Sequential and coordinated control of human plasma cell differentiation by IRF4 and BLIMP1 utilizing a discriminating ISRE/EICE motif lexicon

Reviewer #1 (Public review):

Summary:

This manuscript investigates how IRF4 and BLIMP1 coordinate human plasma cell differentiation. Using a stepwise in vitro culture system starting from primary human naïve B cells, the authors define a developmental window enriched for plasma cell precursors and use stage-specific CRISPR/Cas9 perturbation to examine the roles of IRF4 and PRDM1/BLIMP1 during the transition from plasmablast-like precursors to plasma cells. Single-cell transcriptomic analyses suggest that IRF4 acts early to license plasma cell differentiation, whereas BLIMP1 contributes more prominently to consolidation of the terminal plasma cell program. The authors further combine multiome profiling, CUT&RUN, motif modeling, and EMSA assays to propose the sublet nucleotide variation within ISRE/EICE-like motifs contributes to differential or shared binding by IRF4 and BLIMP1.

Overall, this is a carefully performed and conceptually interesting study. It provides a useful experimental platform for dissecting human plasma cell differentiation and offers a mechanistic model for how two closely connected transcription factors can exert distinct and coordinated genomic functions during terminal B cell differentiation.

Strengths:

A major strength of the study is the establishment and detailed characterization of a human in vitro plasma cell differentiation system. The authors combine phenotypic, functional, and single-cell transcriptomic analyses to define the transition from activated B cells to plasmablst/plasma cell precursor-like cells and then to more mature plasma cells. This system is very useful for future perturbation studies of human plasma cell differentiation.

A second strength is the stage-specific perturbation strategy. By targeting IRF4 or PRDM1 at the precursor-enriched stage, the authors avoid some of the interpretive limitations associated with earlier perturbations that would affect B cell activation, proliferation, and plasma cell commitment simultaneously. The distinct phenotypes observed after IRF4 versus PRDM1 perturbation provide support for a model in which these two factors act in a temporally ordered manner.

A third strength is the integration of multiple genomic and biochemical approaches. The combination of single-cell RNA-seq, chromatin accessibility profiling, CUT&RUN, computational motif analysis, and EMSA assays provides a rich dataset and supports the idea that ISRE/EICE sequence variation contributes to differential IRF4 and BLIMP1 occupancy.

Weaknesses:

While the multi-omic approach and computational modeling are highly impressive, several major assumptions regarding the cellular differentiation model and genomic linkages require more rigorous validation.

First, because CRISPR editing was performed on heterogeneous bulk Day 7 cells rather than purified precursor populations, it remains ambiguous whether the observed developmental blocks are truly specific to the prePC window.

Second, given that IRF4 and BLIMP1 operate within a mutually reinforcing positive feedback loop, the phenotypic divergence between IRF4 KO and PRDM1 KO may reflect differences in protein degradation kinetics or hierarchical dominance rather than a strictly ordered "sequential function".

Lastly, the motif-lexicon model is elegant and supported by biochemical DNA-binding assays, but the link between motif variation and gene regulation in cells remains partly correlative. Direct testing of selected regulatory elements would make the causal claim stronger. Alternatively, the authors should temper the language and present the motif lexicon as a predictive model for differential occupancy rather than as a fully demonstrated mechanism of gene regulation.

Reviewer #2 (Public review):

Summary:

The manuscript by Lau et al. investigates the mechanisms underlying IRF4 and BLIMP1 transcriptional activities during antibody-secreting cell fate decision. Both master regulators of plasma cell differentiation, these two transcription factors have distinct targets and non-overlapping roles. The authors used an in vitro culture system to generate antibody-secreting cells from human naïve B cells, and scRNA-seq, Crispr Cas9 editing, and Cut&Run to dissect the molecular mechanisms defining their specificity.

Strengths:

The experiments are overall well executed, and the manuscript is well written. The in vitro culture model appears to generate genuine human antibody-secreting cells. The identification of non-conserved nucleotides within the binding motifs that induce the specific binding of IRF4 or BLIMP1 is convincing, novel, and exciting.

