IgM+IgD− B cells in human gut-associated lymphoid tissue have memory features and give rise to IgM+ and IgA+ antibody-secreting cells

Bukhari, Ameera M.; Meng, Wenzhao; Rosenfeld, Aaron M.; Prak, Eline T. Luning; Knight, Katherine L.

doi:10.1038/s41598-025-11209-1

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Published: 22 July 2025

IgM⁺IgD⁻ B cells in human gut-associated lymphoid tissue have memory features and give rise to IgM⁺ and IgA⁺ antibody-secreting cells

Ameera M. Bukhari¹^na1,
Wenzhao Meng²^na1,
Aaron M. Rosenfeld²^na1,
Eline T. Luning Prak² &
…
Katherine L. Knight¹

Scientific Reports volume 15, Article number: 26613 (2025) Cite this article

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Abstract

Human IgM⁺ B cells vary in their surface levels of IgD, with the major circulating population of IgM⁺IgD⁺ cells and a minor population (< 5%) of IgM⁺IgD⁻ cells. In contrast, in gut-associated lymphoid tissue (GALT) derived from individuals undergoing tonsillectomy or appendectomy, IgM⁺IgD⁻ B cells constitute ~ 30% of B cells. IgM⁺IgD⁻ cells isolated from both tonsil and appendix lack plasma cell and B1 cell markers, and approximately 50% express the memory marker CD27. Functionally, GALT IgM⁺IgD⁻ cells spontaneously secrete IgM, and class-switch to IgA in response to both T-dependent and T-independent stimulation ex-vivo. Immune repertoire profiling reveals that GALT IgM⁺IgD⁻ cells exhibit lower levels of VH4-34 rearrangements, higher levels of somatic hypermutation, shorter CDR3 sequences and greater clonal overlap with switch memory cells than IgM⁺IgD⁺ cells. Furthermore, clonal lineage analysis reveals that IgM⁺IgD⁻ clones can include class-switched sequence variants. These findings suggest a maturational scheme starting from CD27⁻IgM⁺IgD⁺ B cells to CD27⁺IgM⁺IgD⁺, and then to CD27⁻IgM⁺IgD⁻, and finally to CD27⁺IgM⁺IgD⁻ B cells. In sum, IgM⁺IgD⁻ B cells in the mucosa have memory features, give rise to class-switched memory B cells and antibody-secreting cells, and likely contribute significantly to the IgA repertoire in human GALT.

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Introduction

Gut-associated lymphoid tissue (GALT) is the largest mass of lymphoid tissue in the human body and represents a unique environment in which immune cells are constantly interacting with gut microbes^1,2. B lymphocytes comprise most of the lymphatic tissues of GALT, where they play critical roles in protecting the host from infection, promoting tolerance, and shaping the microbiota through the production of immunoglobulins (Igs)^3,4. Although IgA is the most abundant class of secreted Ig in GALT and attracts much of the attention in the realm of gut health, IgM is also present. IgM plays a substantial role in protecting the host from infection and autoimmunity⁵. In the human gut, secreted IgM was found to coat human microbiota alongside secreted IgA^6,7,8. In some cases, IgM was even more potent than IgA in neutralizing enteric bacteria⁸. IgM can also compensate for the lack of secreted IgA in GALT⁹, as evidenced by elevated levels of serum and salivary IgM in individuals with selective IgA deficiency (sIgAd)^10,11. Moreover, sIgAd patients were found to be less susceptible to infections, allergies, and autoimmune diseases compared to patients who lack IgM, and they can remain asymptomatic for most of their lives if they have an intact IgM response^9,12,13,14.

IgM plasma cells are a major source of secreted IgM in human GALT; however, IgM can also be secreted by B cells that have not differentiated into plasma cells. Data from human cord blood and adult peripheral blood (PB) revealed a B cell population (CD20⁺CD27⁺CD43⁺CD70⁻) that spontaneously and continuously secretes IgM¹⁵. These cells are considered the B1 cell counterpart in humans; however, their defining markers indicate they could also be antigen-experienced B2 cells. IgM^highIgD⁺CD27⁺ B cells were also shown to secrete IgM spontaneously^16,17. These cells were historically considered to be marginal zone (MZ) B cells due to their localization in the splenic MZ and their role in T-independent immune responses^18,19; In contrast, IgM⁺IgD⁻CD27⁻ B cells are less commonly discussed and their classification is less clear. High-throughput V_H sequencing data by Bagnara and colleagues show that IgM⁺IgD⁻CD27⁻ B cells represent a separate population, independent from MZ B cells (IgM⁺IgD⁺CD27⁺) in the human spleen²⁰. Similar findings were reported by Zhao and colleagues, using deep immunophenotypic profiling of B cells in GALT and other tissues, with more emphasis on MZ B cells²¹.

In humans, large B cell clones partition into two broad networks: one that includes the blood, bone marrow, spleen, and lung, and another that is more restricted to tissues within the gastrointestinal tract, including the jejunum, ileum, and colon²². If B cell clones partition into different tissue-spanning regions, perhaps tissue-based B cell responses and subsets will do so as well. Consistent with this idea, Magri and colleagues showed that the human colon and ileum contain a memory B cell population (IgM⁺IgD⁻CD27⁺) that is not seen in the peripheral blood and other human lymphoid organs⁶. These IgM⁺IgD⁻ B cells were shown to be clonally related to the IgM plasma cells in the human gut, but their contribution to other B cell populations in the GALT remains unclear.

