Imunização de camundongos LDLr-/- com peptídeo mimético da LDL eletronegativa: uma potencial estratégia terapêutica para a ateroscleros / Immunization of LDLr - / - mice with electronegative LDL mimetic peptides: a potential therapeutic strategy for atherosclerosis

Introdução: A aterosclerose é uma doença inflamatória crônica decorrente de alterações na parede das artérias de médio e grande calibre e associadas a diversos fatores de risco, dentre os quais destaca-se as hiperlipidemias, ou seja, o aumento plasmático das lipoproteínas, mas também outras comorbidades, como a Síndrome Metabólica. Entre as lipoproteínas, a lipoproteína de baixa densidade (LDL) é de grande relevância na aterosclerose. Diferentes espécies de LDL modificada (LDLm) são originadas através de lipólise, glicação e proteólise, além da oxidação, variando em densidade e eletronegatividade, sendo melhor denominada LDL eletronegativa [LDL (-)]. Considerando as diferenças conformacionais entre a estrutura da ApoB-100 da LDL nativa e da LDL (-), em um estudo inicial, nosso grupo desenvolveu um anticorpo monoclonal (2C7) a partir da imunização de camundongos Balb/c com a LDL (-) humana. Em uma etapa seguinte foi mapeado o epítopo reconhecido pelo anticorpo monoclonal anti-LDL (-) através de phage display. O peptídeo ligante do anticorpo monoclonal anti-LDL (-) foi nomeado p2C7. Esse peptídeo não representa regiões da sequência linear da ApoB-100 humana, mas microdomínios conformacionais de epítopos da ApoB-100 da LDL (-), tornando-os candidatos para a imunomodulação da aterogênese. Portanto, investigar a imunomodulação induzida pelos peptídeo p2C7 miméticos da LDL (-), por representar um epítopo imunodominante da LDL (-), poderá abrir novas perspectivas terapêuticas futuras para a imunomodulação da aterosclerose. Objetivo: Avaliar a imunomodulação promovida pelo p2C7 in vivo, utilizando camundongos C57BL/6 LDLr -/- e amostras de plasma humano. Adicionalmente, no estágio (BEPE) realizado no Instituto Karolinska (dezembro de 2019 a março de 2021), investigou-se o imunometabolismo como mediador nas doenças cardiovasculares. Na parte II-A, estão descritos os resultados do estudo inicialmente proposto. Na parte II-B, apresenta-se os resultados que foram desenvolvidos posteriormente, com ampliação do escopo do projeto, abordando-se a inflamação vascular envolvida no aneurisma de aorta abdominal através de ferramentas de bioinformática. Na parte II-C, são apresentados os resultados do estudo do envolvimento da enzima indolamina 2,3 dioxigenase (IDO) na esteatohepatite não-alcoólica (NASH) e aterosclerose em camundongos ApoE-/- and ApoE/IDO/double-knockout. Metodologia: Foi avaliada a presença de anticorpos anti-p2C7 em amostras de plasma humano de indivíduos com ou sem síndrome metabólica. Realizamos a determinação de TNF circulante nas mesmas amostras e prosseguimos com regressões lineares associando os parâmetros inflamatórios com os níveis de anticorpos anti-p2C7. Camundongos C57BL/6 LDLr -/- foram imunizados com p2C7 e os adjuvantes Alum ou Montanide ISA 720, analisando-se os títulos de anticorpos contra p2C7 e LDL (-), a produção de citocinas (IL-10, IL-4, IL-2, IL-6, IFNγ, IL-17, TNFα) e células secretoras de anticorpos. Camundongos C57BL/6 LDLr -/- foram tolerizados contra os peptídeos mimotopos, com injeções intravenosas (veia caudal) e desafiados com a imunização contendo LDL (-) + Alum. Avaliou-se os títulos de anticorpos contra p2C7 e LDL (-) e a produção de citocinas (TNF-α, IFNγ, IL-12, IL-6, IL-10 e MCP-1). Os camundongos foram mantidos em dieta hipercolesterolêmica por 3 meses para formação da placa aterosclerótica. Após este período, os camundongos foram eutanasiados, avaliando-se a formação de placa aterosclerótica na artéria abdominal e arco aórtico, assim como a produção de citocinas (TNF-α, IFNγ, IL-12, IL-6, IL-10 e MCP-1). Camundongos C57BL/6 LDLr -/- foram imunizados com OVA-p2C7 e, após dieta hipercolesterolêmica de 3 meses para formação de placa aterosclerótica, foram avaliados os parâmetros inflamatórios e avaliada a captação de 18F-FDG no arco aórtico através de PET/CT. Resultados: A imunização com o p2C7 (livre) não foi capaz de induzir resposta humoral, não se observando títulos detectáveis de anticorpos reativos à p2C7 ou LDL (-) em nenhum camundongo imunizado, assim como não foram detectadas células secretoras de anticorpos específicos para a LDL (-). O grupo imunizado com Alum ou Montanide + p2C7 teve aumento significativo na produção de TNF- quando comparado com os demais grupos. O protocolo de tolerização foi realizado com sucesso, visto que os camundongos tolerizados apresentaram títulos de anticorpos inferiores aos controles para o epítopo utilizado. Apenas os camundongos tolerizados com o p2C7 apresentaram aumento significativo na produção de IL-6, IL-12, IL-10, TNF-α, IFNγ e MCP 1 após dieta hipercolesterolêmica. A imunização ativa com OVA-p2C7 foi capaz de reduzir a produção de TNF induzida pela dieta hipercolesterolêmica, assim como reduzir a captação de 18F-FDG. Conclusão: o epítopo p2C7 é altamente expresso na LDL (-) de pacientes com maior risco cardiovascular. Além disso, a imunização ativa com p2C7 também se mostra uma ferramenta promissora para prevenir e regular a inflamação causada pela LDL (-) no curso da aterosclerose

