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Human CD20+CD43+CD27+CD5− B cells generate antibodies to capsular polysaccharides of Streptococcus pneumoniae

2012; Elsevier BV; Volume: 130; Issue: 1 Linguagem: Inglês

10.1016/j.jaci.2012.04.040

1097-6825

Bert Verbinnen, Kris Covens, Leen Moens, Isabelle Meyts, Xavier Bossuyt,

Glycosylation and Glycoproteins Research

Infections caused by Streptococcus pneumoniae (eg, pneumonia, septicemia, and meningitis) are an important cause of mortality and morbidity, especially among young children, the elderly, and immune-compromised patients. Capsular polysaccharides (caps-PSs) are a main determinant of the virulence of S pneumoniae, and antibodies to caps-PSs, which are considered T lymphocyte–independent (TI) type 2 antigens, are protective against infections with S pneumoniae. In mice antibodies to TI antigens have been shown to be produced by a unique B-lymphocyte population, the so-called B-1 cells.1Baumgarth N. The double life of a B-1 cell: self-reactivity selects for protective effector functions.Nat Rev Immunol. 2011; 11: 34-46Crossref PubMed Scopus (692) Google Scholar Moreover, B-1 cells can be subdivided into 2 functionally different subpopulations on the basis of surface expression of CD5. The CD5+ subset (referred to as B-1a cells) is responsible for the constitutive secretion of polyspecific antibodies (mainly IgM isotype), whereas the CD5− subset (B1-b cells) produces only antibodies (IgM, IgA, and IgG isotype) after antigen-specific stimulation, including stimulation with caps-PS antigens of S pneumoniae.2Haas K.M. Poe J.C. Steeber D.A. Tedder T.F. B-1a and B1-b cells exhibit distinct developmental requirements and have unique functional roles in innate and adaptive immunity to S. pneumoniae.Immunity. 2005; 23: 7-18Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 3Haas K.M. Programmed cell death 1 suppresses B-1b cell expansion and long-lived IgG production in response to T cell-independent type 2 antigens.J Immunol. 2011; 187: 5183-5195Crossref PubMed Scopus (45) Google Scholar The identity and even existence of the human counterpart of murine B-1 cells has been in doubt for many years, mainly because of the absence of known cell-surface markers to identify this population. Recently, however, Griffin et al4Griffin D.O. Holodick N.E. Rothstein T.L. Human B1 cells in umbilical cord and adult peripheral blood express the novel phenotype CD20+ CD27+ CD43+ CD70-.J Exp Med. 2011; 208: 67-80Crossref PubMed Scopus (468) Google Scholar claimed the discovery of a small subset of CD20+ B cells in human peripheral blood specifically expressing CD27 and CD43 and lacking CD70 that recapitulates the key functional characteristics of murine B-1 cells. In their work Griffin et al also showed that some, but not all, of the cells that they describe to be human B-1 cells had surface expression of CD5. However, it was not investigated whether similar functional differences as found in their murine counterparts could be attributed to them. Since the discovery of the recently proposed human B-1 cells, no studies have been reported that associate these cells with antibody generation to TI antigens of human pathogens. We here studied whether the CD20+CD27+CD43+CD70− cells are able to generate caps-PS–specific antibodies on vaccination with the caps-PS–based vaccine against S pneumoniae (Pneumo23; Sanofi Pasteur SA, Lyon, France) and whether differences in the CD5+ and CD5− subsets could be detected. We first evaluated the percentage (among CD19+CD20+ cells) and absolute number of the CD5− and CD5+ subsets of the proposed B-1 cells in 5 healthy adult volunteers before and 7 days after vaccination with Pneumo23. Blood cell counts, including leukocyte differentiation, were carried out, as well as flow cytometric phenotyping with fluorescence-labeled antibodies (for the gating scheme, see Fig 1, A). Given the rare nature of the proposed B-1 cells, we applied a stringent gating strategy to exclude doublets on forward scatter H/forward scatter A and CD3+ T cells to discriminate for true B lymphocytes because T cells can still contaminate the proposed B-1 cell population despite doublet exclusion.5Perez-Andres M. Grosserichter-Wagener C. Teodosio C. van Dongen J.J. Orfao A. van Zelm M.C. The nature of circulating CD27+CD43+ B cells.J Exp Med. 2011; 208: 2565-2566Crossref PubMed Scopus (76) Google Scholar, 6Descatoire M. Weill J.C. Reynaud C.A. Weller S. A human equivalent of mouse B-1 cells?.J Exp Med. 2011; 208: 2563-2564Crossref PubMed Scopus (82) Google