transcriptome analysis revealed that the profiles of B cell lineage transcription factors in NMOSD were skewed towards antibody-secreting cells and that IL-2 signaling was upregulated, particularly in naïve B cells. Naïve B cells expressing CD25, a receptor of IL-2, were increased in NMOSD patients and had a higher potential to differentiate into antibody-secreting cells, suggesting CD25+ naïve B cells are committed to differentiate into antibody-secreting cells. CONCLUSIONS: To the best of our knowledge, this is the first study to demonstrate that B cells in NMOSD patients are abnormally skewed towards antibody-secreting cells at the transcriptome level during the early differentiation phase, and that IL-2 might participate in this pathogenic process. Our study indicates that CD25+ naïve B cells are a novel candidate precursor of antibody-secreting cells in autoimmune diseases.

Intravenous Ig Regulates Anti-Desmoglein 3 IgG Production in B220- Antibody-Producing Cells in Mice with Pemphigus Vulgaris.

Intravenous Ig (IVIG) is a treatment option for intractable cases of pemphigus vulgaris (PV), an autoimmune blistering disease caused by autoantibodies against desmoglein 3 (DSG3). To investigate the efficacy of IVIG on autoantibody secretion, we produced PV model mice by adoptive transfer of immunized Dsg3-/- splenocytes to Rag2-/- mice. We found that circulating anti-DSG3 IgG ELISA titer decreased in PV model mice after 5 days of treatment with IVIG compared with PBS-treated mice, whereas the F(ab')2 fragment did not suppress the anti-DSG3 IgG titer. enzyme-linked immunospot assay revealed that IVIG treatment reduced the frequency of anti-DSG3 antibody-secreting cells in the spleen but not in lymph nodes and bone marrow. Moreover, this reduction was observed only in the splenic B220- fraction but not in the B220+ fraction. Furthermore, IVIG decreased the serum levels of anti-DSG3 IgG, even after a significant reduction of its titer, owing to antibody-mediated CD20+ B cell depletion. In addition, IVIG suppressed anti-DSG3 IgG production in B220-CD138+ plasma cells derived from PV model mice ex vivo. These results indicate that IVIG reduced autoantibody production in B220- cells containing plasma cells in PV model mice, and this function may indicate one of the mechanisms of action of IVIG on PV.

Intermittent Fasting Aggravates Lupus Nephritis through Increasing Survival and Autophagy of Antibody Secreting Cells in MRL/lpr Mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which the main contributors to organ damage are antibodies against autoantigens, such as double-stranded DNA (dsDNA). Calorie restriction and intermittent fasting (IF) have been shown to improve autoimmune disease symptoms in patients and animal models. Here, we tested the hypothesis that IF might improve symptoms in MRL/lpr mice, which spontaneously develop an SLE-like disease. Groups of mice were fed every other day (IF) or provided food ad libitum (controls), and various lupus-associated clinicopathological parameters were analyzed for up to 28 weeks. Contrary to expectations, anti-dsDNA antibody levels, immune complex deposition in the kidney, and glomerular injury were higher in the IF group than the control group, although there were no differences in spleen and lymph node weights between groups. Proteinuria was also worsened in the IF group. IF also increased the abundance of B cells, plasmablasts, and plasma cells and elevated autophagy in plasma cells in the spleen and lymph nodes. Secretion of anti-dsDNA antibody by splenocytes in vitro was reduced by chloroquine-induced inhibition of autophagy. These results suggest that IF exacerbates lupus nephritis in MRL/lpr mice by increasing autoantibody immune complex formation.

Detection of antibody-secreting cells specific for the cytomegalovirus and herpes simplex virus surface antigens.

