Several B-cell defects arise in HIV infected patients, particularly in patients with chronic infection and high viral load. Despite several studies, definitive identification of memory B cells based on CD27 surface expression has not been described. Similarly, the rates of cell turnover in different B cell subpopulation from lymphoid and mucosal tissues have not been well documented. In this study, we demonstrate the presence of memory B cell populations and define their distribution, frequency and immunophenotype with regards to activation, proliferation, maturation, and antibody production in normal rhesus macaques from different lymphoid tissues. Thirteen healthy, uninfected rhesus macaques were selected for this study.
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Several B-cell defects arise in HIV infected patients, particularly in patients with chronic infection and high viral load. Despite several studies, definitive identification of memory B cells based on CD27 surface expression has not been described.
Similarly, the rates of cell turnover in different B cell subpopulation from lymphoid and mucosal tissues have not been well documented. In this study, we demonstrate the presence of memory B cell populations and define their distribution, frequency and immunophenotype with regards to activation, proliferation, maturation, and antibody production in normal rhesus macaques from different lymphoid tissues.
Thirteen healthy, uninfected rhesus macaques were selected for this study. All the B cell subpopulation was further characterized phenotypically and their cell turnover rates were evaluated in vivo following bromodeoxyuridine BrdU inoculation.
Increased turnover of tonsillar memory B cells were identified compared to other tissues examined. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
Immunological memory is a crucial feature of adaptive immunity, whereby the first encounter with a pathogen is imprinted indelibly into the immune system .
Memory B cells and long-lived plasma cells are responsible for the long-term humoral immunity elicited by most vaccines  , . Immune responses to T cell-dependent antigen occur within secondary lymphoid tissues. These newly generated memory B cells can re-enter the circulation or remain as resident cells within discrete regions of secondary lymphoid tissue, like marginal zone of spleen or mucosal epithelium of tonsil  ,  ,  ,  , .
Most of the memory B cells information has come from human studies. Presumably because of the constant exposure to antigens, humans have an abundance of memory-like cells, as defined by the marker CD27  ,  , . Surface receptor CD27, a type 1 glycoprotein and a member of tumor necrosis factor receptor family was first reported on a subset of human B cells and was thought that their expression may be acquired late during B cell differentiation  , .
Moreover, memory B cells can be activated to proliferate and differentiate into antibody secreting cells in an antigen-independent fashion by microbial products, cytokines, bystander T-cell help, and, possibly other stimuli yet to be defined  , . Complement receptor 2 CD21 is a cell-surface protein that contains a small cytoplasmic domain and an extracellular domain consisting of a series of short consensus repeats termed complement control protein domains.
CD21, which recognize activated products of complement 3 is predominantly expressed on mature B cells and follicular dendritic cells  and is an important receptor for uptake and retention of immune complexes.
In the absence of CD21 expression, survival of memory B cells is markedly impaired . HIV-induced immune dysfunction includes B-cell activation and the impaired production of antibodies that is partially related to memory B cells  ,  ,  ,  , .
Despite all these studies, identification of specific memory B cell subsets based on CD27 and CD21 surface expression and antibody production has not been well described. Similarly, the rate of cell turnover of B cell subpopulations in different lymphoid and mucosal tissues has not been examined in vivo. In this study, we performed an extensive analysis to characterize memory B cells obtained from peripheral blood PB , axillary lymph node ALN , bronchoalveolar lavage BAL , bone marrow BM , spleen, tonsil and intestinal lamina propria lymphocytes LPL of the jejunum from normal rhesus macaques RMs.
We have characterized memory B cell populations including their distribution, frequency, and immunophenotype with regards to activation, proliferation and maturation in normal macaques.
Furthermore, we examined the levels of turnover of different B cell subsets in lymphoid tissues to determine tissue specific B cell proliferation in RMs. In addition, these DP cells may be important in HIV infection and pathogenesis due to their high cell turnover rate and increased antibody production.
In order to determine the distribution of CD21 surface marker in relation to CD27 expression, we performed extensive analysis of B cells from PB and different lymphoid tissues in normal RMs. Each quadrant shows percentages of specified populations. Statistical significant differences between each group of cells are shown. Unstimulated filled histogram and stimulated open histogram sorted B cell cultures are shown as histogram for expression of anti-CD20, anti-CD, and anti-CD This experiment was repeated independently 3 times and data shown is the mean value of the representative plot.
B-1 cells are a subclass of B cells that express CD5 are produced during fetal development. These cells express greater quantities of IgM, and its receptor shows polyspecificity meaning affinity for immunoglobulins, self-antigens and common bacterial polysaccharides .
