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Genome-wide association studies (GWAS) have identified many genes that are associated with the development of certain autoimmune disorders, but the MHC haplotypes still represent the most prevalent genetic risk factor for many autoimmune diseases. The mechanisms by which MHC-associated genetic susceptibility translates into B cell autoimmunity and the development of autoimmune diseases are complex. There is increasing evidence that the MHC haplotype modulates autoreactive B cell responses in multiple ways. Instead of merely inhibiting the production of IgG autoantibodies and mediating complete immunological tolerance, the non-permitting MHC haplotypes seem to facilitate the production of IgG autoantibodies exhibiting Fc glycosylation patterns that are associated with reduced pathogenicity and a protective cytokine profile of T follicular helper (Tfh) cells. Here, we discuss mechanisms linking MHC haplotypes to the production of pathogenic IgG autoantibodies, which could be relevant for the development of improved diagnosis, particularly in the context of individual medicine.

Acinetobacter baumannii has been recognized as a critical pathogen that causes severe infections worldwide not only because of the emergence of extensively drug-resistant (XDR) derivatives, but also because of its ability to persist in medical environments and colonize compromised patients. While there are numerous reports describing the mechanisms by which this pathogen acquires resistance genes, little is known regarding A. baumannii’s virulence functions associated with rare manifestations of infection such as necrotizing fasciitis, making the determination and implementation of alternative therapeutic targets problematic. To address this knowledge gap, this report describes the analysis of the NFAb-1 and NFAb-2 XDR isolates, which were obtained at two time points during a fatal case of necrotizing fasciitis, at the genomic and functional levels. The comparative genomic analysis of these isolates with the ATCC 19606T and ATCC 17978 strains showed that the NFAb-1 and NFAb-2 isolates are genetically different from each other as well as different from the ATCC 19606T and ATCC 17978 clinical isolates. These genomic differences could be reflected in phenotypic differences observed in these NFAb isolates. Biofilm, cell viability and flow cytometry assays indicate that all tested strains caused significant decreases in A549 human alveolar epithelial cell viability with ATCC 17978, NFAb-1 and NFAb-2 producing significantly less biofilm and significantly more hemolysis and capacity for intracellular invasion than ATCC 19606T. NFAb-1 and NFAb-2 also demonstrated negligible surface motility but significant twitching motility compared to ATCC 19606T and ATCC 17978, likely due to the presence of pili exceeding 2 µm in length, which are significantly longer and different from those previously described in the ATCC 19606T and ATCC 17978 strains. Interestingly, infection with cells of the NFAb-1 isolate, which were obtained from a premortem blood sample, lead to significantly higher mortality rates than NFAb-2 bacteria, which were obtained from postmortem tissue samples, when tested using the Galleria mellonella in vivo infection model. These observations suggest potential changes in the virulence phenotype of the A. baumannii necrotizing fasciitis isolates over the course of infection by mechanisms and cell processes that remain to be identified.

