Comment on “Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination”

Abstract

To the editor: Although recently confirmed in an activation-induced cytidine deaminase (AID) reporter mouse model,1Crouch EE Regulation of AID expression in the immune response.J. Exp. Med. 2007; 204,: 1145-115610.1084/jem.20061952Crossref PubMed Scopus (204) Google Scholar AID expression and immunoglobulin A (IgA) class switch recombination (CSR) in the intestinal lamina propria (LP) remain a matter of debate.2Cerutti A Location, location, location: B-cell differentiation in the gut lamina propria.Mucosal. Immunol. 2008; 1,: 8-1010.1038/mi.2007.8Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar In an article published in the last issue of Mucosal Immunology, Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar revisited AID expression in the human intestine. Barone et al. observed AID expression in intestinal follicles but not LP.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar In addition, Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar detected a proliferation-inducing ligand (APRIL) and its receptors TACI and BCMA in both intestinal follicles and LP.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar As previously published data show that human APRIL elicits AID expression and IgA CSR in the absence of T-cell help to B cells by CD40 ligand,4Litinskiy MB DCs induce CD40-independent immunoglobulin class switching through BLyS and APRIL.Nat. Immunol. 2002; 3,: 822-82910.1038/ni829Crossref PubMed Scopus (1028) Google Scholar, 5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar Barone et al. conclude that APRIL and its receptors are part of a molecular mechanism that promotes IgA CSR in intestinal follicles by both T-cell-dependent (TD) and T-cell-independent (TI) pathways.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Given the additional involvement of APRIL in plasma cell survival,6O'Connor BP BCMA is essential for the survival of long-lived bone marrow plasma cells.J. Exp. Med. 2004; 199,: 91-9810.1084/jem.20031330Crossref PubMed Scopus (767) Google Scholar Barone et al. further suggest that APRIL promotes plasma cell survival in the LP.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Although plausible these conclusions are not demonstrated by the data. Indeed, Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar solely document intestinal expression of APRIL, but provide neither functional nor molecular evidence of APRIL involvement in follicular IgA CSR or LP plasma cell survival. Furthermore, Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar provide neither functional nor molecular evidence to demonstrate the involvement of APRIL, TACI, and BCMA in TD or TI routes of B-cell activation within intestinal follicles. We also question the approach used by Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar to study the expression of AID, a hallmark of ongoing CSR,5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar in the human LP. Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar stained intestinal tissues for AID and APRIL through immunohistochemistry (IHC) and compared the results of these stainings with those obtained by our group through immunofluorescence analysis (IFA).5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar This comparison is inaccurate, because IHC and IFA are fundamentally different methodologies that deal with paraffin-embedded and frozen tissues, respectively. Different tissue processing modalities can affect sensitivity. Indeed, the sensitivity of IHC can be attenuated by antigen retrieval procedures, which modify the antigenic structure of the protein under study due to its exposure to high temperatures. Limited sensitivity explains the detection by Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar of AID in follicular B cells, but not in LP B cells or tonsillar subepithelial B cells. A sensitivity issue may also explain the finding of APRIL in crypt but not lumen-facing epithelial cells.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Although we agree with the former observation,5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar the lack of APRIL in lumen-facing epithelial cells differs not only from our results,5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar but also from the results of Shang et al.7Shang L Toll-like receptor signaling in small intestinal epithelium promotes B-cell recruitment and IgA production in lamina propria.Gastroenterology. 