Suppression of the immune system after the resolution of infection or inflammation is an important process that limits immune- mediated pathogenesis and autoimmunity. Several mechanisms of immune suppression have received a great deal of attention in the past three decades. These include mechanisms related to suppressive cytokines, interleukin (IL)-10 and transforming growth factor (TGF)- β , produced by regulatory cells, and mechanisms related to apoptosis mediated by death ligands, Fas ligand (FasL) and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), expressed by killer or cytotoxic cells. Despite many lines of evidence supporting an important role for B lymphocytes as both regulatory and killer cells in many inflammatory settings, rela- tively little attention has been given to understanding the biology of these cells, their relative importance or their usefulness as therapeutic targets. This review is intended to give an overview of the major mechanisms of immunosuppression used by B lym- phocytes during both normal and inflammatory contexts. The more recent discoveries of expression of granzyme B, programmed death 1 ligand 2 (PD-L2) and regulatory antibody production by B cells as well as the interactions of regulatory and killer B cells with regulatory T cells, natural killer T (NKT) cells and other cell populations are discussed. In addition, new evidence on the basis of independent characterizations of regulatory and killer CD5 + B cells point toward the concept of a multipotent suppressor B cell
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It is established that septic shock is associated with a severe exhaustion and depletion of T lymphocytes . Our data support similar behavior in the B-lymphocyte compartment. We have found that the reduction of circu- lating B cells affects the different B-cell subsets heteroge- neously in septic shock patients and that those different patterns of involvement are observed in survivor and non- survivors. CD23 is a low-affinity receptor for IgE located at the surface of B cells . CD23 is involved in different regulatory functions, such as enhancing antigen presenta- tion, improving cell differentiation and growth and regu- lating IgE synthesis . Some authors have reported that CD23 is expressed on activated B cells, whereas others have suggested that peripheral blood CD23 B cells resem- ble classic memory cells . Our data presented herein show that circulating CD19+CD23+ B lymphocytes are
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Many studies have investigated the fate of adoptively transferred lymphocytes in recipient mice, although little is known of the sites where these transferred cells reside at particular time points. Using flow cytometry, we analyzed the trafficking pattern of adoptively transferred naive B cells into the lymphoid organs of syngeneic B-cell-deficient (µµMT) mice. Within the first 24 h of transfer, the location of B cells was highly dependent on the mode of B-cell transfer. When B cells were injected subcutaneously into µµMT mice, they showed a different trafficking pattern from cells administered into the peritoneal cavity or injected intravenously. After subcutaneous transfer into the thigh, the greatest number of B cells was detected in the popliteal lymph node nearest to the injection site, whereas the lowest number was detected in the axillary lymph node opposite to the injection side. Within the first 24 h of either
T cell help for B cell activation. Mature B cells can be polyclonally activated in a non-MHC restricted fashion by preactivated CD4-positive T cells, or membranes prepared from them (Brian et a l , 1988, Noelle et al.y 1991). In the mouse such contact-dependent T cell help for B cells results in marked proliferation in the presence of IL-4, and differentiation to antibody-secreting cells under the influence of IL-4 and IL-5 (Noelle et al. y 1991). In addition, several studies have shown that neonatal B cells, which fail to proliferate in response to a number of stimuli which activate mature B cells, can be activated by T helper cells (Chang et al. , 1991, Brines and Klaus, in Int. Immunol, in press) We wished to further investigate whether such contact-dependent T cell help would protect the B cell lymphomas against Ig-mediated growth inhibition. To