Weaknesses:

The authors need to correct some overstatements and flaws to improve the manuscript.

In Figure 1f, the authors aimed to determine whether in their culture system the plasma cells emerged from the plasmablasts or directly from the activated B cells. First, it is noticeable that the distinction between plasmablasts and plasma cells relies here only on the expression of CD138. It does not include a higher capacity to secrete antibody or their proliferative state. In Figure 1e, the authors could have strengthened their distinction by showing the Ki67 staining at day 21 for both subpopulations. Second, this question does not seem to be related to IRF4 or Blimp1 activity, and thus one could wonder if it is relevant to this study. Finally, and most importantly, the design of the experiment appears flawed to me. The authors sorted cells at day 7 of culture based on their expression of CD20 and put the two subpopulations back for 14 more days. This culture system is a stepwise system, and it is not specified if the CD20+ cells were put back in the day 7 condition or the day 0 condition with the CD40L stimulation. Have both conditions been tested? This experiment also assumes that all B cells have equal potential to differentiate into antibody-secreting cells. What if it is not the case and some are anergic or have committed to the memory B cell fate during the first 7 days? Then the day 7 CD20+ fraction would be enriched in these cells. Moreover, this experiment didn't show that the plasma cell derived from the plasmablasts in the strict sense of the term, as the CD138+CD20- cells could be a mix of proliferative plasmablasts and immature plasma cells.

In Figure 3a and thereafter, the authors claimed that IRF4 acted earlier than BLIMP1, but both deletions strongly affected differentiation at day 7. IRF4 might have a stronger effect, but it does not mean that it had an earlier effect. To substantiate their claim, the authors would need to demonstrate that, at an earlier time point, deletion of IRF4, but not BLIMP1, results in defective differentiation.

In Figure 3b, the authors stated that in each individual KO the expression of the other transcription factor was lower. Given that there were no cells in the gate, it is puzzling to figure out how these expressions were compared.

In Figure 3c, on the UMAP the bottom right part of the activated B cell cluster does not appear to be attributed to any condition. How can it be? Besides, it is highly surprising that at D9 we cannot see any plasmablast on these UMAP, even in the control. Based on the G1/S and G2/M scores, none of the ASC represented were proliferating. Could the authors explain this strong discrepancy with Figure 1?

Another discrepancy exists between Figure 3b and c: Figure 3b depicted no IRF4- or BLIMP1-expressing cells in either KO, so what were the stunted PC and the BLIMP-KO PC reported in Figure 3c? What are the signature genes defining pre-PC and the score depicted in Supplementary Figure 3d, as the materials and methods only state that they are intermediate between PC and B cells? Could the authors show IRF4, BLIMP1 and some of their known target expression in these populations?

The authors claim that BLIMP1 is not needed to initiate the transition from pre-PC to PC, but in Figure 1, the intracellular staining showed that at day 7 the antibody secreting cells already expressed BLIMP1. This would rather suggest that BLIMP1, unlike IRF4, does not need to be maintained once the cell reaches a certain point.

Author response:

Reviewer #1 (Public review):

Summary:

This manuscript investigates how IRF4 and BLIMP1 coordinate human plasma cell differentiation. Using a stepwise in vitro culture system starting from primary human naïve B cells, the authors define a developmental window enriched for plasma cell precursors and use stage-specific CRISPR/Cas9 perturbation to examine the roles of IRF4 and PRDM1/BLIMP1 during the transition from plasmablast-like precursors to plasma cells. Single-cell transcriptomic analyses suggest that IRF4 acts early to license plasma cell differentiation, whereas BLIMP1 contributes more prominently to consolidation of the terminal plasma cell program. The authors further combine multiome profiling, CUT&RUN, motif modeling, and EMSA assays to propose the sublet nucleotide variation within ISRE/EICE-like motifs contributes to differential or shared binding by IRF4 and BLIMP1.