Infections with the herpes simplex virus (HSV) and the human cytomegalovirus (HCMV) can lead to life-threatening diseases, particularly in immunosuppressed patients. Furthermore, HSV infections at birth (herpes neonatorum) can result in a disseminated disease associated with a fatal multiorgan failure. Congenital HCMV infections can result in miscarriage, serious birth defects or developmental disabilities. Antibody-based interventions with hyperimmunoglobulins showed encouraging results in clinical studies, but clearly need to be improved. The isolation of highly neutralizing monoclonal antibodies is a promising strategy to establish potent therapy options against HSV and HCMV infections. Monoclonal antibodies are commonly isolated from hybridomas or EBV-immortalized B-cell clones. The screening procedure to identify virus-specific cells from a cell mixture is a challenging step, since most of the highly neutralizing antibodies target complex conformational epitopes on the virus surface. Conventional assays such as ELISA are based on purified viral proteins and inappropriate to display complex epitopes. To overcome this obstacle, we have established two full-virus based methods that allow screening for cells and antibodies targeting complex conformational epitopes on viral surface antigens. The methods are suitable to detect surface antigen-specific cells from a cell mixture and may facilitate the isolation of highly neutralizing antibodies against HSV and HCMV.

Hybridoma Technology.

The generation of hybridoma cell lines by the fusion of splenocytes from immunized mice with immortal myeloma cells is a well-established method for the production of monoclonal antibodies. Although other methods have emerged as an effective alternative for the generation of monoclonal antibodies, the use of hybridoma technology remains a viable technique that is accessible to a wide number of laboratories that perform basic cell biological research. Hybridoma technology represents a relatively simple procedure at minimal cost for the continuous production of native whole immunoglobulins. This chapter will describe the materials and methodologies needed for the successful generation of monoclonal antibody (mAb)-producing hybridoma cell lines against target antigens.

Endoplasmic reticulum stress and the unfolded protein response: targeting the Achilles heel of multiple myeloma.

Multiple myeloma is characterized by the malignant proliferating antibody-producing plasma cells in the bone marrow. Despite recent advances in therapy that improve the survival of patients, multiple myeloma remains incurable and therapy resistance is the major factor causing lethality. Clearly, more effective treatments are necessary. In recent years it has become apparent that, as highly secretory antibody-producing cells, multiple myeloma cells require an increased capacity to cope with unfolded proteins and are particularly sensitive to compounds targeting proteostasis such as proteasome inhibitors, which represent one of the most prominent new therapeutic strategies. Because of the increased requirement for dealing with secretory proteins within the endoplasmic reticulum, multiple myeloma cells are heavily reliant for survival on a set of signaling pathways, known as the unfolded protein response (UPR). Thus, directly targeting the UPR emerges as a new promising therapeutic strategy. Here, we provide an overview of the current understanding of the UPR signaling in cancer, and outline its important role in myeloma pathogenesis and treatment. We discuss new therapeutic approaches based on targeting the protein quality control machinery and particularly the IRE1α/XBP1 axis of the UPR.

Cryptic B cell response to renal transplantation.

Transplantation reliably evokes allo-specific B cell and T cell responses in mice. Yet, human recipients of kidney transplants with normal function usually exhibit little or no antibody specific for the transplant donor during the early weeks and months after transplantation. Indeed, the absence of antidonor antibodies is taken to reflect effective immunosuppressive therapy and to predict a favorable outcome. Whether the absence of donor-specific antibodies reflects absence of a B cell response to the donor, tolerance to the donor or immunity masked by binding of donor-specific antibodies to the graft is not known. To distinguish between these possibilities, we devised a novel ELISPOT, using cultured donor, recipient and third-party fibroblasts as targets. We enumerated donor-specific antibody-secreting cells in the blood of nine renal allograft recipients with normal kidney function before and after transplantation. Although none of the nine subjects had detectable donor-specific antibodies before or after transplantation, all exhibited increases in the frequency of donor-specific antibody-secreting cells eight weeks after transplantation. The responses were directed against the donor HLA-class I antigens. The increase in frequency of donor-specific antibody-secreting cells after renal transplantation indicates that B cells respond specifically to the transplant donor more often than previously thought.

Assessment of the immunotoxic potential of trichloroethylene and perchloroethylene in rats following inhalation exposure.