However, no significant differences in CD79a were detected in PB cell subsets. Integrin, alpha L CD11a is involved in cellular adhesion and costimulatory signaling. CD80 B7. CD23, the low-affinity IgE receptor is widely distributed on cells and mediates IgE-related immune responses by enhancing IgE antigen complex presentation, regulating IgE synthesis and influencing cell differentiation and growth of both B and T cells.
BrdU was injected intraperitoneally and tissues were collected 24 hrs after inoculation. The immune activation following SIV infection may also predispose these memory cells to activation-induced apoptosis. The ability of memory B cells to generate a robust and efficient antibody response to secondary challenge by an invading pathogen is a hallmark of immunological memory. IgG production. Bernasconi and his group previously showed that long term serological memory could be generated by antigen independent polyclonal activation and differentiation of memory B cells .
In this study we utilized T cell-independent polyclonal stimulation to define memory B cell function. B-1 CD5 cells are believed to be the primary source of IgM production . By modeling the rate of BrdU uptake in vivo in normal adult RMs, we were able to study the differences in lymphocyte turnover rates among B-cell subsets.
The DP B cells were capable of producing antibody in absence of T cell help, and had increased cell turnover compared to other B cell subpopulations examined. Tonsil has the highest proliferating memory B cell population than any other tissues examined. It is important to monitor the proliferating and functional capabilities of these specific B cell subsets during SIV infection to fully understand the extent of the damage to the memory B cell and correlate these effects with antigen specific humoral immune responses.
All veterinary procedures were performed only with sedated animals. Lymphocytes from the PB, intestine, ALN, and spleen were isolated as previously described  ,  ,  ,  , . Mucus and large debris were removed from the supernatant by filtering through loosely packed glass wool. For enrichment of lymphocytes, supernatants of LPLs were centrifuged over discontinuous Percoll Sigma-Aldrich density gradients followed by washing with phosphate-buffered saline PBS.
For lymphocyte isolation from ALN, tonsil and spleen, tissues were simply minced and gently pressed through um nylon cell strainers. Bronchoalveolar lavage samples were collected in normal saline solution.
Bone marrow cells were filtered, lysed with ACK solution Lonza Biowhittaker and counted for further immunofluorescent staining. Cells from each well were resuspended in staining media and further stained for CD86 activation , CD plasma cell and CD20 phenotyping markers.
Unstimulated cells were kept as negative controls for each experiment and a total of 3 independent experiments were performed with sorted cells. Cell culture supernatants were diluted as necessary and tested in duplicate. Plates were then developed with o-phenylenediamine dihydrochloride OPD Sigma-Aldrich for 5 min and stopped with 2N H 2 SO 4 , and the absorbance was measured on a spectrophotometer at nm with a reference filter of nm.
The specificity of the rhesus monkey IgG detection antibody has been previously demonstrated . The IgG concentrations in cell culture supernatants are presented as micrograms per milliliter. Whole blood, and spleen samples were then lysed and washed using a whole blood lysis protocol as previously described  ,  , . Lymphocytes from ALN, intestinal LPL, BM, and tonsil were stained and processed similar to blood tissues with the omission of the whole blood lysing technique .
Single-stained controls for each fluorochrome were used for compensation settings. At least 30, events were collected from each sample by gating on lymphocytes and data were analyzed using FlowJo software TreeStar Inc.
All monoclonal antibodies were first titrated and tested for their crossreactivity in RMs, for both T and B cells. Results of experimental groups were compared using either a two-tailed Student's paired t-test or nonparametric Mann-Whitney t test using Prism software GraphPad software, SanDiego, CA.
We also thank Drs. Conceived and designed the experiments: AD BP. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Background Several B-cell defects arise in HIV infected patients, particularly in patients with chronic infection and high viral load.
Introduction Immunological memory is a crucial feature of adaptive immunity, whereby the first encounter with a pathogen is imprinted indelibly into the immune system .
Results CD21 expression differs between peripheral blood and lymphoid tissue B cells In order to determine the distribution of CD21 surface marker in relation to CD27 expression, we performed extensive analysis of B cells from PB and different lymphoid tissues in normal RMs.
Download: PPT. Figure 1. Phenotyping B cell subsets in tissues of rhesus macaques. Figure 2. Functional and phenotype properties of B cell subsets. Figure 3. Figure 4. Figure 5. Figure 6. Lymphocyte isolation from tissues Lymphocytes from the PB, intestine, ALN, and spleen were isolated as previously described  ,  ,  ,  , .
Supporting Information. Figure S1. References 1. Zinkernagel RM On immunological memory. View Article Google Scholar 2. Immunity 8: — View Article Google Scholar 3. Science — View Article Google Scholar 4. Ahmed R, Gray D Immunological memory and protective immunity: understanding their relation.
Science 54— View Article Google Scholar 5. Eur J Immunol —
Expression of CD27 and CD23 on peripheral blood B lymphocytes in humans of different ages
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