Memory persistence and differentiation into antibody-secreting cells accompanied by positive selection in longitudinal BCR repertoires
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Evaluation Summary:
By performing homeostatic longitudinal IgH repertoire analysis of human memory B cells and plasma cells, authors draw two major unique conclusions; first, a high degree of clonal persistence in individual memory B cell subsets with inter individual convergence in memory B and plasma cells; second, reactivation of persisting memory B cells with new rounds of affinity maturation during proliferation and differentiation into plasma cells. These conclusions provide a significant insight into how human memory B and plasma cells are generated in a homeostatic condition.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
The stability and plasticity of B cell-mediated immune memory ensures the ability to respond to the repeated challenges. We have analyzed the longitudinal dynamics of immunoglobulin heavy chain repertoires from memory B cells, plasmablasts, and plasma cells from the peripheral blood of generally healthy volunteers. We reveal a high degree of clonal persistence in individual memory B cell subsets, with inter-individual convergence in memory and antibody-secreting cells (ASCs). ASC clonotypes demonstrate clonal relatedness to memory B cells, and are transient in peripheral blood. We identify two clusters of expanded clonal lineages with differing prevalence of memory B cells, isotypes, and persistence. Phylogenetic analysis revealed signs of reactivation of persisting memory B cell-enriched clonal lineages, accompanied by new rounds of affinity maturation during proliferation and differentiation into ASCs. Negative selection contributes to both persisting and reactivated lineages, preserving the functionality and specificity of B cell receptors (BCRs) to protect against current and future pathogens.
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Author Response
Reviewer #1 (Public Review):
Mikelov et al. investigated IgH repertoires of memory B cells, plasmablasts, and plasma cells from peripheral blood collected at three time-points over the course of a year. In order to obtain deep and unbiased repertoire sequences, authors adopted uniquely developed IgH repertoire profiling technology. Based on collected peripheral blood data, authors claim that:
- A high degree of clonal persistence in individual memory B cell subsets with inter-individual convergence in memory and ASCs.
- ASC clonotypes are transient over time and related to memory B cells.
- Reactivation of persisting memory B cells with new rounds of affinity maturation during proliferation and differentiation into ASCs.
- Both positive and negative selection contribute to persisting and reactivated lineages …
Author Response
Reviewer #1 (Public Review):
Mikelov et al. investigated IgH repertoires of memory B cells, plasmablasts, and plasma cells from peripheral blood collected at three time-points over the course of a year. In order to obtain deep and unbiased repertoire sequences, authors adopted uniquely developed IgH repertoire profiling technology. Based on collected peripheral blood data, authors claim that:
- A high degree of clonal persistence in individual memory B cell subsets with inter-individual convergence in memory and ASCs.
- ASC clonotypes are transient over time and related to memory B cells.
- Reactivation of persisting memory B cells with new rounds of affinity maturation during proliferation and differentiation into ASCs.
- Both positive and negative selection contribute to persisting and reactivated lineages preserving the functionality and specificity of BCRs.
The present study provides useful technical application for the analysis of longitudinal B cell repertoires, and bioinformatics and statistical data analysis are impressive. Regarding point 1), clonal persistence of memory B cells is already well known. On the other hand, inter-individual convergence between memory B cells and plasma cells might not be shown in healthy individuals even though the biological significance of circulating plasma cells is questionable.
We thank the reviewer for careful analysis of our manuscript and are grateful for the positive view and all the criticism of our study.
To the best of our knowledge the clonal persistence of memory B cells was previously studied mostly in the contexts of active immune response after natural challenge or after immunization. Here we used the full set of modern experimental and analytical repertoire sequencing approaches to characterize the connection and dynamics of memory and the two antibody-secreting B cell subpopulations during a long period in healthy donors, i.e. in donors without severe inflammatory diseases or who were not experienced intensive response against a natural antigen close to the sample collection time points. In other words, we carefully dissected the repertoire of peripheral blood antigen-experienced B cells in normal state. Thus we believe that our study brings a number of essentially new details to the overall picture of B cell immunity.