address this the Th2 T cell clone D10.G4.1, and a subclone DIO.D, were activated and fixed, or irradiated before being added to WEHI-231 or CH33 in anti-^ Ab growth inhibition assays. Activated, irradiated DIO cells, both D10.G4.1 and DIO.D, partially reversed the growth inhibition in CH33 and CH31, but not in WEHI-231 (Fig. 3.13). A similar protective effect was seen with preactivated DIO cells fixed with paraformaldehyde, indicating that the reversal was not due to secreted cytokines but involved contact-dependent signals. The preactivated T cell clones provided dose dependent protection to the immature B lymphomas (Fig. 3.13), and the effect was induced by T cells activated with two different protocols, over a wide concentration range of stimuli, but was not observed with resting T cells (Fig. 3.14). This is in agreement with studies of contact-dependent T cell help in mature B cells where it is known that the Th cells only become competent to induce B cell activation after de novo protein synthesis (reviewed in Noelle and Snow, 1992). Ales-Martinez et al. (1991) also demonstrated a similar reversal of Ig-mediated growth arrest in CH33 using irradiated D10.G4.1 cells, but concluded that a large
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backgrounds, respectively. In the first model, B cells and DCs were isolated from the spleens of HHDII-DR1 mice as above and co-incubated with either plasmid DNA encoding SSX2 (pTVG-SSX2) or the p103 HLA- A2-restricted nonamer peptide. After 18h, cells from each treatment type were washed and transferred into groups of naïve syngeneic mice. Importantly, DCs were not matured with GM-CSF after treatment with plasmid DNA or peptide in these studies to facilitate antigen uptake and subsequent maturation [35-37]. Immune responses were assessed two weeks after a single cell transfer. As can be seen in Figure 5a, only transfer of B cells co- cultured with plasmid DNA led to antigen-specific IFNγ and IL2 release by CD8 T cells. Immune responses were further evaluated after a 1-week in vitro stimulation, to detect lower frequency responses (Figure 5b). As shown, responses were detectable to both dominant (p103) and subdominant (p41) epitopes following delivery of DNA by B cells, but DC were only able to present the peptide antigen to expand CD8 T cells. CD8 T cells also demonstrated increased CD137 expression, as a marker of antigen-specific activation . In the second model, B cells and DCs were obtained from the spleens of wild type C57/BL6 mice, treated as above by simple co-culture with plasmid DNA encoding ovalbumin or SIINFEKL peptide, and adoptively transferred into naïve age-matched C57BL6 mice. Two weeks later, splenic CD8 T cells were assayed for their ability to secrete IFNγ upon stimulation with the SIINFEKL epitope. Once again, we were able to detect antigen-specific cytokine release only upon B cell presentation of plasmid DNA, and not following DC presentation (Figure 5c). Of note, transfer of B cells co-cultured with even less plasmid DNA (25 µg) was sufficient to elicit the same magnitude of response as observed with traditional intradermal plasmid vaccination using 100 µg DNA (Figure 5c). These data confirm that B lymphocytes, and not dendritic cells, are able to efficiently prime a murine immune response after simple in vitro co- culture with plasmid DNA.
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patient has a failure of B cell maturation at the stage of early B lymphocytes, associated with production of D(mu delta) H chain. The phenotype of his B cells includes: (a) limitation to expression of the mu and delta H chain isotypes, (b) production of mu and delta H chains of reduced size and (c) delayed expression of L chain. Peripheral blood and B cell lines from the patient's mother and sister include 50% cells that express H chain without L chain. B cell lines from the mother and sister produce full-length mu and gamma H chains and truncated mu and delta chains corresponding to the H chains produced by the patient's B cells. Clones with normal and XLA phenotype have been isolated from B cell lines derived from the patient's mother. We conclude that the dimorphism of mother's and sister's B cells results from Lyonization, implying that the gene defect in XLA is intrinsic to B lymphocytes.