The immunotoxic potential of trichloroethylene (TCE) and perchloroethylene (PERC) was assessed after inhalation exposure through the evaluation of the antibody forming cell (AFC) response to sheep red blood cells (SRBC). Female Sprague-Dawley rats were exposed to TCE or PERC vapor at 0, 100, 300, or 1000 ppm for 6 h/day, 5 days/week for 4 weeks (20 exposure days). Additional 0 ppm control groups were included and were dosed with cyclophosphamide via intraperitoneal injection to serve as positive immunosuppressive controls in the SRBC assay. Additional end-points evaluated included liver, kidney, spleen, and thymus weights, hematology, cellular differentials in bronchoalveolar lavage fluid, histopathology of select tissues, and assessment of the phagocytic activity of pulmonary alveolar macrophage (PERC only). Exposure to the high concentration of TCE (1000 ppm) resulted in increases in relative liver and kidney weights and suppression of AFC responses (AFC/spleen and AFC/10(6) spleen cells) by ≈ 70%, while no treatment-related effects were noted at 100 and 300 ppm. Animals exposed to PERC at levels of 300 or 1000 ppm had statistically significant increases in relative liver weights that were accompanied by very slight hypertrophy of the centrilobular hepatocytes. Animals exposed to PERC did not demonstrate a treatment-related effect on the AFC response and no effect was noted on the phagocytic activity of pulmonary alveolar macrophages. The results of these studies indicate that TCE had immunotoxic potential only at high exposure concentrations (1000 ppm), while PERC, at similar exposure concentrations, did not display any evidence of immunotoxicity.

The adjuvant LT-K63 can restore delayed maturation of follicular dendritic cells and poor persistence of both protein- and polysaccharide-specific antibody-secreting cells in neonatal mice.

Ab responses in early life are low and short-lived; therefore, induction of protective immunity requires repeated vaccinations. One of the major limitations in early-life immunity is delayed maturation of follicular dendritic cells (FDCs), which play a central role in mediating the germinal center (GC) reaction leading to production of Ab-secreting cells (AbSCs). We assessed whether a nontoxic mutant of Escherichia coli heat-labile enterotoxin (LT-K63) and CpG1826 as model adjuvants could accelerate FDC maturation and immune response in neonatal mice, using a pneumococcal polysaccharide of serotype 1 conjugated to tetanus toxoid (Pnc1-TT) as a model vaccine. In neonatal NMRI mice, a single dose of Pnc1-TT coadministered with LT-K63 enhanced Pnc1-TT-induced GC reaction. In contrast, CpG1826 had no effect. Accordingly, LT-K63, but not CpG1826, accelerated the maturation of FDC networks, detected by FDC-M2(+) staining, characteristic for adult-like FDCs. This coincided with migration of MOMA-1(+) macrophages into the GCs that can enhance GC reaction and B cell activation. The FDC-M2(+) FDC networks colocalized with enhanced expression of TNF-α, which is critical for the maintenance of mature FDCs and is poorly expressed in neonates. The accelerated maturation of FDC networks correlated with increased frequency and prolonged persistence of polysaccharide- and protein-specific IgG(+) AbSCs in spleen and bone marrow. Our data show for the first time, to our knowledge, that an adjuvant (LT-K63) can overcome delayed maturation of FDCs in neonates, enhance the GC reaction, and prolong the persistence of vaccine-specific AbSCs in the BM. These properties are attractive for parenteral vaccination in early life.

Persistence of immunoglobulin-producing cells in parotid salivary glands of patients with primary Sjögren's syndrome after B cell depletion therapy.

OBJECTIVES: To assess the persistence of immunoglobulin-producing cell populations in the parotid salivary glands of patients with primary Sjögren's syndrome (pSS) after B cell depletion therapy with rituximab. METHODS: Thirteen patients with pSS and four control patients were included in this study. Patients with pSS were treated with rituximab or placebo. Sequence analysis was carried out on IgA- and IgG-encoding transcripts extracted from parotid salivary gland biopsy specimens taken before treatment and at 12-16 and 36-52 weeks after treatment. RESULTS: At baseline, many clonally related sequences were seen in patients with pSS. The number of clonal expansions was significantly higher in patients with pSS than in control patients. Clonal expansions were composed of IgA- and/or IgG-expressing cells. Rituximab did not significantly alter the degree of clonal expansions. Groups of clonally related cells had members which were shared between biopsy specimens taken before and after treatment. Mutation frequencies of immunoglobulin sequences from clonally related cells in patients with pSS were higher after treatment. CONCLUSIONS: Rituximab treatment does not alter the characteristic features of increased clonal expansions seen in the parotid salivary glands of patients with pSS. The presence of clonally related immunoglobulin-producing cells before and after rituximab treatment strongly suggests that immunoglobulin-producing cells persist in salivary glands of patients with pSS despite B cell depletion. The presence of mixed isotype expression within groups of clonally related cells indicates local class switching in salivary glands of patients with pSS. Persistent immunoglobulin-producing cells may underlie disease relapse after treatment.