In this study, we sought to understand the relationship between IgM⁺IgD^- B cells and other B cell subsets in human GALT. We isolated and analyzed four subpopulations of IgM⁺ B cells (IgM⁺IgD⁺ and IgM⁺IgD^- that are either CD27^- or CD27⁺) from two human GALT sites -tonsil and appendix- in individuals ranging in age from 12-hr-old to 50 years. We found that IgM⁺IgD^-CD27^- and IgM⁺IgD^-CD27⁺ B cell populations in human tonsil and appendix spontaneously secrete IgM and are present throughout life. IgM⁺IgD^-CD27^+/- B cells show a mature surface phenotype and class-switch to IgA in response to T-dependent and T-independent stimulation at a significantly higher level than mature naïve IgM⁺IgD⁺CD27^- B cells obtained from the same anatomical site. Analysis of B cell receptor sequences of tonsil and appendix B cell subpopulations reveals that the IgM⁺IgD^-CD27^+/- B cells that are enriched in human GALT, have high numbers of overlapping clones with switched memory IgG and IgA B cells and high levels of somatic hypermutation. They also have lower levels of VH4-34 rearrangements and shorter third complementarity determining region (CDR3) lengths than naïve cells. Taken together, these findings suggest that IgM⁺IgD^- B cells are a memory population that gives rise to class-switched cells and antibody-secreting cells in human GALT.

Results

Demographics

Tonsil samples were obtained from patients undergoing tonsillectomy for tonsillar hypertrophy or recurrent tonsilitis. We analyzed a total of 44 tonsil samples obtained from patients ranging in age from 14 months to 46 years (median = 10 years, mean = 13 years). The female to male ratio for tonsil samples was 1:1.06. Appendix samples were obtained from patients undergoing appendectomy for appendicitis. A total of 39 appendix samples were analyzed from patients ranging in age from 12 h to 50 years (median = 17 years, mean = 22 years) and the female to male ratio was 1:2.27. We excluded all appendix and tonsil samples with known infections. All tonsil and appendix samples were evaluated by a pathologist and only histologically normal tissues were included in this study. Seven tonsil and four appendix samples were included in the BCR sequence analysis. Patient information with sorted subsets for BCR sequence analysis is summarized in (Supplementary Table 1).

Phenotypic characterization of IgM⁺IgD⁻ B cells in human GALT

We used flow cytometry to analyze B cells in GALT, specifically in the tonsil and appendix (Supplementary Fig. 1), and we found that most B cells are mature naive IgM⁺IgD⁺CD27⁻ cells (~ 40–60% of CD19⁺CD38^Low/−) (Supplementary Fig. 2A) or negative for IgM and IgD (double negative-DN) (mean = 30% “tonsil”; 42% “appendix”) (Supplementary Fig. 3A). We also found a large population of CD19⁺CD38^Low/− cells that express only IgM on the surface with little or no IgD (IgM⁺IgD⁻) in both the tonsil and the appendix (~ 5–15% of CD19⁺CD38^Low/− in tonsil and ~ 7–30% of CD19⁺CD38^Low/− in appendix), but not in the peripheral blood (< 5%) (Fig. 1A & B). These IgM⁺IgD⁻ B cells are likely devoid of plasma cells because they are CD38^Low/−.

Further analysis of tonsil and appendix IgM⁺IgD⁻ B cells revealed the majority (~ 90%) of these cells did not stain for the immature and B1-associated B cell marker CD5; the majority (~ 89%) of them were negative for CD10 (early transitional B cells are CD10^hi) and most of these cells did not express the activation and B1 marker CD43 (tonsil > 80%; appendix > 87%) or the plasma cell markers CD38^high and CD138 (> 90%) (Supplementary Fig. 4B & C). In addition, > 90% of the IgM⁺IgD⁻ cells were negative for CD11c, a marker associated with age-associated B cells (ABCs), DN2 B cells and atypical memory B cells^23,24,25. IgM⁺IgD⁻ B cells can express the memory marker CD27, and we found both IgM⁺IgD⁻CD27⁻ and IgM⁺IgD⁻CD27⁺ B cells in tonsil and appendix in subjects of all ages (Supplementary Fig. 2B). From this phenotypic analysis, we conclude that the IgM⁺IgD⁻ B cells do not resemble immature B cells, ABCs, B1-like cells or plasma cells, but correspond most closely to mature B cells.