By assessing the intra- and inter-individual repertoire overlaps we found high reproducibility of B cell memory clones between timepoints, which was just a little bit lower compared to the overlap between replicates. About 5% of largest clonotypes were identical (Fig. 2B left), while the V usage distribution changed more substantially over the time (Fig. 2A left), assuming the impact of non-persistent memory IGH clonotypes. Compared to the intra-individual reproducibility, the number of shared clonotypes between unrelated donors was extremely low, but still detectable, showing impact of convergent clonotypes in antigen-experienced B cells repertoire overlap of unrelated donors. Together, our findings show a high level of individuality of IGH repertoire of antigen-experienced B cells, while common challenges converge it to some extent at the level of most expanded clones, which are extremely stable (persistent) over the time. On the way from naive to the antigen-experienced B cells the germ-line encoded sequence of CDR1 and CDR2 make an impact, which is similar between individuals with similar genetic and environmental context. The latter further supports the previously reported findings on the role of germ-line encoded parts of IGH in the response against specific antigens (Collins et al. DOI: 10.1016/j.coisb.2020.10.011).
Regarding 2), temporal stability of plasma cell clonotypes has been demonstrated already in the bone marrow with serial biopsies over time (Wu et al. DOI: 10.1038/ncomms13838). The Association of clonotypes between memory and plasma cells in the blood of healthy donors might be new, however, again its biological significance is questionable.
Long-term stability of plasma cells was previously shown by a number of studies demonstrating presence of antigen-specific clones or even cells during months and years in human bone marrow and other sites, as well as in mice and primates (Wu et al. DOI: 10.1038/ncomms13838; Landsverk et al. DOI: 10.1084/jem.20161590; Manz et al. DOI: 10.1038/40540; Hammarlund et al. DOI: 10.1038/s41467-017-01901-w; Xu et al. DOI: 10.7554/eLife.59850; Davis et al. DOI: 10.1126/science.aaz8432). We agree that BM samples would add the additional layer to our investigation by describing the interconnection of the B cell memory pool with BM PCs. We also agree that the nature of circulating plasma cells is not fully clear at the moment and the relation of such cells/clones to BM PCs remains to be detailed. However, we cannot agree with the reviewer’s remark about the low (or absent) biological significance of the circulating ASCs. According to modern view, raising from large number of different studies conducted for previous several decades on mice, human and other organisms, the differentiation events in GC after antigen-priming lead to formation of cells switched to antibody-secreting program, and some part of them further reaches the bone marrow as site of residence. The bone marrow niches provide necessary signals required for further differentiation of newly migrated ASC cells to long-living or short-living plasma cells and their further survival in BM. However, the ASCs migrating to BM can be sampled from blood during their migration. The presence of an apoptotic-resistant subset of PCs expressing high-affinity Abs in circulation early after booster immunization in humans was previously shown (Inés González-García et al. DOI: 10.1182/blood-2007-08-108118). Similar in vitro survival ability for transcriptomically different blood ASC subsets was demonstrated by other authors (Garmilla et al. DOI: 10.1172/jci.insight.126732). Recent study, using artificial system modeling the BM niche in vitro, show that peripheral blood ASCs are able to differentiate to LLPC (Joyner et al. DOI: 10.26508/lsa.202101285). Besides, in a number of other studies it was also previously shown the increase of plasmablasts and plasma cells in PB during intensive immune response after primary or secondary immunization/natural challenge (Blink et al. DOI: 10.1084/jem.20042060; Odendahl et al. DOI: 10.1182/blood-2004-07-2507; Lee et al. DOI: 10.4049/jimmunol.1002932) or in active autoimmune condition (Szabo et al. DOI: 10.1111/cei.12703; Jacobi et al. DOI: 10.1002/art.10949). So, we considered ASC subsets in our work as a source of ASCs enriched in recently differentiated antibody-producers different in expression of CD138, which is the marker of LLPC in BM plasma cells and seemingly marks differently differentiated ASCs in circulation. Thus, these ASC subsets complement antigen-primed peripheral blood B cells playing an important role in ongoing immune response and influence to the plasma cells population in the BM. The connection on clonal lineage level between persisting memory B cells and the ASC subsets shown in our study, and findings recently published by Antonio Lanzavecchia’s lab (Phad et al. DOI: 10.1038/s41590-022-01230-1), support the idea that the circulating CD19-/lowCD20-CD27+CD138+/- B cells in PB represent the antibody-producing progeny of reactivated memory.