There is an urgent need for development of new adjuvants to target and drive immune responses in cancer immunotherapies. The E. coli type-II heat-labile enterotoxins (HLEs) LT-IIa, LT-IIb and LT-IIc exhibit strong adjuvant properties with varying capacities to elicit immune responses in mice. The immunomodulatory capabilities of these HLEs depend upon their interactions with specific ganglioside receptors that may vary in expression across immune cell types. Little is known, however, about the lymphoid cells that interact with HLEs in humans. To bridge this gap, human normal PBMCs, isolated by Ficoll density gradient centrifugation, were stained with recombinant B subunits of LT-IIa, LT-IIb, or LT-IIc, and mAbs to CD4, CD8, CD19, CD27, CD38 and CD45RO. Using FACS analysis, we found that LT-IIa, LT-IIb and LT-IIc increasingly bound to CD4+ T cells as the cells transitioned from naive (CD4+CD45RO-CD27+) to memory (CD4+CD45RO+CD27+) cells and from memory cells to effector memory (CD4+CD45RO+CD27-) cells, with LT-IIb exhibiting the most significant increase in binding. Likewise, LT-IIb and LT-IIc increasingly bound to CD8+ T cells as the cells transitioned from naive (CD8+CD45RO-CD27+) to memory (CD8+CD45RO+CD27+) cells and from memory cells to effector memory (CD8+CD45RO+CD27-) cells, with the greatest increase observed for LT-IIb. No major changes in LT-IIa binding were detected during CD8+ T cell differentiation. Finally, LT-IIa and LT-IIc increasingly bound to CD19+ B cells as the cells transitioned from naive (CD19+CD27-) to memory (CD19+CD27+) cells and from memory cells to antibody-secreting (CD19+/-CD27++CD38++) plasma cells, with LT-IIa exhibiting the most significantly increased binding activity. No major changes in LT-IIb binding were observed during B cell differentiation. Taken together, these results suggest that the ganglioside receptors for HLEs (e.g., GD1a, GD1b and GM1) are differentially expressed throughout B and T cell development/differentiation and that binding of HLEs to the various lymphocyte subsets is likely to elicit distinct cellular events associated with immune modulation or immune responsiveness. Understanding of these HLE binding patterns may also prove useful in targeting additional immunomodulating drugs to specific T or B cell subpopulations. Ultimately, characterization of immunological responses to these enterotoxins will lead to a greater understanding of their potential use in targeted cancer immunotherapies for treatment of a wide variety of conditions. Citation Format: Mary-Peyton A. Knapp, Taylor A. Johnson, Madison K. Ritter, Robert O. Rainer, Steven E. Fiester, Jennifer T. Grier, Terry D. Connell, Sergio Arce. Differential binding of E. coli enterotoxins LT-IIa, LT-IIb and LT-IIc to human B and T cell subsets identifies a potential use as adjuvants in cancer immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO024.

Viruses are obligate intracellular parasites, relying to a major extent on the host cell for replication. An active replication of the viral genome results in a lytic infection characterized by the release of new progeny virus particles, often upon the lysis of the host cell. Another mode of virus infection is the latent phase, where the virus is ‘quiescent’ (a state in which the virus is not replicating). A combination of these stages, where virus replication involves stages of both silent and productive infection without rapidly killing or even producing excessive damage to the host cells, falls under the umbrella of a persistent infection. Reactivation is the process by which a latent virus switches to a lytic phase of replication. Reactivation may be provoked by a combination of external and/or internal cellular stimuli. Understanding this mechanism is essential in developing future therapeutic agents against viral infection and subsequent disease. This article examines the published literature and current knowledge regarding the viral and cellular proteins that may play a role in viral reactivation. The focus of the article is on those viruses known to cause latent infections, which include herpes simplex virus, varicella zoster virus, Epstein–Barr virus, human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, Kaposi’s sarcoma-associated herpesvirus, JC virus, BK virus, parvovirus and adenovirus.

T lymphocytes from patients with sarcoidosis respond weakly when stimulated with mitogen or antigen. However, the mechanisms responsible for this anergy are not fully understood. Here, we investigated the protein levels of nuclear transcription factor NF-κB (p50, p65, and p105), IκBα (inhibitor of NF-κB), T-cell receptor (TCR) CD3ζ-chain, tyrosine kinase p56 LCK , and nuclear factor of activated T cells c2 (NF-ATc2) in peripheral blood CD4 + T cells from patients with sarcoidosis. Baseline expression of p65 in these lymphocytes was reduced in 50% of patients. The reduced levels of p65 in sarcoid CD4 + T cells concurred with decreased levels of p50, p105, CD3ζ, p56 LCK , IκBα, and NF-ATc2. Polyclonal stimulation of NF-κB-deficient sarcoid T cells resulted in reduced expression of CD69 and CD154, decreased proliferation, and cytokine (i.e., interleukin 2 [IL-2] and gamma interferon [IFN-γ]) production. The clinical significance of these findings is suggested by the association between low p65 levels and the development of more severe and active sarcoidosis. Although correlative, our results support a model in which multiple intrinsic signaling defects contribute to peripheral T-cell anergy and the persistence of chronic inflammation in sarcoidosis.