Spontaneous secretion of IgM from GALT B cells

To evaluate the functional competence of IgM⁺IgD⁻ cells, we analyzed their ability to secrete antibodies spontaneously. Mature naïve IgM⁺IgD⁺CD27⁻CD38^Low/−, IgM⁺IgD⁻CD27⁻CD38^Low/−, and IgM⁺IgD⁻CD27⁺CD38^Low/− B cells were sorted (Supplementary Fig. 4D and 5) and tested for IgM secretion by ELISpot. We found that both IgM⁺IgD⁻ populations secreted IgM spontaneously after as little as 6 h of incubation (Fig. 2A & B). The frequency of IgM-secreting cells ranged between 100 and 600 spots per 2 × 10⁴ cells. Consistent with mature naïve B cells from peripheral blood¹⁵, none of the immunophenotypically mature naïve IgM⁺IgD⁺CD27⁻ B cells from tonsil or appendix secreted IgM. IgM secretion by the IgM⁺IgD⁻CD27⁻ B cells was at a level comparable to their memory counterpart, IgM⁺IgD⁻CD27⁺, confirming the phenotype data that the IgM⁺IgD⁻CD27⁻ B cells are not immature or transitional B cells (Supplementary Fig. 4), but instead, appear to be antigen-experienced B cells.

IgA class-switch in IgM⁺IgD⁻ B cells in response to T-dependent and T-independent antigens

IgA is the major immunoglobulin isotype in the intestinal mucosa; therefore, we tested the extent to which the GALT IgM⁺IgD⁻ B cells can undergo isotype switch to IgA in response to T-dependent and T-independent stimuli. Tonsil IgM⁺ B cell subpopulations were sorted (Supplementary Fig. 5) and cultured ex-vivo for 1 week and stimulated with CpG (T-independent), or with a cocktail of anti-light chain + anti-CD40 + IL-21 (T-dependent). Culture supernatants were analyzed by cytometric bead array (see methods) and both IgM⁺IgD⁻CD27⁻ and IgM⁺IgD⁻CD27⁺ subpopulations underwent class-switching to IgA in response to T-dependent and T-independent stimulation significantly more than tonsil mature naïve IgM⁺IgD⁺CD27⁻ B cells (Fig. 3). Further, the IgA class-switching of IgM⁺IgD⁻CD27⁺ B cells was nearly 10-fold higher than the classical IgM memory IgM⁺IgD⁺CD27⁺ B cells. We conclude that the IgM⁺IgD⁻CD27⁺ B cells, not the classical IgM memory IgM⁺IgD⁺CD27⁺ B cells, are likely significant contributors to the IgA repertoire in GALT.

Memory repertoire features of IgM⁺IgD^- B cells

Features of the immune repertoire can be used to gain insights into the ontogeny of B cell subsets²⁶. To determine if IgM⁺IgD^- B cells are more similar to naïve or to memory B cell populations present in GALT, we sorted six B cell populations from 7 tonsil and 3 appendix samples IgM⁺IgD⁺CD27^-, IgM⁺IgD⁺CD27⁺, IgM⁺IgD^-CD27^-, IgM⁺IgD^-CD27⁺, IgG⁺ and IgA⁺ (Supplementary Fig. 5, Supplementary Table 1). We then performed separate bulk BCR sequence analysis (metadata provided in Supplementary Table 2) focusing on the antibody heavy chain V region, including the hypervariable third complementarity determining region (CDR3). The number of clones sampled was highest in the IgM⁺IgD⁺ subset and was higher in the tonsil (green bars) than in the appendix (purple bars) (Fig. 4A). To evaluate the clonal landscape, we calculated the D20 index, which represents the fraction of sequence copies in the sequencing library that is occupied by the sum of the copies of the top 20-ranked clones; a lower D20 value indicates the presence of a smaller number of large clones and tends to correspond to a higher overall diversity. Consistent with higher diversity, the D20 index was lowest in the IgM⁺IgD⁺ subset, with values below 0.001 (Fig. 4B). In contrast, IgM⁺IgD^- and switch memory cells had fewer clones and higher D20 indices, consistent with lower diversity. Analysis of VH gene usage in GALT naïve and memory B cell populations revealed that cell subsets clustered with each other more than they did by tissue, with the largest difference occurring between the IgM⁺IgD⁺ subset and the other subsets (Fig. 4C). IgM⁺IgD^- subsets from tonsil and appendix were more similar to memory-switched IgG and IgA B cells than they were to IgM⁺IgD⁺ cells. Of note, IgM⁺IgD⁺ B cells used VH4-34 more frequently than IgM⁺IgD^- or switched memory cells (Fig. 4C). The VH4-34 gene is most likely eliminated during the maturation of the memory response due to self-reactivity²⁷, suggesting that IgM⁺IgD^- B cells are more similar to memory B cells.

Analysis of CDR3 lengths revealed that IgM⁺IgD^- B cells harbored shorter CDR3 sequences (the average CDR3 length is 50.6 nt in tonsil and 50.0 nt in the appendix) which are closer to the average CDR3 length in switch memory B cells than they are to IgM⁺IgD⁺ B cells which had average CDR3 lengths of 53.3 nt in tonsil and appendix (Fig. 4D, Supplementary Table 2). In addition, IgM⁺IgD^- clones tended to have shorter CDR3 sequences overall rather than having a focused collection of ultra-short CDR3 sequences (Supplementary Fig. 6A and inset). Analysis of rearrangements with CDR3s of 6 amino acids or less were often of poor quality with questionable VH gene assignments or poor alignments to VH or JH sequences (data not shown). We therefore focused on short CDR3 sequences that were 7–9 amino acids in length. CDR3 amino acid sequence logos from these shorter CDR3 sequences revealed considerable sequence diversity but also a predominance of canonical (templated) VH and JH-associated sequences, e.g., CAR…DYW (Supplementary Fig. 6B). Most of these short CDR3 amino acid sequences were unique to a single individual rather than being shared by different individuals (Supplementary Fig. 6C). Taken together, these findings suggest that short CDR3 sequences in IgM⁺IgD^- B cells reflected a global process (such as selection or maturation) that occurred independently in different individuals, rather than convergent selection of a specific subset of public clones. Taken together, the clonal diversity, VH gene usage and CDR3 length distribution all support the hypothesis that IgM⁺IgD^- cells are more similar to antigen-experienced, memory-like cells than they are to naïve IgM⁺IgD⁺ B cells.