2008; 135,: 529-53810.1053/j.gastro.2008.04.020Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar and data available in the Swedish Human Proteome resource program (http://www.proteinatlas.org/show_image.php?image_id=1635790). Because of these reasons and the glaring lack of supporting functional data, the lack of APRIL expression by lumen-facing epithelial cells should not be used as an argument to question the role of bacterial Toll-like receptor ligands in APRIL release by intestinal epithelial cells.5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar In this regard, Barone et al.5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar fail to recognize that the flagellin receptor Toll-like receptor-5 promotes APRIL secretion rather than APRIL expression by epithelial cells.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Antigen retrieval procedures can affect not only the sensitivity, but also the specificity of IHC. Indeed, Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar detected AID in CD68+ LP macrophages, and attribute this finding to the poor specificity of an EK2-5G9 antibody to AID (anti-AID) used in some of our studies.5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar We reject this claim, because we used IFA instead of IHC to visualize AID. IFA specifically detected AID in LP-activated B cells, plasmablasts, and plasma cells co-expressing IgA, BSAP (Pax5), IRF4, Blimp-1 and/or CD138,5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar but not CD68 (B. He and A. Cerutti, unpublished data). Importantly, we validated and further extended our IFA-based results by means of fluorescence in situ hybridization (FISH) in both CD40-sufficient and CD40-deficient individuals,5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar whereas Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar did not. Moreover, the specificity of EK2-5G9 for both follicular and extrafollicular B cells, including tonsillar subepithelial B cells, is consistent not only with results by Cattoretti et al.,8Cattoretti G Nuclear and cytoplasmic AID in extrafollicular and germinal center B cells.Blood. 2006; 107,: 3967-397510.1182/blood-2005-10-4170Crossref PubMed Scopus (124) Google Scholar but also with additional data from our group generated with anti-AID antibodies different from EK2-5G9 (B. He and A. Cerutti, unpublished data). Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar back up their claim that there is no AID in the LP by comparing the presence of AID transcripts in microdissected tonsillar germinal centers and LP tissue through quantitative PCR. The sequence of the primers used to amplify AID mRNA is not disclosed. This issue aside, we contend that in this specific setting quantitative PCR is misleading, because based on the false assumption that germinal centers and LP have comparable B-cell densities and AID expression levels. Instead, germinal centers have a much higher B-cell density than the LP, which consists of a mixed population of B cells and non-B cells. Furthermore, the vast majority of germinal center B cells express AID, whereas only a fraction of LP B cells does.5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 9Xu W Epithelial cells trigger frontline immunoglobulin class switching through a pathway regulated by the inhibitor SLPI.Nat. Immunol. 2007; 8,: 294-30310.1038/ni1434Crossref PubMed Scopus (242) Google Scholar Moreover, individual germinal center B cells are likely to express more AID than individual LP B cells. Thus, the observation that germinal centers contain more AID RNA than the LP is completely expected and should not allow one to conclude that the LP lacks AID. Similarly, naive B cells exposed to CD40 ligand and interleukin-4 contain negligible AID RNA compared with germinal center B cells, because only a fraction of naive B cells induce AID and undergo CSR upon in vitro stimulation (B. He and A. Cerutti, unpublished data). A more appropriate method to detect AID in scattered LP B cells is FISH (Figure 1a). Alternatively, one can PCR amplify AID mRNA from purified LP B cells.6O'Connor BP BCMA is essential for the survival of long-lived bone marrow plasma cells.J. Exp. Med. 2004; 199,: 91-9810.1084/jem.20031330Crossref PubMed Scopus (767) Google Scholar At any rate, even the data provided by Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar seem to be consistent with the presence of AID in the LP, because CD20+ B-cell-enriched contain more AID mRNA than CD20+ B-cell-poor LP samples.