Introduction: Atherosclerosis is a chronic inflammatory disease resulting from changes in the wall of medium and large-caliber arteries and associated with several risk factors, among which hyperlipidemias stand out, ie, the increase in plasma lipoproteins, but also other comorbidities, such as Metabolic Syndrome. Among the lipoproteins, low-density lipoprotein (LDL) is of great relevance in atherosclerosis. Different isoforms of modified LDL (LDLm) are originated through lipolysis, glycation and proteolysis, in addition to oxidation, varying in density and electronegativity, being better called electronegative LDL [LDL (-)]. Considering the conformational differences between the ApoB-100 structure of native LDL and LDL (-), in an initial study, our group developed a monoclonal antibody (2C7) from the immunization of Balb/c mice with human LDL (-). In a next step, the epitope recognized by the anti-LDL monoclonal antibody (-) was mapped using phage display. The binding peptide of anti-LDL monoclonal antibodies (-) was named p2C7. This peptide does not represent linear sequence regions of human ApoB-100, but conformational microdomains of LDL (-) ApoB-100 epitopes, making them candidates for the immunomodulation of atherogenesis. Therefore, investigating the immunomodulation induced by p2C7 peptide mimetics of LDL (-) as it represents an immunodominant epitope of LDL (-) could open new future therapeutic perspectives for the immunomodulation of atherosclerosis. Objective: To evaluate the immunomodulation promoted by p2C7 in vivo, using C57BL/6 LDLr -/- mice, and human plasma samples. In addition, in the internship (BEPE), held at the Karolinska Institute (December 2019 to March 2021), immunometabolism as a mediator of Cardiovascular Diseases was studied. In part II-A, the results of the initially proposed study are described. In part II-B, the results that were developed later are presented, expanding the scope of the project, approaching the vascular inflammation involved in the abdominal aortic aneurysm through bioinformatics tools. In part II-C, the results of the study of the involvement of the enzyme indoleamine 2,3 dioxygenase (IDO) in non-alcoholic steatohepatitis (NASH) and atherosclerosis in ApoE-/- and ApoE/IDO/double mice are presented -knockout. Methodology: The presence of anti-p2C7 antibodies in human plasma samples with or without Metabolic Syndrome was evaluated. We measured circulating TNF in the same samples and proceeded with linear regressions associating inflammatory parameters with levels of anti-p2C7 antibodies. C57BL/6 LDLr -/- mice were immunized with p2C7 and the adjuvants Alum or Montanide ISA 720, analyzing the antibody titers against p2C7 and LDL (-), the production of cytokines (IL-10, IL-4, IL -2, IL-6, IFNγ, IL-17, TNFα) and antibody-secreting cells. C57BL/6 LDLr -/- mice were tolerized against mimotope peptides with intravenous injections (caudal vein) and challenged with immunization containing LDL (-) + Alum. Antibody titers against p2C7 and LDL (-) and cytokine production (TNF-α, IFNγ, IL-12, IL-6, IL-10 and MCP-1) were evaluated. The mice were kept on a hypercholesterolemic diet for 3 months for atherosclerotic plaque formation. After this period, the mice were euthanized, evaluating the formation of atherosclerotic plaque in the abdominal artery and aortic arch, as well as the production of cytokines (TNF-α, IFNγ, IL-12, IL-6, IL-10 and MCP -1). C57BL/6 LDLr -/- mice were immunized with OVA-p2C7 and, after a 3-month hypercholesterolemic diet for atherosclerotic plaque formation, inflammatory parameters were evaluated and 18F-FDG uptake was evaluated by PET/CT. Results: Immunization with p2C7 (free) was not able to induce a humoral response, with no detectable titers of antibodies reactive to p2C7 or LDL (-) being observed in any immunized mouse, as well as no detectable antibody-secreting cells for the LDL (-). The group immunized with Alum or Montanide + p2C7 had a significant increase in TNF-α production when compared to the other groups. The tolerance protocol was successfully performed, as the tolerized mice had lower antibody titers than controls for the epitope used. Only mice tolerated with p2C7 showed a significant increase in the production of IL-6, IL-12, IL-10, TNF-α, IFNγ and MCP 1 after a hypercholesterolemic diet. Active immunization with OVA-p2C7 was able to reduce TNF production induced by the hypercholesterolemic diet, as well as to reduce 18F-FDG uptake. Conclusion: the p2C7 epitope is highly expressed in LDL (-) of patients with higher cardiovascular risk. Furthermore, active immunization with p2C7 is also a promising tool to prevent and regulate inflammation caused by LDL (-) in the course of atherosclerosis