IL-5 and IL-17A are critical for the chronic IgE response and differentiation of long-lived antibody-secreting cells in inflamed tissues.

Prolonged survival of long-lived antibody-secreting cells in the BM has been implicated as a key component of long-term humoral immunity. The current study was designed to uncover the extrinsic signals required for the generation and maintenance of ASC in several niches (peritoneum, spleen and bone-marrow). Our results show that protein mixture of the Thalassophryne nattereri venom induced a chronic Th2 humoral response that is characterized by splenic hyperplasia with GC formation and venom retention by follicular DCs. Retention of B1a in the BM were observed. In the late phase (120d) of chronic venom-response the largest pool of ASC into the peritoneal cavity consisted of B220(neg)CD43(high) phenotype; the largest pool of ASC into spleen was constituted by B220 positive cells (B220(high) and B220(low)), whereas the largest pool of ASC into in the BM was constituted by the B220(high)CD43(low) phenotype; and finally, terminally differentiated cells (B220(neg)CD43(high)) were only maintained in the inflamed peritoneal cavity in late phase. After 120d a sustained production of cytokines (KC, IL-5, TNF-α, IL-6, IL-17A and IL-23) and leukocytes recruitment (eosinophils, mast cells, and neutrophils) were induced. IL-5- and IL-17A-producing CD4+ CD44+ CD40L+ Ly6C+ effector memory T cells were also observed in peritoneal cavity. Finally, treatment of venom-mice with anti-IL-5- and anti-IL17A-neutralizing mAbs abolished the synthesis of specific IgE, without modifying the splenic hyperplasia or GC formation. In addition, IL-5 and IL-17A negatively regulated the expansion of B1a in peritoneal cavity and BM, and promoted the differentiation of these cells in spleen. And more, IL-5 and IL-17A are sufficient for the generation of ASC B220(neg) in the peritoneal cavity and negatively regulate the number of ASC B220(pos), confirming that the hierarchical process of ASC differentiation triggered by venom needs the signal derived from IL-5 and IL-17A.

Protective antiviral antibody responses in a mouse model of influenza virus infection require TACI.

Antiviral Abs, for example those produced in response to influenza virus infection, are critical for virus neutralization and defense against secondary infection. While the half-life of Abs is short, Ab titers can last a lifetime due to a subset of the Ab-secreting cells (ASCs) that is long lived. However, the mechanisms governing ASC longevity are poorly understood. Here, we have identified a critical role for extrinsic cytokine signals in the survival of respiratory tract ASCs in a mouse model of influenza infection. Irradiation of mice at various time points after influenza virus infection markedly diminished numbers of lung ASCs, suggesting that they are short-lived and require extrinsic factors in order to persist. Neutralization of the TNF superfamily cytokines B lymphocyte stimulator (BLyS; also known as BAFF) and a proliferation-inducing ligand (APRIL) reduced numbers of antiviral ASCs in the lungs and bone marrow, whereas ASCs in the spleen and lung-draining lymph node were surprisingly unaffected. Mice deficient in transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a receptor for BLyS and APRIL, mounted an initial antiviral B cell response similar to that generated in WT mice but failed to sustain protective Ab titers in the airways and serum, leading to increased susceptibility to secondary viral challenge. These studies highlight the importance of TACI signaling for the maintenance of ASCs and protection against influenza virus infection.

Interferon-α induces unabated production of short-lived plasma cells in pre-autoimmune lupus-prone (NZB×NZW)F1 mice but not in BALB/c mice.