IgM⁺IgD^-CD27^- and IgM⁺IgD^-CD27⁺ B cells exhibit somatic hypermutation and can give rise to class-switched memory cells

To investigate the relationships between IgM⁺IgD^- B cells and other B cell subsets, we analyzed the level of somatic hypermutation (SHM) between and within B cell lineages in each subset. The level (Fig. 4E & F) and distribution (Fig. 4G) of VH gene SHM in IgM⁺IgD^- B cells was intermediate between IgM⁺IgD⁺ and switch memory cells. To further resolve the level of SHM by subset, we carried out a separate analysis in CD27⁺ and CD27^- cells (Fig. 5A & B). This analysis revealed a steady increase in the level of SHM in which IgM⁺IgD⁺CD27^- cells were the least mutated, followed by IgM⁺IgD⁺CD27⁺, then IgM⁺IgD^-CD27^- and finally IgM⁺IgD^-CD27⁺ and even higher levels of SHM in class-switched subsets expressing either IgG or IgA (Fig. 5A & B). This progression of SHM occurred in both tonsil and appendix, with IgA being the most mutated in both tissue sites. Similar to the aggregated data, we also observed that IgM⁺IgD⁺ cells (that were either CD27^- or CD27⁺) exhibited substantial overrepresentation of VH4-34 in both the tonsil (Fig. 5C) and the appendix (Fig. 5D). Both CD27^- and CD27⁺ IgM⁺IgD^- B cells had lower levels of VH4-34 and the tonsil exhibited higher levels of VH3-23 than the appendix.

Next, we analyzed VH mutations in subset-spanning lineages to infer ontogenic relationships between the subsets. String plots of clones that overlap between two or more B cell subsets revealed IgM⁺IgD^- clones that overlapped frequently with class-switched cells, particularly IgA (Fig. 6A). Similarly, Venn diagrams showing all overlapping and non-overlapping clones indicate that the largest intersection of overlapping clones occurs between the two IgM⁺IgD^- B cell populations (CD27^+/-) and the switched memory B cells (IgG⁺ and IgA⁺) in both the appendix and tonsil (Fig. 6B). Selected clonal trees of overlapping clones of IgM⁺IgD^- and class-switched B cell populations, show the evolution of class-switched B cell subsets within lineages that also include IgM⁺IgD^- subsets (Fig. 6C). The high level of SHM and the high number of overlapping clones between IgM⁺IgD^- B cells and class-switched memory B cells indicate that IgM⁺IgD^- B cells have memory features and contribute to the switched memory pool in GALT.

High frequency of IgM⁺IgD^- B cells with low SHM in 12-hr-old infant appendix

In one appendix sample obtained from a 12-hr-old infant, over 75% of the CD19⁺ cells in the tested tissue were negative for IgM and IgD (Fig. 7A), as well as IgG and IgA (data not shown), which might indicate the presence of developing B cells in GALT or a high frequency of CD19⁺ plasma cells. Among IgM⁺ B cells, IgM⁺IgD^- B cells comprised 50% of the B cells, compared to < 20% in adult appendix (Fig. 7A). More than 20% of these IgM⁺IgD^- B cells were positive for the memory marker CD27 (Fig. 7A). VH sequence analysis of IgM⁺ B cell populations showed that SHM levels were very low in all subsets compared to adult rearrangements and that CDR3 lengths were shorter in IgM⁺IgD^- B cells than in the IgM⁺IgD⁺ cells (Fig. 7B & C). In addition, the 12-hr-old donor had shorter CDR3 lengths globally across all subsets (Fig. 7B) when compared to the older children and adult donors (Fig. 4D), Furthermore, the IgM⁺IgD^- B cells in the 12-hr donor exhibited lower frequencies of VH4-34 rearrangements than IgM⁺IgD⁺ B cells (Fig. 7D), similar to our findings in adults (Fig. 4D). Selected lineages of rare large IgM⁺IgD^- clones revealed that trees typically included germline VH gene sequences in addition to the mutated sequences (Fig. 7E). These findings suggest that B cells in GALT are comprised mainly of IgM⁺IgD^- cells in a very young individual and may represent early memory cells. While a single patient sample limits the strength of conclusions, this 12-hr-old infant normal appendix sample offers important baseline data for early neonatal reference and comparison.