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Lymphocyte trafficking is a critical step of the sys- temic immunity (1–3). This process includes migration of T lymphocytes from the bone marrow and thymus to spleen and lymph nodes, as well as the migration of B lymphocytes in the lymph node microenvironment, i.e., from the mantle zones to germinal centers. Con- sidering the promiscuity of chemokines and their receptors, it is still difficult to understand how the cel- lular trafficking is regulated. Although the functions of some chemokines, e.g., stromal cell–derived factor-1 (SDF-1), are well characterized in terms of their ability to recruit B cells into the microenvironment of stroma cells (25), the role of IP-10 and Mig in B-cell trafficking is not defined. In addition to their chemotactic activi- ty for T lymphocytes, IP-10 and Mig share other prop- erties, including inhibition of neovascularization, inhi- bition of progenitor cells, and antitumor effects (4). For these reasons, the expression of CXCR3 may have a dual effect in these B-cell malignancies. Its presence may favor the trafficking of malignant B cells from one site to another site of disease involvement, thus favor- ing the spreading of the disease to peripheral blood, lymph nodes, bone marrow, and other organs. It could also be suggested that the interaction between CXCR3 and relevant chemokines might affect tumor growth by attracting T cells with antitumor functions. In this
Many of the studies that we have ref- erenced above are focused on the role of regulatory and killer B cells in T cell–mediated diseases such as asthma and autoimmunity. There should be lit- tle doubt that immune suppression di- rected by B lymphocytes is an important factor in regulating T-cell functions and therefore in modulating allergic and au- toimmune pathogenesis. Impairments in B cell–mediated immune regulation or resistance to their suppressive functions may turn out to be critical contributing factors to T cell–mediated diseases. It will be of interest to determine whether any of the known genetic and environ- mental risk factors that have been iden- tified in these diseases play a role in the survival and function of suppressive B cells. Finding pharmacological or bio- logical methods for reversing defects in B cell–mediated immune suppression may lead to novel therapeutics for many diseases. It is also possible that func- tional immune-suppressive B cells may contribute to autoimmune pathogenesis in some cases. Elevated IL-10 levels have been linked to increased pathogen- esis in SLE in patients and mouse mod- els, yet B cell–specific IL-10 production and T-cell suppression was impaired in a cohort of SLE patients (44,157–159). These seemingly conflicting results highlight the need for a deeper under- standing not only of the direct functions of suppressive B cells but also their in- teractions with other effector and regu- latory cell populations. It may matter whether immune suppression is domi- nated by T cells or B cells in certain clin-
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The complex biantennary N-linked oligosaccharide, such as those found at the Fc region in IgG have more than 30 different glycoforms (Parekh, et al., 1985). p 1,4-GalTase, from a number of different sources, readily galactosylated biantennary oligosaccharides where the GlcNAc residues were exposed on both branches, acting primarily on the M anal,3 arm with successive galactosylation on the M anal,6 branch occurring at a much slower rate (Blanken, et al., 1984; Paquet, et al., 1984; Morita, et al., 1988). The presence of a bisecting GlcNAc molecule, did not alter the branch specificity of bovine milk or thymus p 1,4-GalTase, but decreased the galactosylation of the Man a 1,3 branch by 78%, probably as a result of steric hindrance (see below) (Blanken, et al., 1984; Narasimhan, et al., 1985). Purified p 1,4-GalTase maintained the same substrate specificities throughout each step of its purification, suggesting that the same enzyme was responsible for the galactosylation of each branch (Morita, et al., 1988). However, in bovine IgG only the non-bisected sugar chains were preferentially galactosylated on the M anal,3 arm, whilst the bisected structures were predominantly galactosylated on the M anal,6 arm (Fujii, et al., 1990). Highly-ordered branch specificity has also been observed with p 1,4-GalTase in the synthesis of blood-group I structures, where galactosylation of the p i ,6 branched GlcNAc residue of the trisaccharide GlcNAcp 1,3(GlcNAcp 1,6)Gal preceeded that of P 1,3 linked GlcNAc (Blanken et al., 1982). Hence p 1,4-GalTase exhibits very high branch specificity which alters depending on the oligosaccharide acceptor, species and glycoprotein. Time course studies revealed that B cells from controls continued to galactosylate AsAg-IgG without saturation for up to 6 hours, whilst B cells from RA patients had reached saturation by 2 hours (Furukawa, et al., 1990). It would be worthwhile if the galactosylation efficiencies seen in the B cells between the RA and control resulted in different branch specificities on the AsAg-IgG substrate.