IFN-α is known to play a critical role in the pathogenesis of systemic lupus erythematosus (SLE), but the mechanisms remain unclear. We previously showed that within weeks, exposure to IFN-α in vivo induces lupus in pre-autoimmune lupus-prone NZB×NZW F1 (NZB/W) but not in BALB/c mice. In the current study, we show that in vivo expression of IFN-α induces sustained B-cell proliferation in both BALB/c and NZB/W mice. In NZB/W but not BALB/c mice, B-cell proliferation was accompanied by a rapid and unabated production of autoantibody-secreting cells (ASCs) in secondary lymphoid organs, suggesting that a B-cell checkpoint is altered in the autoimmune background. The majority (>95%) of ASCs elicited in IFN-α-treated NZB/W mice were short-lived and occurred without the induction of long-lived plasma cells. A short course of cyclophosphamide caused a sharp drop in IFN-α-elicited short-lived plasma cells, but the levels recovered within days following termination of treatment. Thus, our work provides new insights into effectiveness and limitations of the current SLE therapies.

Increased frequency of immunoglobulin (Ig)A-secreting cells following Toll-like receptor (TLR)-9 engagement in patients with Kawasaki disease.

Kawasaki disease (KD) is an acute vasculitis affecting mainly infants and children. Human B cells express Toll-like receptor (TLR)-9, whose natural ligands are unmethylated cytosine-guanine dinucleotide (CpG) motifs characteristic of bacterial DNA. The aim of this study was to clarify the pathogenesis of KD analysing the activation status of peripheral blood mononuclear cells (PBMC), focusing on B lymphocyte activation and functions. Ten patients and 10 age-matched healthy donors were recruited from the Bambino Gesù Hospital of Rome, Italy and enrolled into this study. We determined phenotype profile and immunoglobulin (Ig) production of PBMC from KD patients and age-matched controls. We found that the frequency of CD19(+) B lymphocytes and CD19(+) /CD86(+) activated B lymphocytes from KD patients during the acute phase before therapy was increased significantly. Moreover, B lymphocytes of acute-phase KD patients were more prone to CpG oligodeoxynucleotide (ODN) activation compared with the age-matched controls, as assessed by a significant increase of the number of IgA-secreting cells (SC). In the same patients we found a marked increase of IgM, IgG, interleukin (IL)-6 and tumour necrosis factor (TNF)-α production compared with the control group. In addition, in two convalescent KD patients, conventional treatment with intravenous immunoglobulin (IVIG) restored the normal frequency of CD19(+) B cells, the number of IgA-, IgM- and IgG-SC and the production of IL-6 and TNF-α. Our findings indicate that the percentages of peripheral B lymphocytes of acute-phase KD patients are increased and are prone to bacterial activation in terms of increased numbers of IgA-SC and increased production of IL-6 and TNF-α inflammatory cytokines. Thus, our data support the hypothesis of an infectious triggering in KD.

Homeostatic expansion of autoreactive immunoglobulin-secreting cells in the Rag2 mouse model of Omenn syndrome.

Hypomorphic RAG mutations, leading to limited V(D)J rearrangements, cause Omenn syndrome (OS), a peculiar severe combined immunodeficiency associated with autoimmune-like manifestations. Whether B cells play a role in OS pathogenesis is so far unexplored. Here we report the detection of plasma cells in lymphoid organs of OS patients, in which circulating B cells are undetectable. Hypomorphic Rag2(R229Q) knock-in mice, which recapitulate OS, revealed, beyond severe B cell developmental arrest, a normal or even enlarged compartment of immunoglobulin-secreting cells (ISC). The size of this ISC compartment correlated with increased expression of Blimp1 and Xbp1, and these ISC were sustained by elevated levels of T cell derived homeostatic and effector cytokines. The detection of high affinity pathogenic autoantibodies toward target organs indicated defaults in B cell selection and tolerance induction. We hypothesize that impaired B cell receptor (BCR) editing and a serum B cell activating factor (BAFF) abundance might contribute toward the development of a pathogenic B cell repertoire in hypomorphic Rag2(R229Q) knock-in mice. BAFF-R blockade reduced serum levels of nucleic acid-specific autoantibodies and significantly ameliorated inflammatory tissue damage. These findings highlight a role for B cells in OS pathogenesis.