Discussion

Here we describe a B cell population that is enriched in human appendix and tonsil that expresses high levels of IgM and little to no IgD on the surface (IgM⁺IgD⁻). Although some previous studies have described IgM⁺IgD⁻ B cells as immature or transitional cells, particularly when CD27⁻^28,29, our data show that in the GALT, CD27⁺ and CD27⁻ IgM⁺IgD⁻ cells harbor characteristics of antigen-experienced cells. In addition to exhibiting spontaneous Ig secretion, IgM⁺IgD⁻ cells have higher levels of SHM, as well as altered usage of VH genes and shorter CDR3 lengths than mature naïve IgM⁺IgD⁺ cells. Furthermore, IgM⁺IgD⁻ B cell clones overlapped with IgG and IgA class-switched memory B cells, suggesting that these cells are memory-like B cells. IgM⁺IgD⁻ cells also had reduced frequencies of VH4-34 usage compared to IgM⁺IgD⁺ cells. Their more similar VH gene usage to class-switched IgG and IgA memory B cells suggests that IgM⁺IgD⁻ B cells underwent a similar selection process to that of switch memory cells in vivo, further supporting the idea that they are memory-like B cells.

How IgM⁺IgD⁺ and IgM⁺IgD⁻ B cells fit into the developmental schema of memory B cell maturation has been the subject of other papers in the literature over the years. In an early description of CD27⁺ circulating B cell subsets, IgM⁺IgD⁺ and IgM⁺IgD⁻ cells were proposed to have a germinal center origin based on the presence of somatic hypermutation in both subsets³⁰. However, in this same study, CD27⁻IgM⁺IgD⁺ cells were considered to be pre-germinal center. Subsequent studies by the same authors and others revealed substantial clonal overlap between CD27⁻ and CD27⁺ cell subsets^31,32,33. Furthermore, bulk transcriptomic analysis on sorted subsets revealed very few significant gene changes between CD27⁺IgM⁺IgD⁺ and CD27⁺IgM⁺IgD⁻, calling into question the degree to which any of these IgMIgD subsets is truly functionally distinct³⁴. Efforts to place different B cell subsets into a developmental continuum have revealed plasticity and interconnectedness between CD27⁺ and CD27⁻ B cell subsets³³.

Our study differs from these studies in two important respects. First, we separately characterize CD27⁺ and CD27⁻ IgM⁺ B cell subsets into four populations and show, by somatic hypermutation and repertoire analysis that they fall into a clear and reproducible developmental pattern with gradually increasing SHM levels within the same individuals:

CD27⁻IgM⁺IgD⁺→CD27⁺IgM⁺IgD⁺→CD27⁻IgM⁺IgD⁻→CD27⁺IgM⁺IgD⁻.

In other words, the antibody isotype pattern is more important than CD27 status for the developmental ordering, if one relies on SHM as a “gold standard” molecular clock. IgM⁺IgD⁻ B cells also have lower frequencies of VH4-34 than IgM⁺IgD⁺ cells, irrespective of CD27 level, further supporting their more mature status. Second, we analyzed these four B cell populations in two different tissue sites, rather than in the peripheral blood. This analysis not only confirms the earlier work in peripheral blood, but strengthens its generalizability to the locations where germinal centers actually reside. Furthermore, unlike the blood, tissue sites have fewer naïve cells, far more abundant populations of IgM⁺IgD⁻ cells and cells undergo less mixing, enabling more rigorous evaluations of clonal overlap. By analyzing two separate tissue sites and showing similar results in both, this study provides a robust foundation for this developmental scheme.

We also present functional data in support of this developmental scheme in which IgM⁺IgD⁻ B cells are more mature than IgM⁺IgD⁺ cells, irrespective of CD27 status. Our in vitro stimulation assays showed that class-switching of IgM⁺IgD⁻ B cell populations in response to T-dependent and T-independent antigens was ten times higher than their IgM⁺IgD⁺ counterparts. One possible explanation for the large difference in the magnitude of the response to stimulation could be the difference in IgD expression. Noviski et al. showed that IgD is less sensitive to autoantigen stimulation than IgM and have suggested that IgD maintains quiescence of autoreactive B cells³⁵. Although we do not know if these IgM⁺IgD⁻ B cells are autoreactive, in vitro and BCR sequencing data support the idea that IgD can reduce the stimulation signal received through surface IgM^36,37.

Our in vitro data also showed a propensity of IgM⁺IgD⁻ B cells to undergo class-switching to IgA in response to T-dependent and T-independent antigens. Of note, we did not detect appreciable levels of secreted IgG in these stimulation cultures. However, BCR sequence analysis of tonsil and appendix B cells showed a number of shared clones between IgM⁺IgD⁻ B cells and both IgG⁺ and IgA⁺ memory cells, which indicates that IgM⁺IgD⁻ B cells can also class-switch to IgG. This discrepancy between the class-switch tendencies of the in vivo vs. the in vitro data could be attributed to the absence of environmental factors that drive IgG class-switching in culture, such as the presence and the levels of T helper cytokines that support class-switching e.g., IL-2, IL-4 and IL-5. In vivo, there may be multiple T-dependent and T-independent stimulation signals that drive IgG class-switching. Another non-mutually exclusive possibility is that it is easier for IgM⁺IgD⁻ cells to differentiate into IgA⁺ antibody-secreting cells than it is for them to differentiate into IgG⁺ ASCs. In a recent extensive mass cytometry analysis of B cells from GALT and tonsil, Zhao et al. showed that the IgM⁺IgD⁻CD27⁻ cells are phenotypically similar to IgA-expressing cells²¹.