Caracterização imunológica da formulação vacinal baseada em VLP (Virus Like Particle) da Proteína Circumsporozoíta de Plasmodium vivax / Immunological characterization of the VLP (Virus Like Particle) vaccine formulation fused to Circumsporozoite Protein of Plasmodium vivax

O Plasmodium vivax é a espécie mais comum de parasita causador da malária humana encontrada fora da África, com maior endemicidade na Ásia, América Central e do Sul e Oceania. Embora o Plasmodium falciparum cause a maioria do número de mortes, o P. vivax pode levar à malária grave e resultar em morbimortalidade significativa. O desenvolvimento de uma vacina protetora será um passo importante para a eliminação da malária. Recentemente, uma formulação contendo as três variantes alélicas da proteína circumsporozoíta de P. vivax (PvCSP - All epitopes) induziu proteção parcial em camundongos após desafio com esporozoíto híbrido Plasmodium berghei (Pb), no qual as repetições centrais do PbCSP foram substituídas por repetições PvCSP-VK210 (esporozoítos Pb/Pv). No presente estudo, a proteína quimérica PvCSP contendo as variantes alélicas (VK210, VK247 e P. vivax-like) fusionadas com a proteína de nucleocapsídeo do vírus da caxumba (formando partículas semelhantes a nucleocapsídeos ou do inglês, NLP - Núcleo Like Particles) na ausência (NLP-CSPR) ou na presença do domínio C-terminal (CT) conservado da PvCSP (NLP-CSPCT). Para a realização do estudo selecionamos os adjuvantes Poly (I:C), um RNA sintético de dupla fita, agonista do receptor Toll do tipo 3 (TLR3) ou o adjuvante Montanide ISA 720, uma emulação óleo em agua. Para obter uma forte resposta imune, a levedura Pichia pastoris foi usada para expressar as proteínas recombinantes na forma de NLPs. Camundongos foram imunizados com cada uma das proteínas recombinantes em combinação com os adjuvantes citados. Embora ambas as NLPs tenham sido capazes de gerar uma forte resposta imune, com altos níveis de títulos e longevidade, apenas a formulação contendo a proteína NLP-CSPCT na presença do adjuvante Poly (I:C) foi selecionada para ser explorada em experimentos futuros. Esta proteína em combinação com o adjuvante Poly (I:C) induziu alta frequência de células secretoras de anticorpos específicas para o antígeno homólogo nos dias 5 e 30, no baço e na medula óssea, respectivamente. Altos títulos de IgG contra as 3 variantes de PvCSP foram detectados nos soros. Posteriormente camundongos imunizados com NLP-CSPCT foram desafiados com esporozoítos Pb/Pv e a parasitemia no 5º dia demonstrou proteção estéril em 30% dos camundongos desafiados. Portanto, a formulação vacinal gerada neste estudo tem potencial para ser explorada no desenvolvimento de uma vacina universal contra a malária causada por P. vivax

Plasmodium vivax is the most common species of human malaria parasite found outside Africa, with high endemicity in Asia, Central and South America, and Oceania. Although Plasmodium falciparum causes the majority of deaths, P. vivax can lead to severe malaria and result in significant morbidity and mortality. The development of a protective vaccine will be a major step toward malaria elimination. Recently, a formulation containing the three allelic variants of the P. vivax circumsporozoite protein (PvCSP--All epitopes) showed partial protection in mice after a challenge with the hybrid Plasmodium berghei (Pb) sporozoite, in which the PbCSP central repeats were replaced by the VK210 PvCSP repeats (Pb/Pv sporozoite). In the present study, the chimeric PvCSP allelic variants (VK210, VK247, and P. vivax-like) were fused with the mumps virus nucleocapsid protein (assembling into nucleo like particles - NLP) in the absence (NLP-CSPR) or presence of the conserved C-terminal (CT) domain of PvCSP (NLP-CSPCT). To carry out the study, we selected the adjuvants Poly (I:C), a synthetic double-stranded RNA, Toll-like receptor 3 (TLR3) agonist or Montanide ISA 720 adjuvant, an oil-water emulation. To elicit stronger immune response, Pichia pastoris yeast was used to produce the NLPs. Mice were immunized with each recombinant protein in combination with above. Although both NLPs were able to generate stronger immune response, with high antibodies titer levels and longevity, formulation containing NLP-CSPCT in the presence of Poly (I:C) was selected to be explored in future experiments. NLP-CSPCT with Poly (I:C) adjuvant presented a high frequency of antigen-specific antibody-secreting cells (ASCs) on days 5 and 30, respectively, in the spleen and bone marrow. Moreover, high IgG titers against all PvCSP variants were detected in the sera. Later, immunized mice with NLP-CSPCT were challenged with Pb/Pv sporozoites. Sterile protection was observed in 30% of the challenged mice. Therefore, this vaccine formulation use has the potential to be a good candidate for the development of a universal vaccine against P. vivax malaria.