Expansion of immunoglobulin-secreting cells and defects in B cell tolerance in Rag-dependent immunodeficiency.

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs, but does not abrogate, V(D)J recombination activity. In spite of a severe block at the pro-B cell stage and profound B cell lymphopenia, significant serum levels of immunoglobulin (Ig) G, IgM, IgA, and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP-keyhole limpet hemocyanin were severely impaired, even after adoptive transfer of wild-type CD4(+) T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell-activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire, which is associated with defects in central and peripheral checkpoints of B cell tolerance, is an important, previously unrecognized, aspect of immunodeficiencies associated with hypomorphic RAG mutations.

Intraclonal competition inhibits the formation of high-affinity antibody-secreting cells.

Protective immunity requires a diverse, polyclonal B cell repertoire. We demonstrate that affinity maturation of the humoral response to a hapten is impaired when preexisting clonally restricted cells recognizing the hapten are dominant in the B cell repertoire. B1-8i(+/-) mice, which feature a high frequency of B cells with nitrophenyl (NP)-binding specificity, respond to NP-haptenated proteins with the production of NP-specific Abs, but affinity maturation is impaired due to insufficient generation of high-affinity Ab-producing cells. We manipulated the frequency of NP-specific B cells by adoptive transfer of B1-8 B cells into naive, wild-type recipients. Remarkably, when 10(4) B1-8 B cells were transferred, these cells supported efficient affinity maturation and plasma cell differentiation. In contrast, when 10(6) B1-8 cells were transferred, affinity maturation did not occur. These data indicate that restricting the frequency of clonally related B cells is required to support affinity maturation.

Reduced interleukin-5 production by peripheral CD4+ T cells in common variable immunodeficiency patients.

We evaluated the capacity of peripheral CD4+ T helper cells in four Common Variable Immunodeficiency (CVI) patients to secrete interleukin-4 (IL-4) and IL-5. While in control CD4+ T cells, stimulated via CD3 and cultured in presence of IL-2 or IL-15, a 10 fold increased production of IL-5 (146 +/- 30; 142 +/- 25 pg/ml) was found, a 4 fold increment of this cytokine was, instead, detected in 3 out of 4 CVI patients (34 +/- 13; 39 +/- 12 pg/ml) (p < 0.05). In conclusion, the reduction of IL-5, involved in the late regulation of B cell differentiation into Ig-secreting plasma cells, may contribute to the defective antibody production in CVI patients.

The role of human glioma-infiltrating microglia/macrophages in mediating antitumor immune responses.

Little is known about the immune performance and interactions of CNS microglia/macrophages in glioma patients. We found that microglia/macrophages were the predominant immune cell infiltrating gliomas ( approximately 1% of total cells); others identified were myeloid dendritic cells (DCs), plasmacytoid DCs, and T cells. We isolated and analyzed the immune functions of CD11b/c+CD45+ glioma-infiltrating microglia/macrophages (GIMs) from postoperative tissue specimens of glioma patients. Although GIMs expressed substantial levels of Toll-like receptors (TLRs), they did not appear stimulated to produce pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin 1, or interleukin 6), and in vitro, lipopolysaccharides could bind TLR-4 but could not induce GIM-mediated T-cell proliferation. Despite surface major histocompatibility complex class II expression, they lacked expression of the costimulatory molecules CD86, CD80, and CD40 critical for T-cell activation. Ex vivo, we demonstrate a corresponding lack of effector/activated T cells, as glioma-infiltrating CD8+ T cells were phenotypically CD8+CD25-. By contrast, there was a prominent population of regulatory CD4 T cells (CD4+CD25+FOXP3+) infiltrating the tumor. We conclude that while GIMs may have a few intact innate immune functions, their capacity to be stimulated via TLRs, secrete cytokines, upregulate costimulatory molecules, and in turn activate antitumor effector T cells is not sufficient to initiate immune responses. Furthermore, the presence of regulatory T cells may also contribute to the lack of effective immune activation against malignant human gliomas.