Overall, this is a carefully performed and conceptually interesting study. It provides a useful experimental platform for dissecting human plasma cell differentiation and offers a mechanistic model for how two closely connected transcription factors can exert distinct and coordinated genomic functions during terminal B cell differentiation.

Strengths:

A major strength of the study is the establishment and detailed characterization of a human in vitro plasma cell differentiation system. The authors combine phenotypic, functional, and single-cell transcriptomic analyses to define the transition from activated B cells to plasmablast/plasma cell precursor-like cells and then to more mature plasma cells. This system is very useful for future perturbation studies of human plasma cell differentiation.

A second strength is the stage-specific perturbation strategy. By targeting IRF4 or PRDM1 at the precursor-enriched stage, the authors avoid some of the interpretive limitations associated with earlier perturbations that would affect B cell activation, proliferation, and plasma cell commitment simultaneously. The distinct phenotypes observed after IRF4 versus PRDM1 perturbation provide support for a model in which these two factors act in a temporally ordered manner.

A third strength is the integration of multiple genomic and biochemical approaches. The combination of single-cell RNA-seq, chromatin accessibility profiling, CUT&RUN, computational motif analysis, and EMSA assays provides a rich dataset and supports the idea that ISRE/EICE sequence variation contributes to differential IRF4 and BLIMP1 occupancy.

Weaknesses:

While the multi-omic approach and computational modeling are highly impressive, several major assumptions regarding the cellular differentiation model and genomic linkages require more rigorous validation.

First, because CRISPR editing was performed on heterogeneous bulk Day 7 cells rather than purified precursor populations, it remains ambiguous whether the observed developmental blocks are truly specific to the prePC window.

We agree that CRISPR/Cas9 editing of bulk D7 cultures complicates interpretation because this population contains both activated B cells and PB/prePCs. We will therefore revise the text to distinguish phenotypic effects measured across the bulk D7 culture from the downstream single-cell analysis focused on cells along the prePC-to-PC trajectory. In particular, our interpretation of IRF4 and BLIMP1 function in prePCs is based primarily on the D9 scRNA-seq analysis, in which cells arrested in the activated B cell compartment are not used to define the perturbed PC-trajectory states. We will clarify this analytic design in a future revision and temper language implying that all effects arise exclusively within prePCs.

Second, given that IRF4 and BLIMP1 operate within a mutually reinforcing positive feedback loop, the phenotypic divergence between IRF4 KO and PRDM1 KO may reflect differences in protein degradation kinetics or hierarchical dominance rather than a strictly ordered "sequential function".

We agree that the divergence between IRF4 and PRDM1 perturbations could reflect differences in protein turnover, or hierarchical dominance, in addition to developmental timing. We will revise the Discussion to state that our data support a temporally ordered model in which IRF4 acts early to license the prePC-to-PC transition and BLIMP1 consolidates the terminal state, but that the current experiments do not exclude alternative explanations related to hierarchical dominance or degradation kinetics. We will also note in the revised Discussion that degron-based perturbations, rescue experiments, and gain-of-function analyses would be needed to resolve the functional ordering of IRF4 and BLIMP1 with higher temporal precision.

Lastly, the motif-lexicon model is elegant and supported by biochemical DNA-binding assays, but the link between motif variation and gene regulation in cells remains partly correlative. (1) Direct testing of selected regulatory elements would make the causal claim stronger. (2) Alternatively, the authors should temper the language and present the motif lexicon as a predictive model for differential occupancy rather than as a fudlly demonstrated mechanism of gene regulation.

We agree that the current data support the motif lexicon primarily as a predictive model for differential TF occupancy rather than as a fully causal mechanism of gene regulation. We will therefore revise the relevant text in the Results and Discussion. The EMSA data directly test nucleotide-dependent binding preferences, and the CUT&RUN/multiome analyses show that these motif variants are differentially associated with IRF4- or BLIMP1-bound DEG-linked OCRs. However, direct causal testing of endogenous regulatory elements, for example by base editing of selected ISRE/EICE variants, will be required to determine whether these variants are sufficient to predictably alter gene activity in differentiating plasma cells.