Combined immunomagnetic cell sorting and ELISPOT assay for the phenotypic characterization of specific antibody-forming cells.

A combination of immunomagnetic cell sorting and ELISPOT techniques has been evaluated to permit enrichment and characterization of antibody-secreting cells (ASC). Cell suspensions containing putative ASC were first incubated with magnetic microbeads coated with antibodies specific for a given cell surface marker. After separation of bead-cell clusters and free cells, the resulting cell populations were examined for the presence of ASC by an ELISPOT assay. As a model system, the expression of selected cell differentiation markers by human circulating ASC has been evaluated after parenteral tetanus vaccination and during the course of a Leishmania infection. Prior treatment of blood MNC with beads coated with antibodies to CD38, HLA-DR or CD19 permitted the isolation of virtually all blood ASC. Further, prior immunomagnetic removal of T (CD2+) cells from blood MNC, followed by isolation of CD38+ cells facilitated the detection of Leishmania major-specific ASC in all six patients examined, whereas parasite-specific ASC among unfractionated blood mononuclear cells could only be detected in 3 out of these six patients. Simple and rapid, this approach provides not only accurate estimates of the frequency of ASC within a given B cell population or subpopulation, but can also efficiently enrich functional ASC from complex cell suspensions and thus should be particularly useful in situations where ASC are present at low frequencies.

Polyreactive antigen-binding B cells in the peripheral circulation are IgD+ and B7-.

Polyreactive antibodies are naturally occurring antibodies, primarily of the IgM isotype, that are capable of reacting with a wide variety of different self and non-self antigens. Previously, we reported that a B cell capable of making polyreactive antibody has Ig receptors on its surface that can bind different antigens. The present investigation was initiated to characterize these polyreactive antigen-binding B cells further. A panel of fluorescein isothiocyanate-labeled antigens (insulin, IgG Fc fragment or beta-galactosidase) served as probes to select polyreactive antigen-binding B cells by cell sorting. Our experiment revealed that these polyreactive antigen-binding B cells were mainly of the IgD isotype. They expressed high levels of CD40 and major histocompatibility complex class II molecules, but little or no B7-1, B7-2, or Fas. In contrast to the binding of antigens to monoreactive receptors (usually high affinity), the binding of antigens to polyreactive receptors (usually moderate or low affinity) did not up-regulate the expression of B7-1 or B7-2. Antigens that bound to polyreactive receptors, however, were internalized and degraded, although not as efficiently as antigens that bound to monoreactive receptors. Despite the ability of these B7- cells to process antigens, they were not able to activate T cells in a mixed leukocyte reaction. It is concluded that polyreactive antigen-binding B cells have properties that are consistent with the ability to induce immunological tolerance.

The phenotype and fate of the antibody-forming cells of the splenic foci.

The first product of the humoral response to antigen is low-affinity antibody, produced by extrafollicular foci of antibody-forming cells (AFC) in organs such as spleen and lymph node. These cells proliferate rapidly but then undergo an equally rapid decline, so that they are present in only small numbers 14 days after immunization. We have used 6-parameter flow cytometry to isolate and examine the characteristics of (4-hydroxy-5-nitrophenyl)acetyl-specific AFC, looking in particular for those markers that might differentiate them from cells of the intrafollicular (germinal center) arm of the T-dependent immune response. At day 7 of the primary response, most AFC were found to express surprisingly low levels of B220, high levels of syndecan, and retain significant levels of surface IgG1. We then used enzyme-linked immunospot assays to demonstrate that the rapid decline of these cells was not likely to be due to migration to organs such as the bone marrow. Their decline could, however, be explained by apoptosis in situ, which was demonstrated immunohistologically by nick-end labeling.

Normal uterine cervix: characterization of isolated lymphocyte phenotypes and immunoglobulin secretion.