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After subcutaneous injection of adult mice with MMTV (SW), infected B cells can be detected only in their draining lymph nodes after day 2 to 3, even when highly sensitive meth- ods such as PCR are used (15). The number of infected B cells increases due to the Sag response over a period of 6 days, and cell division of infected B cells is needed to allow detection of the reverse-transcribed viral genome (1, 13–16). In parallel, there is a strong expansion of Sag-reactive V b 6-specific T cells. We monitored these two parameters (infection and T-cell re- sponse) in the in vitro infection system. A similar kinetics of infection was observed in splenocytes infected in vitro, as in the draining lymph nodes after in vivo injection of MMTV(SW) (Fig. 1). A PCR signal was monitored and indicated a peak at day 4 after infection (Fig. 1A). As in the in vivo model, no infection was measurable during the first day, confirming the low rate of infection ( , 1/1,000 cells) in vivo (15). After 4 days of culture, the percentage of B cells started to decrease due to preferential death (data not shown). On day 9, B lymphocytes represented only 20% of the total cells compared to 50 to 60% at the onset of culture.
into contact with tumor cells and acquire increased metastatic behavior. This phenomenon is associated with the activation of the extracellular signal-regulated kinase pathway in melanoma cells . B-cell lineage in tumor issues and tumor-draining lymph nodes are clonally and functionally related to each other. The physiological relationship between the two sources of B lymphocytes may be relative to tumor-specific immune responses in breast cancer patients . B cells and downstream myeloid-based pathways regulate represented tractable targets for combinatorial therapy in SCC; hence, B-cell depletion may be relatively straightforward for achieving clinical benefits . Moreover, B cells may be positively correlated with the poor prognosis of patients harboring metastatic carcinomas. For example, when recruited by chemokine CXCL13, B cells can produce lymphotoxin to promote the progression of castration-resistant prostate cancer , mainly through activating of the IKKa-BMI1 pathway in cancerous prostate stem cells . This finding further indicates that B cells may be a potent biomarker in speculating the prognosis of cancer patients. B cells are also known to induce decreased response to chemotherapy. Recently, Shalapour et al. discovered that B cells can down-regulate the response to low-dose oxaliplatin (an immunogenic chemotherapeutic agent) in mouse prostate cancer models; specifically, B cells can induce immunogenic cell apoptosis to promote tumor-directed cytotoxic lymphocyte activation. Such immunosuppressive B cells are plasmocytes that express IgA, IL-10, and PD- L1, depending mostly on TGF-β receptor signaling . Similarly, B-cell depletion by administrating of anti-CD20 monoclonal Abs can significantly improve the response of patients to chemotherapy . Therefore, we suggest that the elimination or inhibition of TIL-Bs may be crucial to the successful immunotherapy of tumors.
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Differences in the expression of Leu-1 (CD5) define two populations of recovering B cells after human marrow transplantation, Leu-1+ and Leu-1- B cells. The Leu-1+ B cells were polyclonal, of donor origin, and did not express detectable interleukin 2 receptor. Leu-1+ B cells generally appeared 2-4 wk after marrow grafting and often preceded the recovery of Leu-1- B cells. Acute and chronic graft vs. host disease (GvHD) resulted in the recovery of significantly fewer Leu-1+ B cells, whereas Leu-1- B cells were only decreased in acute GvHD. Multivariate analysis showed no significant effect of age, disease, prednisone or azathioprine, or ex vivo treatment of the marrow with anti-Leu-1 and complement on recovery of Leu-1+ and Leu-1- B cells, independent of the effects of GvHD. Leu-1+ B cells are a major lymphocyte population posttransplant. They may reflect a stage of differentiation of normal B cells or a separate B cell lineage.