Scholar As shown in Fig 1, B, we found a significant increase in both the percentage (among CD19+CD20+ cells; P = .031, Wilcoxon test) and absolute number (P = .031, Wilcoxon test) of the proposed B-1 cells 1 week after the vaccination. Interestingly, before vaccination, the majority of the proposed B-1 cells were CD5+ (65.6% ± 5.9%), whereas after vaccination, the majority were CD5− (68.0% ± 7.4%, Fig 2).Fig 2Polysaccharide (PS)–specific antibody secretion by CD5− and CD5+ subsets of the proposed B-1 cell and by naive and memory B cells after vaccination with Pneumo23. Healthy subjects were vaccinated with Pneumo23, and 1 week later, their CD5+ and CD5− proposed B-1 cells (CD19+CD20+CD27+CD43+CD70−), as well as their naive B cells (CD19+CD20+CD27−CD43−) and memory B cells (CD19+CD20+CD27+CD43−), were analyzed for their capacity to secrete IgA, IgM, and IgG anti–caps-PS antibodies by using ELISpot. Cells were plated at 2 × 104 cells per well and incubated for 12 hours at 37°C. A, Representative ELISpot wells from 1 donor are shown for PS1. B, The number of spots for PS1 and PS4 for each B-cell subset is shown for IgA, IgM, and IgG. >>, Excess of spots; —, not done.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, we compared the capacity of the CD5+ and CD5− subsets of the proposed B-1 cells with that of naive cells (defined as CD20+CD27−CD43−CD70−), conventional memory B cells (defined as CD20+CD27+CD43−CD70−), plasmablasts (defined as CD19+CD20−CD138−), and plasma cells (defined as CD20−CD138+) to produce caps-PS–specific IgA, IgG, and IgM antibodies in response to Pneumo23 by using an ELISpot assay. Because of the limited number of proposed B-1 cells that we could isolate from vaccinated volunteers, we focused our ELISpot assay on 2 prevalent serotypes of S pneumoniae: serotype 1 and serotype 4. In brief, PBMCs were isolated from 5 healthy subjects 1 week after vaccination with Pneumo23, and the different B-cell subpopulations were isolated by using a combination of magnetic selection (CD19 and CD138 microbeads; Miltenyi Biotec, Bergisch Gladbach, Germany) and fluorescence-activated cell sorting on a FACSAriaII (BD Biosciences, San Jose, Calif). Postsort analysis confirmed that the purity of the different populations was greater than 95%. For each condition, 20,000 cells per well were plated, and Fig 2, A, shows representative ELISpot wells. An overview of the number of specific antibody-secreting cells per 20,000 cells of CD5− and CD5+ proposed B-1 cells, naive cells, and memory B cells for each donor is shown in Fig 2, B. From this figure, it is clear that caps-PS–specific antibody-secreting cells are found within the CD5− subpopulation of the proposed B-1 cell population. In addition to CD5− proposed B-1 cells, we also observed strong specific antibody production by plasmablasts and plasma cells (data not shown). Very little caps-PS–specific antibody-secreting cells were found in the CD5+ proposed B-1 cell population, and almost no such cells were observed within the naive and memory B-cell subpopulation. Further characterization revealed that CD20+CD43+CD27+CD5− B cells expressed CD38 and high levels of CD27. Expression of CD38, CD27, and CD43 was highest on plasmablasts. Finally, we investigated the presence of the CD5− and CD5+ subsets of the proposed B-1 cells in a 5-year-old patient with a selective polysaccharide antibody deficiency (SPAD). This is a rare syndrome that is defined as an absent or diminished antibody response to caps-PS antigens after vaccination with Pneumo23 in patients older than 2 years of age. Patients with SPAD have recurrent pneumococcal infections and other pyogenic infections, although their immunoglobulin and immunoglobulin subclass levels and responses to protein antigens are normal. Interestingly, although we could find the proposed B-1 cells in the peripheral blood of this patient, these cells were almost exclusively CD5+ (96.9% of the proposed B-1 cells). The IgM, IgA, and IgG subclass levels of the patient with SPAD were normal. Five age-matched control subjects (age, 2-5 years) all showed higher levels of the CD5− subset of proposed B-1 cells (28.8% ± 12.5% of total proposed B-1 cells) and had normal antibody responses on Pneumo23 vaccination.7Jeurissen A. Moens L. Raes M. Wuyts G. Willebrords L. Sauer K. et al.Laboratory diagnosis of specific antibody deficiency to pneumococcal capsular polysaccharide antigens.Clin Chem. 2007; 53: 505-510Crossref PubMed Scopus (38) Google Scholar The results of the patient with SPAD, as well as those of the age-matched