Cholera toxin (CT), LT-IIa, and LT-IIb are potent adjuvants which induce distinct T-helper (Th)-cell cytokine profiles and immunoglobulin G (IgG) subclass and IgA antibody responses. To determine if the distinct immune regulatory effects observed for LT-IIa, LT-IIb, and CT are elicited by binding of the enterotoxins to their cognate ganglioside receptors, the lineages of lymphoid cells that interact with the three enterotoxins and their effects on various lymphocyte responses in vitro were evaluated. Binding patterns of LT-IIa, LT-IIb, and CT to several lymphoid cell populations were distinctive for each enterotoxin. LT-IIa and CT, but not LT-IIb, induced apoptosis in CD8+T cells. LT-IIa(T34I), a mutant with no detectable binding to gangliosides, did not induce apoptosis. Blockade of GM1on the surface of CD8+T cells by LT-IIa(T14I), a mutant that binds only to GM1but does not induce apoptosis, did not inhibit induction of apoptosis by LT-IIa. Mitogen-induced proliferation of CD8+T cells was abrogated by treatment with CT, while resting CD8+T cells which were sensitive to LT-IIa-induced apoptosis became more resistant to apoptosis after mitogen activation. Exposure to CT, but not to LT-IIa or LT-IIb, inhibited mitogen-driven CD4+T-cell proliferation and expression of CD25 and CD69. In mitogen-stimulated B cells, CT, but not LT-IIa or LT-IIb, enhanced expression levels of CD86, while only CT induced B-cell differentiation into plasma cells. Thus, LT-IIa, LT-IIb, and CT exhibit distinguishable immunomodulatory properties which are likely dependent upon their capacities to recognize different ganglioside receptors on lymphocytes.

(NZB x NZW)F1 (NZB / W) mice develop a disease similar to human systemic lupus erythematosus (SLE), including autoantibody production, hypergammaglobulinaemia and inflammation of the kidneys. It is known that large numbers of lymphocytes infiltrate the kidneys of these mice. Here, we compare the roles of bone marrow, spleen and inflamed kidneys of NZB / W mice in the activation of B cells and the persistence of antibody-secreting cells (ASC). ASC are present in the kidneys of NZB / W mice with full-blown disease, as many as in the spleen and bone marrow. The specificity of the ASC in the inflamed kidneys is not restricted to self-antigens. After immunization of NZB / W mice with ovalbumin (OVA) the OVA-specific ASC are found initially in the spleen. Weeks later, OVA-specific ASC are found in high numbers in the bone marrow and the kidneys of these mice, but no longer in the spleen. As determined by FACS, B cells with a germinal center phenotype (B220(+) / PNA(+)) are found only in very low numbers in the kidneys, but in high numbers in the spleen of NZB / W mice. Germinal centers could not be detected in the kidneys, but in the spleen, and plasma cells appear to be scattered over the tissue. These data suggest that in autoimmune NZB / W mice, plasma cells generated in immune reactions of secondary lymphoid organs, later accumulate and persist in the inflamed kidneys, were they enhance the local concentrations of Ab and immunocomplexes. These experiments identify the inflamed kidneys of NZB / W mice as a site of prime relevance for the homeostasis of plasma cells, irrespective of their specificity.