PROBLEM: Isolation of viable cervical lymphocyte populations and characterization of their function in healthy tissue is necessary to understand immunity in the genital tract. METHODS: Normal, cervical tissue was digested using a multi-enzymatic digestion procedure. Lymphocytes were characterized using FACS analysis and ELISPOT analysis for immunoglobulin secreting cells. RESULTS: Following the digestion procedure, 0.16 x 10(6) +/- 0.8 cells/g of tissue with a viability of 90-98% were isolated from normal cervical tissue. FACS analysis determined that B lymphocytes were the predominant cell type in normal cervical tissue representing a significantly higher percentage than that found in peripheral blood (P = 0.015). T lymphocytes and NK cells represented a significantly lower percentage than that found in peripheral blood (P = 0.0001 and 0.026, respectively). The largest percentage of immunoglobulin secreting cells isolated were secreting IgG followed by IgA. A limited number of IgM secreting cells were detected. IgA2 secreting cells represented 34.46 +/- 4.6% of the total number of IgA plasma cells. CONCLUSION: These studies represent the first analysis of viable mononuclear cells isolated from normal cervical tissue. The results form a baseline from which it will now be possible to compare changes that occur at the cervical squamocolumnar junction in response to infection or neoplasia.

Analysis of human IgG and IgA subclass antibody-secreting cells from localized chronic inflammatory tissue.

The chronic inflammatory diseases in humans have been intensively investigated, however the immune mechanisms underlying diseases such as rheumatoid arthritis (RA), inflammatory bowel disease, and periodontal disease (PD) remain elusive. In this study, we have analyzed the distribution of IgM, IgG, and IgA secreting cells with emphasis on the IgG and IgA subclasses among mononuclear cell populations isolated from gingiva at different stages of PD. Surgically removed tissues were treated with Dispase to gently dissociate cells and the Ficoll-Hypaque gradient centrifugation was used to enrich for viable mononuclear cells rich in lymphocytes, macrophages, and plasma cells. The total numbers of plasma cells increased with the severity of disease. Immunofluorescence analysis showed that most Ig-containing cells were of the IgG isotype; however, significant numbers of IgA-positive cells but few IgM-positive cells were seen. This isolation procedure allowed analysis, at the single cell level, of the distribution of IgG and IgA subclasses of antibody-secreting cells with monoclonal antibodies to human IgG and IgA subclasses. For this, we selected four monoclonal anti-IgG subclass (anti-gamma 1, -gamma 2, -gamma 3, and -gamma 4) antibodies with no subclass cross reactivity for use in the enzyme-linked immunospot assay. Analysis of slight, moderate, and advanced stages of PD showed a progressive increase in spotforming cells (SFC) numbers, and the major isotype of SFC was IgG followed by IgA. The major IgG subclass SFC seen was IgG1 followed by IgG2 whereas similar numbers of IgG3 and IgG4 SFC were observed, a pattern also seen with cells from synovium of RA patients and in mitogen-triggered spleen and PBMC. In terms of the IgA subclass distribution, IgA1 predominated in moderate stages, whereas a selective increase in IgA2 SFC were seen in the more advanced stage of PD. These results show that significant numbers of viable plasma cells/Ig-secreting cells can be isolated from inflamed gingival tissues. Further, careful analysis has shown that IgG subclass responses in gingiva are similar to those found in synovia of RA subjects, and in stimulated PBMC and spleen. However, it should be noted that the number of IgG4- and IgA2-secreting cells increased in the advanced stage of PD.

Inverse relationship between proliferation and differentiation in a human TNP-specific B cell line. Cell cycle dependence of antibody secretion.

Studies of an EBV-transformed and TNP-specific human B cell line revealed that, unlike myeloma or hybridoma cell lines that consist mainly of fully differentiated cells, most of the cloned EBV-transformed cells were not fully differentiated, as judged by inability to bind TNP-SRBC and to secrete anti-TNP antibody. The minority of more differentiated cells were selected by TNP-SRBC rosetting. They were found to proliferate to a lesser extent than nonrosetting cells and to contain increased numbers of antibody-secreting cells. This inverse relationship between proliferation and differentiation was also shown to be cell cycle related in that the TNP-SRBC rosetting cells resided, to a greater extent than the nonrosetting cells, in the G1 phase of the cell cycle. The finding that the G1 phase of the cell cycle was associated with differentiation into anti-TNP secreting cells was confirmed by demonstrating that treatment with hydroxyurea, which arrests the cells in G1, resulted in decreased proliferation and an increased proportion of antibody-secreting cells. Similarly, addition of phorbol ester resulted in increased antibody secretion and decreased proliferation, suggesting a role for protein kinase C in this differentiation pathway. The strategy of increasing the number of antibody-producing cells in this human EBV line, by promoting differentiation of the cells in G1, may be relevant to the large scale production of specific human mAb for the treatment and diagnosis of human diseases.

The influence of exogenous antigenic stimulation on the specificity repertoire of background immunoglobulin-secreting cells of different isotypes.