The short CDR3 length and higher level of somatic hypermutation (SHM) observed in IgM⁺IgD⁻ than IgM⁺IgD⁺ B cells suggest that IgM⁺IgD⁻ B cells may be further along the differentiation pathway toward the germinal center (GC) stage. Consistent with a possible GC origin, some IgM⁺IgD⁻ B cells express low levels of CD38⁶, in contrast to the IgM⁺IgD⁺CD27⁻ B cells which tend to be uniformly negative for CD38 (Supplementary Fig. 4B & C). Additional stratification and BCR analysis of these populations based on CD38 expression may provide deeper insights into their maturation process into GC B cells. Moreover, although the majority of IgM⁺IgD⁻ cells did not express markers typically associated with immature, activated, or tissue-resident B cells (Supplementary Fig. 4), a subset of these cells did exhibit expression of markers such as CD5, CD10, and CD43. This underscores the heterogeneity of IgM⁺IgD⁻ B cells in GALT and highlights the need for more refined characterization in future studies, incorporating both the markers used in this study and those previously applied to peripheral blood and tissue B cell subsets^38,39,40. Of note, MZ B cells are another population which characteristically also expresses high IgM and low IgD. MZ B cells may contribute to the observed phenotype and share some BCR features^21,41. In mice, epigenetic and single cell studies have partitioned B cell maturational trajectories into subsets that can undergo GC-dependent and GC-independent development into memory cells or plasma cells⁴². The trajectories of human B cells are less well understood, in part because of limitations in experimental manipulation and genetic variation, but also because of variable methods for immunophenotypic subset classification³⁹. Single cell multiomics approaches are helping to overcome some of these difficulties and, coupled with machine learning classifiers, are showing promise in further defining the phenotypic and functional heterogeneity within human B cell subsets⁴³. Further analysis of the cellular context and tissue architecture, via spatial transcriptomics and tissue mass cytometry approaches, is beginning to generate further insights into these maturation processes^21,44.

The differentiation of IgM⁺IgD⁻ cells to IgA⁺ ASCs may be an important feature of protective immunity at mucosal sites. Magri et al. showed that IgM⁺IgD⁻CD27⁺ B cells are enriched in the ileum and are low or absent in other compartments of the gastrointestinal tract including the colon and other parts of the small intestine, suggesting that there are regional differences in how mucosal memory responses are established⁶. Similar to what has been reported by Zhao et al.²¹, we found an abundance of IgM⁺IgD⁻ B cells in both the appendix and tonsil, despite regional differences between these anatomic sites.

One intriguing property of antibodies produced by IgM⁺IgD⁻ B cells is their short CDR3 length. Recently Baumgarth and colleagues described a population of murine B1-derived plasma cells that have evidence for public clones and short CDR3 sequences⁴⁵. We looked for public clones in IgM⁺IgD⁻ B cells and did not find convincing evidence for shared CDR3 amino acid sequences that were encoded by different nucleotide sequences, which would have been suggestive of convergent selection⁴⁶. Rather than finding an abundance of shared CDR3 amino acid sequences between different individuals, each individual had their own private collection of short CDR3-bearing IgM⁺IgD⁻ clones. The shift in CDR3 length also was not focused on a few large clones with very short CDR3 sequences but rather represented a decrease across the population of clones, which is more consistent with a global selection process. Also, unlike B1 B cells, most IgM⁺IgD⁻ B cells did not express B1-associated markers CD5 or CD43. However, IgM⁺IgD⁻ B cells were abundant in early life.

IgM⁺IgD⁻ B cells were particularly frequent in an appendix sample from a 12-hr- old newborn suggesting that they may be precursors to more mature (e.g., switch memory) B cell populations. Clonal lineage analysis further supports this idea. In the 12-hr-old, IgM⁺IgD⁻ lineages tended to have lower levels of SHM and often retained the germline VH gene. Retention of germline VH gene sequences is also observed in B cell clonal lineages isolated from bronchus-associated lymphoid tissue of pediatric organ donors⁴⁷. In contrast, in older individuals, IgM⁺IgD⁻ lineages had higher levels of SHM with fewer germline VH gene sequences. IgM⁺IgD⁻ lineages also had more mutated clones and higher levels of SHM than IgM⁺IgD⁺ B cell lineages. Finally, some IgM⁺IgD⁻ clones exhibited overlap with switch memory cells, including more mutated sequence variants within clones that corresponded to cells having a switch memory phenotype. Although conclusions drawn from a single patient sample are inherently limited, this sample from a normal 12-hr-old infant provides valuable reference data for early neonatal B cell development in GALT. The large number of IgM⁺IgD⁻ B cells with some SHM in neonatal GALT reminds us of GALT B cells found in species such as chicken, rabbit and sheep^48,49. In these species, GALT B cells undergo somatic diversification early in ontogeny, form the preimmune repertoire, and can be considered memory-like. While little somatic diversification is found in the human GALT B cells 12 h after birth, it would be important to examine SHM in GALT B cells from infancy to adulthood. These IgM⁺IgD⁻ B cells in neonatal human GALT could be similar to GALT B cells in chickens, rabbits and sheep.