The diagnosis of canine heartworm infection is based upon the presence of circulating Dirofilaria immitis microfilariae or on techniques for the detection of serum antibodies or antigens. In the first of these, discrimination between D. immitis, D. repens and Acanthocheilonema dracunculoides microfilariae is based upon the acid phosphatase histochemical stain. In this paper, we propose an alternative technique for histochemical staining using a commercial kit test of naphthol-AS-OL (Leucognost-SP). This offers the advantages of speed and simplicity as compared to the standard Barka procedure.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is constitutively expressed at high levels in healthy alveolar macrophages, in contrast to other tissue macrophages and blood monocytes. PPARgamma ligands have been shown to down-regulate IFN-gamma-stimulated inducible NO synthase (iNOS) in macrophages. Because NO is an important inflammatory mediator in the lung, we hypothesized that deletion of alveolar macrophage PPARgamma in vivo would result in up-regulation of iNOS and other inflammatory mediators. The loss of PPARgamma in macrophages was achieved by crossing floxed (+/+) PPARgamma mice and a transgenic mouse containing the CRE recombinase gene under the control of the murine M lysozyme promoter (PPARgammaKO). Alveolar macrophages were harvested by bronchoalveolar lavage (BAL). Lymphocytes (CD8:CD4 ratio = 2.8) were increased in BAL of PPARgammaKO vs wild-type C57BL6; por = 0.0001. Both iNOS and IFN-gamma expression were significantly elevated (por = 0.05) in BAL cells. Th-1 associated cytokines including IL-12 (p40), MIP-1alpha (CCL3), and IFN inducible protein-10 (IP-10, CXCL10) were also elevated. IL-4 and IL-17A were not detected. To test whether these alterations were due to the lack of PPARgamma, PPARgamma KO mice were intratracheally inoculated with a PPARgamma lentivirus construct. PPARgamma transduction resulted in significantly decreased iNOS and IFN-gamma mRNA expression, as well as reduced BAL lymphocytes. These results suggest that lack of PPARgamma in alveolar macrophages disrupts lung homeostasis and results in a Th1-like inflammatory response.

Cholera toxin (CT) and the type II heat-labile enterotoxins (LT-IIa and LT-IIb) are potent immunological adjuvants which are hypothesized to enhance the production of antibody (Ab)-secreting cells, although their mechanisms of action are not fully understood. The treatment of splenic cells with concanavalin A (ConA) plus CT enhanced the production of immunoglobulin A (IgA) and IgM by dividing cells that expressed high levels of major histocompatibility complex class II (MHC-II), CD19, and CD138 and low levels of B220 a phenotype characteristic of plasma blasts. LT-IIa or LT-IIb moderately enhanced IgA and IgM production without enhancing plasma blast differentiation. CT up-regulated CD25, CD69, CD80, CD86, and MHC-II in isolated B cells but failed to induce proliferation or differentiation. The treatment of unfractionated splenic cells with ConA plus CT induced B-cell proliferation and differentiation, but the elimination of CD4+T cells inhibited this effect. CT treatment of ConA-activated CD4+T cells up-regulated CD134 and CD154, whereas the blockage of CD40-CD154 interactions inhibited the induction of plasma blasts and Ig synthesis. The treatment of unfractionated splenic cells with CT, LT-IIa, or LT-IIb enhanced the production of interleukin-6 (IL-6) and IL-10, whereas the production of gamma interferon was inhibited in both CD4+and CD8+T cells mostly by CT. Thus, major regulatory effects of CT on lymphocytes are likely exerted early during the induction of immune responses when B and T cells initially encounter antigen. Neither LT-IIa or LT-IIb had these effects, indicating that type II enterotoxins augment Ab responses by other mechanisms.

