TO THE EDITOR: Although recently confirmed in an activation-induced cytidine deaminase (AID) reporter mouse model,1 AID expression and immunoglobulin A (IgA) class switch recombination (CSR)

in the intestinal lamina propria (LP) remain a matter of debate.2 In an article published in the last issue of _Mucosal Immunology_, Barone _et al._3 revisited AID expression in the human

intestine. Barone _et al._ observed AID expression in intestinal follicles but not LP.3 In addition, Barone _et al._3 detected a proliferation-inducing ligand (APRIL) and its receptors TACI

and BCMA in both intestinal follicles and LP.3 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

Although plausible these conclusions are not demonstrated by the data. Indeed, Barone _et al._3 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._3 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._3 to study the expression of AID, a

hallmark of ongoing CSR,5 in the human LP. Barone _et al._3 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).5 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._3 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.3

Although we agree with the former observation,5 the lack of APRIL in lumen-facing epithelial cells differs not only from our results,5 but also from the results of Shang _et al._7 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.5 In this regard, Barone _et al._5 fail to recognize that the flagellin receptor Toll-like receptor-5 promotes APRIL secretion rather than APRIL expression by

epithelial cells.3 Antigen retrieval procedures can affect not only the sensitivity, but also the specificity of IHC. Indeed, Barone _et al._3 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.5 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 CD138,5 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,5 whereas Barone _et al._3 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._,8 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._3 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.5, 9

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.6 At any rate, even the data provided by Barone _et al._3 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.3 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._3 do

not discuss recent studies showing LP AID expression and both TD and T1 LP IgA CSR events in an AID-reporter mouse model.1 In addition, Barone _et al._3 do not mention the fact that APRIL

triggers IgA1 CSR in addition to IgA2 CSR,5 which may explain the presence of APRIL not only in IgA2-rich areas such as the colon LP,5 but also in IgA1-rich lymphoid areas such as mucosal

follicles.3, 5, 10 Finally, Barone _et al._3 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.5, 11, 12, 13, 14 This evidence is consistent with the local presence of clonally expanded B cells in the LP15

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.16 In summary, we contend that Barone _et al._3 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._3 neither allow an accurate detection of LP AID nor can be compared with

approaches successfully used in earlier works.1, 5, 17, 18 DISCLOSURE The authors declared no conflict of interest. REFERENCES * Crouch, E.E. _et al_. Regulation of AID expression in the

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(2001). Article  Google Scholar  Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Pathology and Laboratory Medicine of Weill Medical College of Cornell

University and Weill Graduate School of Medical Sciences of Cornell University, New York, New York, USA B He, W Xu & A Cerutti Authors * B He View author publications You can also search

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THIS ARTICLE CITE THIS ARTICLE He, B., Xu, W. & Cerutti, A. Comment on “Gut-associated lymphoid tissue contains the molecular machinery to support T-cell-dependent and T-cell-independent

class switch recombination”. _Mucosal Immunol_ 3, 92–94 (2010). https://doi.org/10.1038/mi.2009.125 Download citation * Published: 16 December 2009 * Issue Date: January 2010 * DOI:

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