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar This difference would be visually more evident in a graphic lacking germinal center data. An additional problem relates to the inaccurate discussion of the literature on the inductive function of the LP. For example, Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar do not discuss recent studies showing LP AID expression and both TD and T1 LP IgA CSR events in an AID-reporter mouse model.1Crouch EE Regulation of AID expression in the immune response.J. Exp. Med. 2007; 204,: 1145-115610.1084/jem.20061952Crossref PubMed Scopus (204) Google Scholar In addition, Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar do not mention the fact that APRIL triggers IgA1 CSR in addition to IgA2 CSR,5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar which may explain the presence of APRIL not only in IgA2-rich areas such as the colon LP,5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar but also in IgA1-rich lymphoid areas such as mucosal follicles.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 10Chiu A Hodgkin lymphoma cells express TACI and BCMA receptors and generate survival and proliferation signals in response to BAFF and APRIL.Blood. 2007; 109,: 729-73910.1182/blood-2006-04-015958Crossref PubMed Scopus (163) Google Scholar Finally, Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar do not discuss earlier evidence showing that APRIL cooperates with antigen, cytokines, and microbial molecular patterns to induce not only direct IgM-to-IgA1 CSR, but also sequential IgA1-to-IgA2 CSR and B-cell proliferation.5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 11Gupta M A proliferation-inducing ligand mediates follicular lymphoma B-cell proliferation and cyclin D1 expression through phosphatidylinositol 3-kinase-regulated mammalian target of rapamycin activation.Blood. 2009; 113,: 5206-521610.1182/blood-2008-09-179762Crossref Scopus (44) Google Scholar, 12He B Lymphoma B cells evade apoptosis through the TNF family members BAFF/BLyS and APRIL.J. Immunol. 2004; 172,: 3268-327910.4049/jimmunol.172.5.3268Crossref PubMed Scopus (246) Google Scholar, 13Cerutti A The regulation of IgA class switching.Nat. Rev. Immunol. 2008; 8,: 421-43410.1038/nri2322Crossref PubMed Scopus (487) Google Scholar, 14Ozcan E Transmembrane activator, calcium modulator, and cyclophilin ligand interactor drives plasma cell differentiation in LPS-activated B cells.J. Allergy Clin. Immunol. 2009; 123,: 1277-1286.e510.1016/j.jaci.2009.03.019Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar This evidence is consistent with the local presence of clonally expanded B cells in the LP15Yuvaraj, S. et al. Evidence for local expansion of IgA plasma cell precursors in human Ileum. J. Immunol. PMID 19786537 (2009).Google Scholar and with the expression of growth-associated nuclear proteins such as Ki-67 (http://www.proteinatlas.org/show_image.php?image_id=282924) and γ-H2AX (Figure 1b) in some LP B cells. Of note, the histone γ-H2AX targets AID-induced double-strand switch DNA breaks in B cells transiting through the G1 phase of the cell cycle.16Petersen S AID is required to initiate Nbs1/γ-H2AX focus formation and mutations at sites of class switching.Nature. 2001; 414,: 660-66510.1038/414660aCrossref PubMed Scopus (433) Google Scholar In summary, we contend that Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar provide neither functional nor molecular evidence to demonstrate that APRIL signals IgA CSR in B cells from intestinal follicles through TD and TD pathways involving TACI and BCMA. We also question the conclusion that the human intestinal LP lacks AID, because the experimental approaches used by Barone et al.3Barone F Patel P Sanderson J Spencer J Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent class switch recombination.Mucosal. Immunol. 2009; 2,: 495-50310.1038/mi.2009.106Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar neither allow an accurate detection of LP AID nor can be compared with approaches successfully used in earlier works.1Crouch EE Regulation of AID expression in the immune response.J. Exp. Med. 2007; 204,: 1145-115610.1084/jem.20061952Crossref PubMed Scopus (204) Google Scholar, 5He B Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.Immunity. 2007; 26,: 812-82610.1016/j.immuni.2007.04.014Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 17Tsuji M Requirement for lymphoid tissue-inducer cells in isolated follicle formation and T cell-independent immunoglobulin A generation in the gut.Immunity. 2008; 29,: 261-27110.1016/j.immuni.2008.05.014Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 18Fagarasan S Kinoshita K Muramatsu M Ikuta K Honjo T In situ class switching and differentiation to IgA-producing cells in the gut lamina propria.Nature. 2001; 413,: 639-64310.1038/35098100Crossref PubMed Scopus (360) Google Scholar The authors declared no conflict of interest. Download .ppt (.61 MB) Help with ppt files PowerPoint slide for Fig. 1