The heat-labile enterotoxins, such as cholera toxin (CT), and the labile toxins types I and II (LT-I and LT-II) of Escherichia coli have been extensively studied for their immunomodulatory properties, which result in the enhancement of immune responses. Despite superficial similarity in structure, in which a toxic A subunit is coupled to a pentameric binding B subunit, different toxins have different immunological properties. Administration of appropriate antigens admixed with or coupled to these toxins by oral, intranasal, or other routes in experimental animals induces mucosal IgA and circulating IgG antibodies that have protective potential against a variety of enteric, respiratory, or genital infections. These include the generation of salivary antibodies that may protect against colonization with mutans streptococci and the development of dental caries. However, exploitation of these adjuvants for human use requires an understanding of their mode of action and the separation of their desirable immunomodulatory properties from their toxicity. Recent findings have revealed that adjuvant action is not critically dependent upon the enzymic activity of the A subunits, and that the isolated B subunits may exert different effects on cells of the immune system than do the intact toxins. Interaction of the toxins with immunocompetent cells is not exclusively dependent upon their conventional ganglioside receptors. Immunomodulatory effects have been observed on dendritic cells, macrophages, CD4+and CD8+T-cells, and B-cells. Numerous factors—including the precise form of the toxin adjuvant, properties of the antigen, whether and how they are coupled, route of administration, and species of animal model—affect the outcome, whether this is enhanced humoral and cellular immunity, or specific induced tolerance toward the antigen.

LT-IIa and LT-IIb, the type II heat-labile enterotoxins ofEscherichia coli, are closely related in structure and function to cholera toxin and LT-I, the type I heat-labile enterotoxins ofVibrio choleraeandE. coli, respectively. Recent studies from our group demonstrated that LT-IIa and LT-IIb are potent systemic and mucosal adjuvants. To determine whether binding of LT-IIa and LT-IIb to their specific ganglioside receptors is essential for adjuvant activity, LT-IIa and LT-IIb enterotoxins were compared with their respective single-point substitution mutants which have no detectable binding activity for their major ganglioside receptors [e.g., LT-IIa(T34I) and LT-IIb(T13I)]. Both mutant enterotoxins exhibited an extremely low capacity for intoxicating mouse Y1 adrenal cells and for inducing production of cyclic AMP in a macrophage cell line. BALB/c female mice were immunized by the intranasal route with the surface adhesin protein AgI/II ofStreptococcus mutansalone or in combination with LT-IIa, LT-IIa(T34I), LT-IIb, or LT-IIb(T13I). Both LT-IIa and LT-IIb potentiated strong mucosal and systemic immune responses against AgI/II. Of the two mutant enterotoxins, only LT-IIb(T13I) had the capacity to strongly potentiate mucosal anti-AgI/II and systemic anti-AgI/II antibody responses. Upon boosting with AgI/II, however, both LT-IIa(T34I) and LT-IIb(T13I) enhanced humoral memory responses to AgI/II. Flow cytometry demonstrated that LT-IIa(T34I) had no affinity for cervical lymph node lymphocytes. In contrast, LT-IIb(T13I) retained binding activity for T cells, B cells, and macrophages, indicating that this immunostimulatory mutant enterotoxin interacts with one or more unknown lymphoid cell receptors.

Despite the important role immunoglobulin G (IgG)-secreting plasma cells play in memory immune responses, the differentiation and homeostasis of these cells are not completely understood. Here, we studied the differentiation of human IgG-secreting cells ex vivo and in vitro, identifying these cells by the cellular affinity matrix technology. Several subpopulations of IgG-secreting cells were identified among the cells isolated from tonsils and bone marrow, particularly differing in the expression levels of CD9, CD19, and CD38. CD38 low IgG-secreting cells were present exclusively in the tonsils. A major fraction of these cells appeared to be early plasma cell precursors, as upon activation of B cells in vitro, IgG secretion preceded up-regulation of CD38, and on tonsillar sections, IgG-containing, CD38 low cells with a plasmacytoid phenotype were found in follicles, where plasma cell differentiation starts. A unitary phenotype of migratory peripheral blood IgG-secreting cells suggests that all bone marrow plasma cell populations share a common precursor cell. These data are compatible with a multistep model for plasma cell differentiation and imply that a common CD38 low IgG-secreting precursor gives rise to a diverse plasma cell compartment.