The total number of spontaneously occurring ("background") IgM-, IgG-, and IgA-secreting cells and the frequency of antigen-specific IgM-, IgG-, and IgA-secreting cells were determined in germ-free BALB/c mice fed a chemically defined ultrafiltered diet (GF-CD), in specific pathogen-free BALB/c mice fed an autoclaved natural ingredient diet (SPF-NI), and in conventional BALB/c mice fed nonautoclaved natural ingredients (CV-NI). This was done by means of the ELISA-plaque assay. The results did not show differences among the various groups of mice with regard to the total numbers of IgM-secreting cells in the various lymphoid organs. Also the frequencies of IgM-secreting cells specific for DNP27-BSA and the anti-idiotypic monoclonal antibodies Ac38 and Ac146 did not differ significantly among GF-CD, SPF-NI, and CV-NI mice. GF-CD mice, however, did show substantially decreased numbers of IgG- and IgA-secreting cells in their lymphoid organs. Furthermore, there were striking differences in the frequencies of antigen-specific IgG- and IgA-secreting cells between GF-CD mice and the two other groups of mice. These results indicate that exogenous antigenic stimulation has a great effect on both the total numbers and the specificity repertoires of background IgG- and IgA-secreting cells. Such an influence could not be detected with regard to the background IgM-secreting cells. This suggests two distinct compartments of background Ig-secreting cells: a very stable, endogenously regulated compartment consisting mainly of IgM-secreting cells, and another compartment, consisting mainly of IgG- and IgA-secreting cells, whose numbers and specificity repertoire appeared to be influenced by exogenous antigenic stimulation.

Mucosal subclass distribution of immunoglobulin G-producing cells is different in ulcerative colitis and Crohn's disease of the colon.

As a marked local immunoglobulin G (IgG) response has previously been found to be the most prominent immunopathological feature of both ulcerative colitis and Crohn's disease, the subclass distribution of colonic IgG-producing immunocytes was examined. This study included tissue specimens from 10 patients with ulcerative colitis and 8 with Crohn's colitis. Paired immunofluorescence staining was performed with subclass-specific murine monoclonal antibodies combined with a rabbit antibody reagent of IgG; the proportion of cells belonging to each subclass could thereby be determined in relation to the total number of mucosal IgG immunocytes. A significantly higher median proportion of IgG1 immunocytes was found in ulcerative colitis (81.3%) than in Crohn's colitis (66.5%). Conversely, the median proportion of IgG2 immunocytes was significantly higher in Crohn's colitis (24.9%) than in ulcerative colitis (9.4%). This disparity in the local IgG subclass response might reflect dissimilar mucosal exposure to mitogenetic or antigenic stimuli or genetically determined immunoregulatory differences in the two categories of patients.

In vitro regulation of IgA subclass production. III. Selective transformation of IgA1 producing cells by Epstein-Barr virus.

In past experiments, using limited dilution analysis, we have demonstrated that a high percentage of immunoglobulin-secreting clones derived from Epstein-Barr virus- (EBV) stimulated lymphocytes secrete IgA. To further characterize the IgA produced by these clones, the IgA subclass of supernatants from clones stimulated 4 to 6 wk previously with EBV was determined by radioimmunoassay. All of 17 IgA-producing clones secreted IgA1; none secreted IgA2. Because we have shown that surface IgM+ (sIgM+) B cells are an enriched source of IgA2 plasma cell precursors, panning techniques were used to purify sIgM+ B cells from tonsils. Of 103 clones derived from these sIgM+ B cells, 102 secreted IgA1 and only one secreted IgA2. The relative absence of IgA2-producing clones could not be attributed to an absence of EBV receptors on IgA2 cells. A mean of 84 +/- 4% of freshly isolated IgA2 B cells and 78 +/- 6% of IgA1 B cells could be stained with a monoclonal antibody binding the EBV receptor; and there was no failure of EBV to infect IgA2 plasma cells precursors. Of IgA2 plasma cells derived from peripheral blood lymphocytes stimulated 7 days previously with EBV, 54 +/- 7% were positive for the EBV nuclear antigen, compared with 54 +/- 18% of IgA1 plasma cells from the same cultures. Seven days after EBV stimulation, a mean of 25% of the total IgA plasma cells were positive for cytoplasmic IgA2, whereas by 21 days after stimulation only 7% were positive for IgA2. This shift in the proportions of IgA1 and IgA2 plasma cells could be attributed to a failure of the IgA2 plasma cell number to increase after 10 days in culture. There was no evidence for selective suppression of IgA2 production by T cells or selective lysis of IgA2 plasma cells by infectious EBV particles. These results demonstrate that although precursors for both IgA1- and IgA2-producing cells can be stimulated to differentiate in response to EBV, there is preferential transformation of IgA1-producing cells.

Different subclass distribution of IgA-producing cells in human lymphoid organs and various secretory tissues.

A highly reproducible paired immunofluorescence staining method was used to map the relative distribution of IgA1- and IgA2-producing cells in peripheral lymphoid organs and various secretory tissues. Spleen, peripheral lymph nodes, and tonsils all contained a marked predominance (91 to 95%) of IgA1 immunocytes. However, striking variations were demonstrated among the secretory tissues with regard to the median proportion of IgA1-producing cells: nasal mucosa, 96%; lacrimal glands, 81%; major salivary glands, 66%; mammary glands, 63%; gastric and proximal small intestinal mucosa, 84 to 77%; ileum, 55%; and large bowel, 41%. Thus, IgA2 production is relatively enhanced mainly in the distal gut and in mammary and salivary glands, in that order.