control subjects, are shown in Table I. These results underscore again the importance of the CD5− subset of proposed B-1 cells for antibody production to polysaccharide antigens.Table IPercentage of CD5− proposed B-1 cells before Pneumo23 vaccination, PS-specific antibody levels for 3 different serotypes of S pneumoniae (before and 3 weeks after vaccination), and total immunoglobulin isotype levels for the patient with SPAD and 5 age-matched control subjectsCD5− B-1 cells (%)Pn type 3 (mg/L)Pn type 4 (mg/L)Pn type 9N (mg/L)IgG (g/L)IgG1 (g/L)IgG2 (g/L)IgG3 (g/L)IgG4 (g/L)IgM (g/L)IgA (g/L)BeforeAfterBeforeAfterBeforeAfterSPAD3.100.530.640.740.740.540.646.444.750.750.380.080.400.99C125.61.57.561.433.772.3315.315.25NA0.600.25NA0.510.46C228.50.439.391.2315.910.479.6310.38.020.820.070.540.892.30C341.80.863.191.43>20.50.6223.629.987.950.850.400.180.940.99C438.4>12>120.9019.230.3913.994.173.181.000.160.150.430.38C59.90.431.320.616.520.472.877.075.950.270.210.05NANANA, Not available; Pn, pneumococcal. Open table in a new tab NA, Not available; Pn, pneumococcal. Our results are in line with those of Barrett et al,8Barrett D.J. Sleasman J.W. Schatz D.A. Steinitz M. Human anti-pneumococcal polysaccharide antibodies are secreted by the CD5- B cell lineage.Cell Immunol. 1992; 143: 66-79Crossref PubMed Scopus (17) Google Scholar who reported that CD5− B cells were responsible for producing caps-PS–specific antibodies after vaccination with Pneumo23. However, no further distinction between different B-cell subsets was made at that time. Moreover, it has been reported that IgM memory B cells are responsible for controlling infections caused by S pneumoniae.9Kruetzmann S. Rosado N.M. Weber H. Germing U. Tournilhac O. Peter H.H. et al.Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen.J Exp Med. 2003; 197: 939-945Crossref PubMed Scopus (532) Google Scholar However, in this study the distinction between B-1 and memory B cells based on the expression of CD43 was not known. It is clear that the population of IgM memory B cells (as defined by CD27 expression) contained the B-1 cells. It should be noted that some controversy has arisen about the nature of the human B-1 cells. Perez-Andres et al5Perez-Andres M. Grosserichter-Wagener C. Teodosio C. van Dongen J.J. Orfao A. van Zelm M.C. The nature of circulating CD27+CD43+ B cells.J Exp Med. 2011; 208: 2565-2566Crossref PubMed Scopus (76) Google Scholar and Descatoire et al6Descatoire M. Weill J.C. Reynaud C.A. Weller S. A human equivalent of mouse B-1 cells?.J Exp Med. 2011; 208: 2563-2564Crossref PubMed Scopus (82) Google Scholar have raised the possibility that CD27+CD43+ B cells might be activated cells on their way to plasma cell differentiation. However, these concerns were subsequently refuted by Griffin et al4Griffin D.O. Holodick N.E. Rothstein T.L. Human B1 cells in umbilical cord and adult peripheral blood express the novel phenotype CD20+ CD27+ CD43+ CD70-.J Exp Med. 2011; 208: 67-80Crossref PubMed Scopus (468) Google Scholar by stating that B-1 cells are identified on CD20 expression and not on CD19 expression. CD20 should exclude plasmablasts and plasma cells. We ourselves found that after vaccination, the CD20+CD43+CD27+ B-cell population showed high levels of CD27 expression and expressed CD38 (as do plasmablasts and plasma cells) and found that plasmablasts and plasma cells also produced anti–caps-PS antibodies. Our data suggest that antigen-specific CD20+CD43+CD27+CD5− B cells (which are labeled as B-1 cells by Griffin et al4Griffin D.O. Holodick N.E. Rothstein T.L. Human B1 cells in umbilical cord and adult peripheral blood express the novel phenotype CD20+ CD27+ CD43+ CD70-.J Exp Med. 2011; 208: 67-80Crossref PubMed Scopus (468) Google Scholar) become activated, expand, undergo class-switching, and differentiate in response to immunization with Pneumo23. This is in line with a recent study of B-1 cells in mice in which it was shown that B-1b cells (which are CD5−) become activated, expand, undergo class-switching, and differentiate into antibody-secreting cells in response to TNP-Ficoll, a TI-2 antigen.3Haas K.M. Programmed cell death 1 suppresses B-1b cell expansion and long-lived IgG production in response to T cell-independent type 2 antigens.J Immunol. 2011; 187: 5183-5195Crossref PubMed Scopus (45) Google Scholar Taken together, our results have demonstrated an important role for the human CD5− subset of CD20+CD43+CD27+ B cells in antibody production against caps-PS of S pneumoniae. The exact nature of these cells remains to be elucidated.