Recent results suggest that plasma cell longevity is not an intrinsic capacity, but depends on yet unknown factors produced in their environment. In this study, we show that the cytokines IL-5, IL-6, TNF-α, and stromal cell-derived factor-1α as well as signaling via CD44 support the survival of isolated bone marrow plasma cells. The cytokines IL-7 and stem cell factor, crucially important for early B cell development, do not mediate plasma cell survival, indicating that plasma cells and early B cells have different survival requirements. As shown in IL-6-deficient mice, IL-6 is required for a normal induction, but not for the maintenance of plasma cell responses in vivo, indicating that the effects of individual survival factors are redundant. Optimal survival of isolated plasma cells requires stimulation by a combination of factors acting synergistically. These results strongly support the concept that plasma cell survival depends on niches in which a combination of specific signals, including IL-5, IL-6, stromal cell-derived factor-1α, TNF-α, and ligands for CD44, provides an environment required to mediate plasma cell longevity.

Summary Recent results on the biology of plasma cells have shown that these cells can survive as long as memory B cells. Possibly, such long-lived plasma cells are also involved in the production of autoantibodies. Here, we discuss the potential involvement of long-lived plasma cells in the pathogenesis of autoimmune disease and the consequences it has for the development of effective therapeutic strategies.

Summary In atopic patients and patients with hyper-IgE syndrome (HIE) highly elevated IgE serum levels can be detected. Due to their very low frequency little is known about IgE-producing plasma cells (PC) in peripheral blood. We used CD138 MACS microbeads to enrich plasma cells from peripheral blood of normal donors, atopic patients and one HIE patient. CD138+ cells were mainly CD45+, CD44++, CD19dim, CD38++, CD27++, CD86+, HLA-DR+/++, CD71dim, VLA-4+, VLA-5–, CD28–, CD25–, CD69–, CLA–, CD20–, CD21– and CD22–. They show weak expression of surface Ig but high levels of intracellular Ig and they secrete Ig in culture. Thus CD138+ cells from peripheral blood show characteristics of early plasma cells. IgE+ CD138+ plasma cells could be detected in 19 of 24 normal donors with an average frequency of 0·06% IgE+ cells among CD138+ cells. Higher frequencies were detected in atopic patients, atopic patients with markedly elevated serum IgE levels and the hyper-IgE patient with an average of 0·32%, 7·21% and 6·54%, respectively. Additionally, using the recently developed cellular affinity matrix technology, we were able to detect IgE secreting plasma cells and thereby could demonstrate that most of the IgE secreting cells express CD138. The frequency of IgE+ CD138+ cells among PBMC correlated highly significantly with serum IgE titres (r = 0·8532***), indicating that IgE secreting CD138+ cells in peripheral blood are directly related to the plasma cell pool contributing to the IgE titre.

Plasma blasts formed during memory immune responses emigrate from the spleen to migrate into the bone marrow and into chronically inflamed tissues where they differentiate into long-lived plasma cells. In this study, we analyze the chemokine responsiveness of plasma blasts formed after secondary immunization with OVA. Starting from day 4 and within ∼48 h, OVA-specific plasma blasts emigrate from spleen and appear in the bone marrow. Although these migratory cells have lost their responsiveness to many B cell attracting chemokines, e.g., CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant), they migrate toward CXCL12 (stromal cell-derived factor 1α), and toward the inflammatory chemokines CXCL9 (monokine induced by IFN-γ), CXCL10 (IFN-γ-inducible protein 10), and CXCL11 (IFN-inducible T cell α chemoattractant). However, the responsiveness of plasma blasts to these chemokines is restricted to a few days after their emigration from the spleen, indicating a role for these molecules and their cognate receptors, i.e., CXCR3 and CXCR4, in the regulation of plasma blast migration into the bone marrow and/or inflamed tissues.