In summary, we identified a B cell population that is enriched in human GALT. These cells are IgM⁺IgD⁻, show characteristics of antigen-experienced B cells and may represent an early memory population. Further, these cells class switch to IgA 10-fold more than IgM⁺IgD⁺CD27⁺ B cells, suggesting that they are major contributors to the IgA repertoire in GALT. Much remains unknown about these cells, including their developmental origin, BCR specificity, contribution to mucosal and peripheral immunity, and mode of renewal. These are important topics for future studies.

Materials and methods

Human tissue and blood samples

Histologically normal tonsil and appendix surgical tissue specimens were obtained from the Pathology Department at Loyola University Medical Center with approval by the institutional review board at Loyola University Medical Center. The age of tissue donors ranged from birth (12-hr-old) to 50 years. The ratio of males to females was 1:1 for tonsil samples and 2:1 for appendix samples. Tonsil and appendix mononuclear cells were teased from fresh tissue pieces (~ 1–3 cm³) and passed through a 100 μm cell strainer (Corning – Millipore Sigma - Cat: CLS431752). Peripheral blood mononuclear cells were isolated from buffy coats on lymphocyte separation medium (Corning 25072CV – Fisher Scientific – Cat: MT25072CV).

Flow cytometry and cell sorting

After 30 min of FcR block (Biolegend – Cat: 422302), cells were stained by standard methods with the following fluorescent antibodies: anti-CD19 (HIB19), anti-IgM (MHM-88), anti-IgD (IA6-2), anti-IgG (HP6017), anti-IgA (IS11-8E10), anti-CD5 (L17F12), anti-CD10 (HI10a), anti-CD43 (CD43-10G7), anti-CD38 (HIT-2), anti-CD138 (DL-101), anti-CD11c (3.9), and anti-CD27 (LG.3A10, or O323). Analysis of T-bet (4B10) was performed on fixed and permeabilized cells (Biolegend – Cat#: 424401). Our most complex flow cytometry panel utilized a combination of the following fluorophores: FITC, PerCP-Cy5.5, Pacific Blue, AmCyan, Qdot 655, Qdot 705, Qdot 800, Alexa Fluor 700, APC, APC-Cy7, PE, PE-Texas Red, and PE-Cy7. Cells were analyzed using the LSRFortessa; dead cells were excluded with fixable cell viability dye (eBioscience – eFluor 780), and data were analyzed using FlowJo V10 software. Gating schemes are shown in Supplementary Figs. 1, 4 and 5. Antibodies and fluorochromes used for cell sorting (Supplemental Fig. 5) are as follows: Fixable cell viability dye (eBioscience—eFluor 780 – APC-Cy7), CD19 (Pacific Blue), IgM (FITC), IgD (PE-Texas Red), CD27 (PE-Cy7), IgG (PerCP-Cy5.5), IgA (APC).

ELISpot

After blocking (5% BSA) and coating (10 µg/ml anti-L-chain – Biolegend – Clone: MHL-38 and MHK-49) 96-well PVDF membrane plates (Millipore Sigma), sorted tonsil and appendix B cells were plated (20,000 cells/well) and incubated at 37 °C, 5% CO₂ for 6 h. Gating strategy for cell sorting (Supplementary Fig. 5). Cells with high CD38 expression, characteristic of plasma cells⁶, were excluded. Doublets were excluded using FSC-A vs. FSC-H plots to ensure only single cells are sorted, and post-sort purity analysis was performed after each sort to ensure the purity > 97%. IgM secretion was measured using biotin-labeled anti-µ heavy chain (MHM-88) and HRP-conjugated streptavidin (Biolegend – Cat: 405210). Spots were developed using the HRP substrate - TMB (MABTECH- Cat: 3651-10) and counted using an S6 FluoroCore M2 ELISpot plate reader (ImmunoSpot, CTL). All poorly defined spots (diffused faint staining of the spots) were considered background noise and were excluded in the spot quantification. Gating strategy for cell sorting (Supplementary Fig. 5).

In vitro B cell stimulation and quantitation of IgA

Flow cytometry-sorted tonsil B cells were seeded at 10⁵ cells/well in 96-well U-bottom plates (Thermo Fisher) for 6–7 days in complete RPMI 1640 medium (Thermo Fisher) supplemented with 10% FBS, 500 ng/ml BAFF (Peprotech), and 100 ng/ml APRIL (Peprotech). Cells were stimulated with 1 µg/ml CpG ODN-2006 (Invivogen), or 200 ng/ml anti-L-chain (κ + λ) (10H6E5 and 3C12D7) + 200 ng/ml anti-CD40 (5C3) + 500 ng/ml IL-21 (Peprotech). IgA secretion was measured in the culture supernatant by cytometric bead array (BD – Cat: 558683) following the manufacturer’s instructions. Gating strategy for cell sorting (Supplementary Fig. 5).

BCR sequencing and data QC

DNA was extracted from sorted cells (Supplementary Fig. 5). using a Gentra Puregene Cell kit (Qiagen, catalog no. 158046). Immunoglobulin heavy-chain family–specific PCRs were performed on genomic DNA samples using primers in FR1 and JH as described previously⁵⁰. Two biological replicates were run on all samples. Sequencing was performed using an Illumina 2 × 300-bp paired-end kit (Illumina MiSeq Reagent Kit v3, 600-cycle, Illumina MS-102-3003) in the Human Immunology Core at the University of Pennsylvania.