Ontogenic studies on the appearance of two classes of immunoglobulin-forming cells in the spleen of the Aleutian skate, Bathyraja aleutica, a cartilaginous fish.

We identified the presence in the Aleutian skate, Bathyraja aleutica, of two classes of immunoglobulins (Ig), a high molecular weight Ig analogous to mammalian IgM and a low molecular weight Ig, similarly to the spiny rasp skate, Raja kenojei, (Kobayashi, K. et al., Mol. Immunol. 1984. 21: 397), using an immunological cross-reaction with the specific antisera to the spiny rasp skate Ig components. The antigenic similarity of the heavy chains of the Ig of the Aleutian skate and those of the spiny rasp skate was less than that between their light chains. Two types of Ig-producing cells, one producing the high molecular weight and the other forming the low molecular weight Ig, were present in the spleen of embryos and adults in the Aleutian skate at a ratio of 5-6:4-5. The number of these Ig-producing cells increased with advancing development of the embryos but was 1/20 to 1/50 of those of adults. Cells, each of which were capable of forming both classes of Ig, were found in the spleen of embryos but not in that of adults. These results suggested that the spleen of the Aleutian skate is the primary lymphoid organ for B lymphocyte differentiation and proliferation, possibly equivalent to the bursa of birds.

In vivo isotype regulation of humoral responses to dextran B1355 in BALB/c mice. Double-class IgG3/IgM producers as a function of age.

This report shows that, in 8- to 10-month-old BALB/c mice immunized intraperitoneally with dextran B1355, approximately 75% of IgG3 anti-alpha (1----3) polyglucan (anti-dex) plaque-forming cells (PFC) detected in the spleen were identified as double-Ig class producers secreting simultaneously IgG3 and IgM antibodies with the same specificity for the dex epitope. Under the same conditions of immunization, however, IgA anti-dex PFC were mostly single-class secretors. IgA PFC developed in the spleen in highest numbers (equal to IgM), but in Peyer's patches IgA PFC were sevenfold more numerous than IgM. Furthermore, spleen IgG3 anti-dex PFC responses were low compared with spleen IgA and IgM anti-dex PFC responses and appeared only late in ontogeny. The possibility is discussed whether a TH dependence of the IgA anti-dex response and a TH-independent generation of the IgG3 response are responsible for the different pattern of isotype expression.

Specificity of plasma cells in the rheumatoid synovium. I. Immunoglobulin class of antiglobulin-producing cells.

Plasma cells synthesizing rheumatoid factors (RF) were identified by fluorescent staining of sections of synovium and macrophage-depleted cells from dispersed synovial tissue. The latter avoided problems related to sampling errors in studying tissue sections and in the uncertainty raised by the staining of macrophages with intracellular complexes. Plasma cells producing IgG predominated, and seropositive patients had a higher proportion of IgM producers than seronegative subjects. None the less, in both groups of patients more than 90% of the IgM plasma cells were synthesizing RF, whereas the corresponding figure for IgG was between 50% and 60%. Only around 10% of IgA plasma cells were positive for RF. The high percentage of IgM plasma cells making RF would tend to argue for an IgG-specific response and against direct polyclonal activation as the stimulus. The percentage of IgG-producing cells positive for RF is also consistent with a dominant response to IgG. Accepting the difference in the relative proportion of total IgM- to IgG-producing plasma cells in seropositive as against seronegative patients, the close similarity between the two groups in the fraction of cells making RF favours the view that the two groups have a comparable underlying immunopathology dependent on IgG autosensitization. From the technical standpoint, the dispersed cell method gives results in line with those obtained with sections but which are easier to read, whereas the fluorescent techniques described give clear and reproducible results for the detection of RF of different heavy-chain isotype.

Human in vivo antigen-induced lymphoblastoid B cells are capable of cyclical antibody production in vitro.

In vivo immunization of normal volunteers with tetanus toxoid induces the formation of a circulating B cell subset that has the capacity to secrete specific antibody in vitro without the need for T cell help or mitogen stimulation. From earlier studies it was not clear whether the spontaneous antibody-secreting lymphoblastoid (LB) B cell was at a terminal stage of differentiation or if it had the capacity to give rise to additional B cell subsets, such as memory cells or more fully mature antibody-producing cells. In this study we have shown that at least two distinct waves of spontaneous antibody secretion can occur in vitro when cultures are initiated with the lymphocytes from individuals immunized 6 days earlier. The first production of antibody was completed by 3 days of in vitro culture and the second production of antibody did not initiate until day 7 or 8 of culture and was completed by day 12. The B cells responsible for the second stage of antibody production appeared derived from a portion of the antibody-secreting cells present on day 3 in that 1) treatment of the cultures on day 0 with BuDr and light equally inhibited the first and second rounds of antibody synthesis; 2) when isolated from the blood, both B cell subsets were in the large cell fraction after 1 X G sedimentation; and 3) under conditions of limiting numbers of cells, the cells responsible for the second wave of antibody production were almost exclusively found in cultures positive for a B cell that had produced antibody on days 1 to 3. Although only a portion (10 to 30%) of the LB B cells present on day 0 had the capacity to again produce antibody on days 8 to 12, the two cells were capable of producing similar quantities of antibody on a per cell basis. These results indicate that the mature circulating LB cell induced in vivo by immunization is not terminally differentiated, but under appropriate conditions has the capacity to give rise to additional antibody-secreting cells.