A selected fraction of plasmablasts enters the compartment of nondividing, long-lived plasma cells to maintain humoral antibody memory. In accord with a current model for lymphocyte homeostasis, the lifetime of long-lived plasma cells is probably regulated by competition for a limited number of survival niches present in splenic red pulp, bone marrow and inflamed tissue. Plasma cells secreting autoantibodies specific for some, but not all, self-antigens are probably 'allowed' to enter the compartment of long-lived plasma cells and provide antibody-mediated 'autoimmune memory' that is resistant to conventional therapies.

Background NZB/W mice develop a disease similar to human systemic lupus erythematosus (SLE), including autoantibody production, hypergammaglobulinaemia and inflammation of the kidneys. Large numbers of lymphocytes infiltrate the kidneys of these mice but the role of this organ for the production of antibodies is not clear. Objectives Here, we compare the role of bone marrow, spleen and inflamed kidneys of NZB/W mice for the activation of B cells and for the persistence of antibody secreting cells (ASC). Methods NZB/W mice were immunised i.p.i with Ovalbumin and then boosted 1 month later. The number of antigen-specific PC present in spleen, bone marrow and kidneys was determined by Elispot. The frequency of different cell types was determined by flow-cytometry. The distribution of PC in the different organs was investigated by microscopy. Results ASC are present in the kidneys of mice with full blown disease, as many as in the spleen and bone marrow, and 50 times more than in the kidneys of normal mice. In the kidneys, ASC are located mainly in the outer medulla, close to B- and T cell infiltrates. The specificity of the ASC in the inflamed kidneys is not restricted to self-antigens. After immunisation of NZB/W mice with Ovalbumin (OVA), the antigen-specific ASC are found initially exclusively in the spleen. Weeks later, during a period of at least 3 month, OVA-specific ASC are found in stable and high numbers within the bone marrow and the kidneys of these mice, but no longer in the spleen. As determined by FACS, B cells with a germinal centre phenotype (B220+/PNA+) are found only in very low numbers in the kidneys, but in high numbers in the spleen of NZB/W mice. Germinal centres could not be detected in the kidneys, but in the spleen. The lack of B cell activation and the kinetics of the appearance of OVA-specific ASC in the kidneys suggest that in autoimmune NZB/W mice, plasma cells generated during an immune reaction in secondary lymphoid organs, later accumulate and persist in the inflamed kidneys, like in bone marrow. Conclusion These experiments identify the inflamed kidneys of NZB/W mice as site of prime relevance for the homeostasis of plasma cells, irrespective of their specificity.

NZB/W mice develop a disease similar to human systemic lupus erythematosus (SLE), including autoantibody production, hyper-gammaglobulinaemia and inflammation of the kidneys. It is known that large numbers of lymphocytes infiltrate the kidneys of these mice but the role of this organ for the production of antibodies is not clear. Here, we compare the role of bone marrow, spleen and inflamed kidneys of NZB/W mice for the activation of B cells and for the persistence of antibody secreting cells (ASC). ASC are present in the kidneys of mice with full blown disease, as many as in the spleen and bone marrow, and 50 times more than in the kidneys of normal mice. In the kidneys, ASC are located mainly in the outer medulla, close to B- and T cell infiltrates. The specificity of the ASC in the inflamed kidneys is not restricted to self-antigens. After immunization of NZB/W mice with Ovalbumin (OVA), the antigen-specific ASC are found initially exclusively in the spleen. Weeks later, during a period of at least 3 months, OVA-specific ASC are found in stable and high numbers within the bone marrow and the kidneys of these mice, but no longer in the spleen. As determined by FACS, B cells with a germinal center phenotype (B220+/PNA+) are found only in very low numbers in the kidneys, but in high numbers in the spleen of NZB/W mice. By histology, germinal centers could not be detected in the kidneys, but in the spleen. The lack of B cell activation and the kinetics of the appearance of OVA-specific ASC suggest that in autoimmune NZB/W mice kidneys, plasma cells generated during an immune reaction in secondary lymphoid organs, later accumulate and persist, like in bone marrow. These experiments identify the inflamed kidneys of NZB/W mice as site of prime relevance for the homeostasis of plasma cells, irrespective of their specificity, suggesting that chronically inflamed tissue attracts plasma cells as such and extends the overall capacity of the body for plasma cells, allowing autoreactive plasma cells to survive for long times within the inflamed tissue and to provide exorbitant titers of antibodies locally.