Resulting reads were filtered as described previously^50,51. Briefly, sequences with an average Phred quality score of less than 30 were discarded, individual bases with a quality score less than 30 were masked with an N to indicate uncertainty, and short reads were discarded. Sequences passing this quality control step were aligned and annotated with IgBLAST v1.17.0⁵². The annotated sequences were then imported into ImmuneDB v0.29.11⁵³ for clonal inference, lineage construction, and downstream processing. Related sequences for each individual were grouped into clones if they shared the same IGHV gene, IGHJ gene, CDR3 length, and had 85% or higher amino acid sequence similarity in the CDR3.

BCR repertoire analysis

Immune repertoire profiling data were visualized using ImmuneDB⁵³. All downstream analyses included only productive clones with at least two copies within an individual subject. For general repertoire analysis, samples from the 12-hr-old individual were excluded except for the short CDR3 sequence analysis. To determine the degree of overlap across subsets in tonsil and appendix samples, sequences that were present in 1 copy per subset were removed (in addition to the initial subject-level data filtering) to minimize the potential contribution of barcode hopping. These further filtered data were then aggregated by subset across all of the donors for a given tissue. Each unique CDR3 amino acid sequence was then tracked through every subset for the string plot in the main manuscript figure and grouped into major subset fractions (IgM⁺IgD⁺, IgM⁺IgD⁻ and class-switched) for the Venn diagram. Clonal lineages were further filtered based on copy number of individual sequence variants (minimum of 10 copies per node) for ease of visualization. Lineages were plotted using ete3⁵⁴. Aggregated VH usage across all individuals was plotted by tissue and major subset. Each unique clone was counted only once per subset, normalization was performed by subset (row in the heatmap). VH genes with < 1% usage in all subsets were excluded from the heatmaps. For donors > 1 day old, sample lineages were chosen from those that overlapped between IgM⁺IgD⁻ and either IgG⁺ or IgA⁺ B cells. For the 12-hr-old donor, lineages were selected from those occurring in IgM⁺IgD⁻. For the analysis of short CDR3-containing sequences, all clones with CDR3 sequences in the specified size range (7–9 amino acids, inclusive) were analyzed without down-sampling.

Statistical analyses

For immune repertoire profiling data, SciPy v1.9.3 was used to perform all tests of significance. Mann-Whitney U-tests were performed to compare SHM and CDR3 length distributions between subsets and within each subset by tissue. The frequency of IGHV4-34 clones in IgM⁺IgD⁺ samples was compared to IgM⁺IgD^- and class-switched samples using one-sided binomial tests. For the 12-hr-old donor, CDR3 lengths of clones in IgM⁺IgD^- samples were compared to those of clones in IgM⁺IgD⁺ samples using a one-sided Mann-Whitney U-test. For non-immune repertoire profiling data analyses, statistical significance was determined by an unpaired, two-tailed Student’s t-test using GraphPad Prism software (GraphPad Prism 9.5.1 - La Jolla, CA).

Data availability

Raw immune repertoire sequencing data for all individuals and subsets is available on SRA under BioProject PRJNA1291145.

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Acknowledgements

This work was supported by NIH grants AI40254 and AI140132 and P30AI1045008. The authors declare no competing financial interests.

Funding

This work was supported by NIH grants AI140254, AI140132 and P30AI1045008.

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Ameera M. Bukhari, Wenzhao Meng and Aaron M. Rosenfeld contributed equally to this work.

Authors and Affiliations

Department of Microbiology and Immunology, Loyola University Chicago, 2160 S. First Ave, Maywood, IL, 60153, USA
Ameera M. Bukhari & Katherine L. Knight
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, 405B Stellar Chance Labs, 422 Curie Blvd., Philadelphia, PA, 19104, USA
Wenzhao Meng, Aaron M. Rosenfeld & Eline T. Luning Prak

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Ameera M. Bukhari
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Wenzhao Meng
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Aaron M. Rosenfeld
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Contributions

Ameera Bukhari conducted the human tissue and blood sample collection and preparation, flow cytometry and cell sorting, ELISpot, class-switch analysis, Wenzhao Meng and Aaron Rosenfeld conducted the BCR Sequencing and BCR Repertoire Analysis. Ameera Bukhari wrote the introduction, methods, results, and discussion sections related to her experiments. Eline Luning Prak wrote the methods, results, and discussion sections regarding the BCR sequencing and repertoire analysis. All authors read and approved the final manuscript. All authors have agreed to authorship and order of authorship for this manuscript and all authors have the appropriate permissions and rights to the reported data.

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Correspondence to Eline T. Luning Prak or Katherine L. Knight.

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Bukhari, A.M., Meng, W., Rosenfeld, A.M. et al. IgM⁺IgD⁻ B cells in human gut-associated lymphoid tissue have memory features and give rise to IgM⁺ and IgA⁺ antibody-secreting cells. Sci Rep 15, 26613 (2025). https://doi.org/10.1038/s41598-025-11209-1

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Received: 07 December 2024
Accepted: 08 July 2025
Published: 22 July 2025
DOI: https://doi.org/10.1038/s41598-025-11209-1