Regarding 3) and 4), it is hard to generalize observations from the presented data because the analysis was based on just four donor cases with different health conditions, i.e. a combination of healthy and allergic. The cell number of plasmablasts and plasma cells isolated from peripheral blood is extremely low compared to memory B cells, and in fact, the vast majority of ASCs reside in the tissues such as lymphoid organs, bone marrow, and mucosal tissues rather than in circulating blood (Mandric et al. DOI: 10.1038/s41467-020-16857-7). As the most critical problem, direct pieces of evidence to claim points, 3) and 4) are missing.
We fully agree that our study has a set of limitations and added more detailed discussion of them to the revised version (lines 582-600). We agree that our cohort group is not large, nevertheless our observations demonstrate reproducibility among different donors and hold statistical significance for detected differences. To justify our generalization of this cohort group, combined from healthy and allergic donors, we added more detailed analysis as a Supplementary Note, showing that within our study design we observe no difference between healthy and allergic donors both on the level of the clonal repertoire and the level of clonal lineages.
The number of sampled plasmablasts and plasma cells compared to memory B cells in our study reflects the ratio between the subpopulations in the peripheral blood of middle aged donors and corresponds to the previous estimations published by the others. According to the fact that about 15% of the most abundant clonotypes on average were reproducible between parallel samples (replicates), the sampled numbers of PBL and PL allowed us to reach a relatively high reproducibility of the clone sampling at the level of cells. This as well as the diversity estimations point out that we sequenced the representative number of ASCs in peripheral blood to characterize their clonal repertoire and their connection with the B cell memory pool. Indeed the vast majority of plasma cells reside in different tissues, mostly in the bone marrow, but we believe that the ASCs in circulation represent the pool of newly generated and/or migrating between sites ASCs at different stages of differentiation. However, the further studies showing clonal relationship between memory B cells and ASCs in circulation and tissue-resident ASCs are still required to provide a more detailed view to this aspect.
We agree that we cannot provide much direct evidence to support points 3) and 4), however we revealed a bunch of indirect ones, which are very consistent between each other supporting the points on memory reactivation and clonal selection claimed:
From the biological sense, rapid increase of frequency of LBmem lineages and its’ perfect reproducibility between replicates (Supplementary Figure S7E), indicate increase in the number of the sampled cells, i.e. lineage expansion, occurred due to proliferation after antigen challenge or migration between tissues of residence due to some other signals. Predominance of ASC phenotype indicates their involvement in ongoing immune response.
Large G-MRCA distance in LBmem lineages together with low inter-lineage genetic divergence points out on that the observed clonotypes of LBmem lineages diverged recently, originate from some mature clonotype and represent only a single clade of full lineage phylogeny.
Most of LBmem lineages (47 out of 52) includes Bmem clonotypes, showing interconnection of LBmem cluster to Bmem subset. For 38 out of 52 LBmem lineages we detected Bmem clonotype at the time point prior to lineage expansion.
Significant difference in SHM patterns between HBmem and LBmem lineages reflects difference in selection forces, affecting their evolution. In evolutionary genomics, it is rarely possible to study evolution directly, and most often changes in genetic sequences are the only type of data available. Therefore, we are inclined to trust the conclusions drawn from the use of tools designed for this type of problem. If negative selection is expected in the evolution of any protein, positive selection is much more tricky to detect. Thus the presence of its signs suggests new rounds of affinity maturation or presence of some mechanism, leading to reactivation of the best-fitted representatives of the lineage.
In addition to the indirect evidence, we found direct and clear example of memory reactivation inside the clonal lineage (Fig. 4F). We added alignment of the CDR3 region of this lineage as Supplementary Figure S7 to confirm that both its’ HBmem - like and LBmem - like parts originate from the same recombination event.
These findings lead to the conclusion that most of the LBmem lineages in analysis originated from some pre-existing memory. However we can not say for sure that in all the cases the memory is similar in properties to the persistent memory of HBmem cluster. The one exemplary clonal lineage shows that at least some of LBmem lineages represent re-activation of persistent HBmem lineages. The most recent study in the field published by Phad et al. (DOI: 10.1038/s41590-022-01230-1) have also demonstrated clonal relatedness of peripheral blood plasmablasts to the persistent memory. It should also be noted that in the present study we focused on the most expanded clones and clonal lineages, while the mechanisms determining the power of expansion are well not defined and thus the behavior of not so large clones can be different. To conclude, we believe that our findings can be generalized while probably representing only a part of the whole complex picture describing the behavior of B cell memory in normal state.