The most important link between the immune network theory and clinically useful therapies so far is the use of human intravenous immunoglobulin (IVIg) in the treatment of autoimmune diseases. Although still controversial, one of the main mechanisms that has been postulated for the in vivo effects of IVIg, is the selection of immune repertoires through idiotypic interactions. We describe here anti-idiotype IgG monoclonal antibodies (MAbs), which were obtained by immunization of syngeneic mice (Balb/c) with an anti-ganglioside antibody. These anti-idiotype MAbs show multiple idiotypic connections and share some of the properties of the IVIg pool. The antiidiotype (Ab2) MAbs B7 and 34B7 showed heterogeneous binding with the idiotypes of several anti-ganglioside antibodies, MAbs obtained from splenocytes of nonimmunized newborn mice, F(ab')2 fragments of IgG human myeloma proteins, and nonimmunoglobulin antigens. The recognition pattern of the B7 MAb to the idiotypes of human immunoglobulins was also studied using a phage display library obtained from the variable region genes of an asymptomatic AIDS patient and also F(ab')2 fragments obtained from an IVIg pool of healthy human donors. We also demonstrated that these MAbs produced some of the in vitro effects reported for the human IVIg pool, such as the inhibition of cell proliferation of human B and T cell lines and of normal human lymphocytes activated with different mitogens. Another striking property of the MAb B7 was its ability to induce a dose-dependent specific antibody T-cell response in vivo in syngeneic mice. Both anti-idiotype MAbs showed anti-metastatic effect in vivo when injected intravenously to mice inoculated with MB16-F10 melanoma cells. The antimetastatic effect of the antiidiotype MAbs was not observed in athymic mice inoculated with the same tumor. This kind of antibody can become an interesting tool for further exploration of the role of idiotypic network connections in the regulation of the immune system and to study the effects of interventions on network connectivity in experimental autoimmune disease, using a reagent better chemically defined than the IVIg pool.

The duration of specific antibody titers ranges from a few weeks up to several years. While the formation of antibody secreting plasma cells takes place in secondary lymphoid tissues, long-lasting antibody responses are provided by bone marrow plasma cells. The majority of plasma cells initially formed remains in secondary lymphoid tissues and die within a few days. Plasma cells that migrate into the bone marrow or into chronically inflamed tissues can survive for much longer periods of time. The capacity of plasma blasts to migrate into these tissues is regulated by chemokine receptors. Expression of CXCR4 is important for plasma cell migration into the bone marrow. CXCR3 ligands expressed in large quantities in chronically inflamed tissues (e.g. in effected kidneys in systemic lupus erythematosus) and can attract plasmablasts, thus mediating plasma cell accumulation at those sites. Here we analyzed the regulation of chemokine receptor expression during plasma cell differentiation in culture. Purified CXCR3-negative B cells were activated T dependently in a two-step culture system, for 3 days by CD40 ligand together with IL-2 and IL-10 and for 5 more days with IL-2 and IL-10. Alternatively, cells were stimulated T independently with CpG, IL-2 and IL-10. Addition of IFN-γ or monocyte/T-cell culture supernatant in the initial culture, but not at late stages, induced the expression of CXCR3 on plasma cells, thus suggesting that the regulation of this receptor is an early event during plasma cell differentiation. In contrast, CXCR4 was present on all plasma cells, formed under any stimulation conditions and even when CXCR4-negative B cells were activated to form plasma cells. These data indicate that expression of CXCR4 and, as a consequence, the potential to migrate into the bone marrow is generally associated with differentiation into plasma cells, whereas the expression of CXCR3 must be induced by the inflammatory cytokine IFN-γ.