Coculture of bovine PBMC with endothelial cells did not give the same results for active replication by RPV as those of PBMC on matrix components. Both lymphocytic and mono- cytic cells were seen microscopically to adhere to endothelial cells; the removal of leukocytes required only gentle pipetting. As for the viability of lymphocytes, there was an increase in the number of viable B cells and g / d T cells after contact with endothelial cells, but there was no influence on other T-lym- phocyte populations. When the capacity to support active rep- lication of RPV was analyzed, this also was increased in all lymphocyte populations. The increase was most apparent in B-cell and, particularly, CD4 1 T-lymphocyte susceptibility to RPV. Consequently, in contrast to contact with matrix compo- nents, intercellular contact between lymphocytes and endothe- lial cells increased the capacity of the former to support active replication by RPV. Whether the mechanism involved is that reported for human immunodeficiency virus and MV, through modulation of LFA-1, remains to be determined. What is clear is that increased lymphocyte viability alone does not explain the susceptibility to RPV; otherwise, the greatest increase in this susceptibility would have been seen with g / d T cells and B lymphocytes after adherence to endothelial cells. Active sig- nalling between lymphocytes and endothelial cells is probably
mutated Ig genes has been described [14-16], which is ele- vated in patients with systemic lupus erythematosus (SLE) . Abnormalities in the frequencies of peripheral blood memory B cells have been reported in SLE , and Sjögren's syndrome (SS) . However, in RA the data on possible dis- turbances of peripheral blood B cell distributions have not been delineated as well. Part of this could relate to differences in disease duration and therapy of the cohorts studied [19-21]. Treatment with TNF blockers ameliorates the signs and symp- toms of RA and disease progression [22-25]. Recently, a study of peripheral blood and tonsilar biopsies from RA patients undergoing treatment with the combined TNF and lymphotoxin α (LTα) antagonist, etanercept, suggested that part of the success of this therapy in RA could be linked to a disruption of follicular dendritic cell (FDC) networks in second- ary lymphoid organs, thus impairing germinal centre formation, and decreasing the number of CD27 + memory B cells in the
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cytotoxicity. It was found that (a) lymphocytes from patients with AH are cytotoxic to Chang liver cells compared to controls (P less than 0.001); (b) lymphocytes from patients with acute and chronic hepatitis are less cytotoxic when incubated with autologous and homologous HB2Ag-positive and -negative AH, CAH, and CPH are as cytotoxic as normal controls when stimulated with a nonspecific mitogen such as Con A; and (d) lymphocytes from patients with CAH while on prednisone therapy showed marked depression of cytotoxicity when stimulated with Con A. Thus these studies show that patients with AH have circulating T lymphocytes which are capable of causing the destruction of Chang liver cells. There is no defect in T-cell function as measured by Con A-stimulated cytotoxicity. There is a serum […]
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First, like other members of the Ig-ITIM family, PECAM-1 may function to feedback inhibit or set thresholds for cellular activation resulting from sig- naling cascades that emanate from ITAM-containing receptors. For example, Fc receptors (40) and a newly recognized platelet collagen receptor (41) signal via ITAMs. Might their activating and/or adhesive func- tions be modulated by PECAM-1 in cells that express them? The T-cell receptor complex also contains mul- tiple ITAM-bearing subunits (the ζ chain and the CD3 ε, δ, and γ subunits), the signal- ing patterns of which are thought to be regulated, in part, by the CTLA-4 and KIRs, each of which belong to the Ig-ITIM family (Table 1). Why should T cells express still another inhibitory receptor? The answer may lie within the extracellular domain: the homophilic nature of the PECAM-1 extracellular domain (42, 43) may serve to target phos- phatase activity to specific sites within the cell that are not served by these other inhibitory recep- tors. It is interesting to note that CTLA-4–deficient mice have con- stitutively activated T cells and a lymphoproliferative disorder (44, 45). It might be instructive to see, via the generation of double Table 2