Reviewer #2 (Public Review):
The findings in this manuscript have been properly hypothesized and adequately demonstrated, and have some levels of practical guidance. The authors performed a detailed longitudinal analysis of a subset of immune-experienced B cells from donors without severe pathology. They selected a comprehensive analytical framework for BCR clonal lineage from these data and suggested interconnected B-cell clone-level subsets, B-cell memory fusion in donor-independent, and long-term persistent peripheral blood memory-enriched clonal lineages. Lastly, their evolutionary results analyzing the B-cell clonal lineage plus annotation suggest that activating B-cell subsets of preexisting memory-B cells is accompanied by the maturation of new rounds of affinity.
We thank the Reviewer for careful analysis and positive view on our study.
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Evaluation Summary:
By performing homeostatic longitudinal IgH repertoire analysis of human memory B cells and plasma cells, authors draw two major unique conclusions; first, a high degree of clonal persistence in individual memory B cell subsets with inter individual convergence in memory B and plasma cells; second, reactivation of persisting memory B cells with new rounds of affinity maturation during proliferation and differentiation into plasma cells. These conclusions provide a significant insight into how human memory B and plasma cells are generated in a homeostatic condition.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Mikelov et al. investigated IgH repertoires of memory B cells, plasmablasts, and plasma cells from peripheral blood collected at three time-points over the course of a year. In order to obtain deep and unbiased repertoire sequences, authors adopted uniquely developed IgH repertoire profiling technology. Based on collected peripheral blood data, authors claim that; 1) A high degree of clonal persistence in individual memory B cell subsets with inter-individual convergence in memory and ASCs. 2) ASC clonotypes are transient over time and related to memory B cells. 3) Reactivation of persisting memory B cells with new rounds of affinity maturation during proliferation and differentiation into ASCs. 4) Both positive and negative selection contribute to persisting and reactivated lineages preserving the …
Reviewer #1 (Public Review):
Mikelov et al. investigated IgH repertoires of memory B cells, plasmablasts, and plasma cells from peripheral blood collected at three time-points over the course of a year. In order to obtain deep and unbiased repertoire sequences, authors adopted uniquely developed IgH repertoire profiling technology. Based on collected peripheral blood data, authors claim that; 1) A high degree of clonal persistence in individual memory B cell subsets with inter-individual convergence in memory and ASCs. 2) ASC clonotypes are transient over time and related to memory B cells. 3) Reactivation of persisting memory B cells with new rounds of affinity maturation during proliferation and differentiation into ASCs. 4) Both positive and negative selection contribute to persisting and reactivated lineages preserving the functionality and specificity of BCRs.
The present study provides useful technical application for the analysis of longitudinal B cell repertoires, and bioinformatics and statistical data analysis are impressive. Regarding point 1), clonal persistence of memory B cells is already well known. On the other hand, inter-individual convergence between memory B cells and plasma cells might not be shown in healthy individuals even though the biological significance of circulating plasma cells is questionable.
Regarding 2), temporal stability of plasma cell clonotypes has been demonstrated already in the bone marrow with serial biopsies over time (Wu et al. DOI: 10.1038/ncomms13838). The Association of clonotypes between memory and plasma cells in the blood of healthy donors might be new, however, again its biological significance is questionable.
Regarding 3) and 4), it is hard to generalize observations from the presented data because the analysis was based on just four donor cases with different health conditions, i.e. a combination of healthy and allergic. The cell number of plasmablasts and plasma cells isolated from peripheral blood is extremely low compared to memory B cells, and in fact, the vast majority of ASCs reside in the tissues such as lymphoid organs, bone marrow, and mucosal tissues rather than in circulating blood (Mandric et al. DOI: 10.1038/s41467-020-16857-7). As the most critical problem, direct pieces of evidence to claim points, 3) and 4) are missing.
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Reviewer #2 (Public Review):
The findings in this manuscript have been properly hypothesized and adequately demonstrated, and have some levels of practical guidance. The authors performed a detailed longitudinal analysis of a subset of immune-experienced B cells from donors without severe pathology. They selected a comprehensive analytical framework for BCR clonal lineage from these data and suggested interconnected B-cell clone-level subsets, B-cell memory fusion in donor-independent, and long-term persistent peripheral blood memory-enriched clonal lineages. Lastly, their evolutionary results analyzing the B-cell clonal lineage plus annotation suggest that activating B-cell subsets of preexisting memory-B cells is accompanied by the maturation of new rounds of affinity.
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