An idiotype-specific helper population that bears immunoglobulin, Ia, and Lyt-1 determinants.

A helper cell population with phenotypic characteristics of both B and T cells is described. This helper population, called BH, is present in normal unprimed C57BL/6 mice and preferentially helps the expression of NPb idiotype-bearing plaque-forming B cells in the absence of T helper cells. Its surface phenotype is Lyt-1.2+, Ig+, Lyb-3+, Thy-1.2-, Lyt-2.2-. The helper activity of the BH population is IgH restricted and BH cells selectively bind NPb idiotypic determinants. Collectively the data demonstrate that this unique subpopulation can regulate the response of antibody-secreting B cells through specific recognition of idiotypic determinants.

Adjuvants influence the immunoglobin subclass distribution of immune responses in vivo.

Mice were immunized against fluorescein isothiocyanate (FITC)-labelled human gamma globulin (HGG) in the absence or presence of different adjuvants. The immune response was assayed every other day with regard to both total Ig-secreting cells and FITC-specific plaque-forming cells (PFC). The adjuvants influenced the type of immune response induced to the same antigenic determinant. Thus, addition of Freund's complete (FCA) or incomplete (FIA) adjuvant preferentially led to the secretion of IgG1 PFC of an average high affinity. Most newly appearing IgG-secreting cells were also detected as FITC-specific PFC. The use of lipopolysaccharide (LPS) as an adjuvant resulted in the induction of both IgM and IgG, particularly of the IgG3 and IgG2b subclasses. However, these antibodies had relatively low affinity, and a large number of total IgG-secreting cells induced by LPS had no detectable FITC specificity. The FCA/FIA- and LPS-induced responses to FITC-HGG were additive when injected together, indicating that they act on distinct subpopulations of B lymphoid cells. The adjuvant response to LPS, but not the response to FCA/FIA, was totally absent in mice of the C3H/Hej strain, which are non-responders to the polyclonal activating properties of LPS. Finally, the response induced by FCA or FIA was T-cell-dependent and the LPS response T-cell-independent as assayed in nude mice.

Isotype specificity of helper T cell clones. Peyer's patch Th cells preferentially collaborate with mature IgA B cells for IgA responses.

The nature of the IgA B cell precursors that receive preferential help from selected clones of T helper cells from mouse Peyer's patches (PP Th A) were studied. Activation of the PP Th A clones required the presence of antigen, sheep erythrocytes (SRBC), in a culture system supporting development of antibody-secreting plasma cells. Two types of PP Th A cells were used. Both gave vigorous IgA responses; the first also supported low IgM, and the second low IgM and IgG subclass antibody responses. Removal of sIgA+ B cells from either splenic or PP B cell cultures selectively depleted precursors of IgA antibody producers. Cultures of purified sIgA+ B cells, cloned PP Th A cells and SRBC, selectively yielded IgA antibody producers. Finally, PP Th A cells did not support IgA responses in B cell cultures derived from spleens of young mice (days 1-25), and full IgA responses were not seen until the donor mice were 6-7 wk of age. These results suggest that cloned T helper cells can recognize and collaborate with mature, IgA committed B cells.

In vitro immunoglobulin production by peripheral blood mononuclear cells from multiple myeloma patients and patients with benign monoclonal gammopathy. Regulation by cell subsets.

Peripheral blood mononuclear cells (PBM) from four patients with IgG myeloma and four patients with benign monoclonal gammopathy (BMG) were stimulated with pokeweed mitogen (PWM), and the in vitro immunoglobulin production over 7 days was measured with an enzyme-linked immunosorbent assay. All myeloma patients were sufficiently treated with cytostatic drugs. Their PBM did not produce monoclonal Ig in vitro, as opposed to PBM from two patients with BMG. Unseparated PBM from myeloma patients produced smaller amounts of polyclonal Ig than unseparated cells from normal donors. However, macrophage-depleted PBM from myeloma patients produced amounts of Ig comparable to those of normal donors when autologous or allogeneic adherent cells were returned in defined numbers. T cells from three of the four myeloma patients could provide help for the Ig production by B cells from healthy donors. These results indicate that functionally normal polyclonal B cells circulate in the blood of myeloma patients. The circulating T-cell population also has no obvious defect. In contrast, blood macrophages seemed to be altered with respect to their regulating function for polyclonal Ig production. The results obtained by using cell populations from patients with BMG did not differ from those of healthy people.