Cytotoxicity assay. The cytotoxicity assay used is similar to the one previously described (11), with slight modifications. For preparation of effector cells, mice were sacrificed 10 days after primary infection and spleen single-cell suspensions were prepared with RPMI 1640 containing 10% heat-inactivated fetal calf serum, 100 m g of streptomycin sulfate per ml, 100 U of penicillin per ml, 2 mM L - glutamine, 2.5 mM sodium pyruvate, 20 mM N-2-hydroxyethylpiperazine-N9-2- ethanesulfonic acid (HEPES), and 30 mM 2-mercaptoethanol (complete me- dium) and mixed in a 2:1 ratio with stimulator cells. The latter, obtained from spleens of nonimmune animals, had been previously incubated (10 7 cells per ml
Multicolor ﬂ ow cytometry for analysis or for sorting was performed on an LSR Fortessa-5 or FACS Aria (BD Biosciences), respectively. Single-cell suspensions of splenocytes were blocked with anti- CD16/32 mAb (clone 2.4G2), followed by staining with the follow- ing antibodies: anti-B220 (clone RA3-6B2) and anti-CD45.2 (clone 104) from BD Biosciences. HEL-binding B cells were stained as described previously (Chan et al, 2009). For cell cycle analyses, spleen cells were ﬁ rst stained for extracellular antigens and then were analysed with 10 μ g/ml DAPI staining using a Cyto ﬁ x/ Cytoperm kit (BD Biosciences) or PFA and Tween-20. Cell cycle was calculated by FlowJo Dean/Jett/Fox algorithm or by setting gates manually. The Click-iT EdU Alexa Fluor 488 Imaging kit and CaspGLOW Fluorescein Active Caspase Staining kit (both from Thermo Fisher Scienti ﬁ c) were used according to the manufac- turer ’ s instructions. Data were analysed with FlowJo software (Tree Star).
In this chapter I have discussed the rationale for assessing the effects of oncogenes in B-CLL by gene transfer and described the design of five expression vectors suitable for such transfection studies. O f the final oncogene expression vectors prepared, the c-myc oncogene seemed likely to be the most informative following gene transfer experiments as chromosomal translocations involving the c-myc locus as well as deregulated c-myc protein levels are found in several B lineage neoplasias (Cole 1986). Transfection studies using human EBV transformed B cell lines also provide evidence for a role of the Ha-ras gene in B lymphocytes (Nasi et al. 1990, Seremetis et al. 1989). Both genes were subcloned into a vector containing the powerful CM V-LTR gene regulatory sequences and tested for expression and function in NIH3T3 cells. The availability of multiple vectors also allows the effects of oncogene cooperation to be studied. The hypothesis that more than one gene is involved in the generation of some B cell malignancies is supported by experiments with transgenic mice, where overexpression of the c-myc gene in B lymphocytes was achieved using tissue specific gene regulatory elements (Adams et al. 1985). Although overexpression of c-myc leads to B cell tumours the overall frequency is low. Up to 100- fold greater frequencies were obtained when c-myc overexpressing cells were co transfected with other oncogenes (Adams and Cory 1991, Alexander et al. 1989a, Alexander et al. 1989b).
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amounts of resting Treg are required for eliciting a clin- ical response. In addition, we cannot rule out that the transferred Treg might also be affected by low amounts of CTX still circulating in the body of the recipients, though we performed Treg transfers five days after the last CTX injection to avoid interferences with CTX. Nevertheless, a significant increase of the Treg fre- quency compared to the levels before transfer, and higher levels of CD4+Foxp3+ Treg were detectable in the peripheral blood up to four weeks after transfer, implicating that a reasonable number of these cells sur- vived in recipients despite the insufficient availability of IL-2. Concerning the therapeutic durability of the Treg treatment, we suggest that repetition of the Treg trans- fers, for example, in intervals of two weeks, could sus- tain remission for a longer period of time. In addition, the efficacy of Treg transfers could probably be increased by simultaneous provision of IL-2 in vivo to facilitate their survival and homeostasis in the IL-2-defi- cient recipients .
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