Reviewer #2 (Public review):

Summary:

The manuscript by Lau et al. investigates the mechanisms underlying IRF4 and BLIMP1 transcriptional activities during antibody-secreting cell fate decision. Both master regulators of plasma cell differentiation, these two transcription factors have distinct targets and non-overlapping roles. The authors used an in vitro culture system to generate antibody-secreting cells from human naïve B cells, and scRNA-seq, Crispr Cas9 editing, and Cut&Run to dissect the molecular mechanisms defining their specificity.

Strengths:

The experiments are overall well executed, and the manuscript is well written. The in vitro culture model appears to generate genuine human antibody-secreting cells. The identification of non-conserved nucleotides within the binding motifs that induce the specific binding of IRF4 or BLIMP1 is convincing, novel, and exciting.

Weaknesses:

The authors need to correct some overstatements and flaws to improve the manuscript.

In Figure 1f, the authors aimed to determine whether in their culture system the plasma cells emerged from the plasmablasts or directly from the activated B cells. First, it is noticeable that the distinction between plasmablasts and plasma cells relies here only on the expression of CD138. It does not include a higher capacity to secrete antibody or their proliferative state. In Figure 1e, the authors could have strengthened their distinction by showing the Ki67 staining at day 21 for both subpopulations.

Second, this question does not seem to be related to IRF4 or Blimp1 activity, and thus one could wonder if it is relevant to this study.

Finally, and most importantly, the design of the experiment appears flawed to me. The authors sorted cells at day 7 of culture based on their expression of CD20 and put the two subpopulations back for 14 more days. This culture system is a stepwise system, and it is not specified if the CD20⁺ cells were put back in the day 7 condition or the day 0 condition with the CD40L stimulation

We agree that CD138 alone does not fully define terminal PC maturation. In the revised manuscript, we will clarify that CD138 was interpreted in the context of a broader maturation profile, including CD20 downregulation, ICAM2 upregulation, IRF8 loss, IRF4/BLIMP1 expression, Ki-67 loss, and antibody secretion. The D7 PB population was proliferative and CD138^-, whereas D21 CD20^- cells were largely Ki-67^- and included CD138⁺ cells, supporting their progressive maturation. We will include Ki67 analysis in CD138^- and CD138⁺ cells at D21 in the revision.

We agree that the motivation and culture conditions for this experiment required a clearer explanation. The purpose of the D7 sort-and-reculture experiments was to identify the developmental window enriched for cells competent to generate PCs, thereby defining the stage at which IRF4 and PRDM1 should be perturbed. Sorted D7 CD20⁺ actB cells and CD20^-CD38⁺CD27⁺ PBs were both placed into the same D7-D14 differentiation conditions, allowing a direct comparison of their PC-generating competence under identical culture conditions. We will clarify this design in the Results and Methods. We have not tested whether returning D7 CD20⁺ cells to D0 conditions involving CD40L stimulation restores PC differentiation, and we will now acknowledge in the revision that the CD20⁺ fraction may contain cells with distinct intrinsic differentiation potential, including cells differentiating into non-PC states.

What if it is not the case and some are anergic or have committed to the memory B cell fate during the first 7 days? Then the day 7 CD20⁺ fraction would be enriched in these cells.

We agree that the CD20⁺ D7 cells may contain anergic or memory B cell precursors. However, this does not alter the interpretation that the CD20^- (CD38⁺/CD27⁺) PBs contain a PC precursor population. Even if memory B cells are generated in the CD20⁺ fraction by D7, based on the sorting experiments, their presence would have little-to-no effect on developing PC precursor populations.

Moreover, this experiment didn't show that the plasma cell derived from the plasmablasts in the strict sense of the term, as the CD138⁺CD20- cells could be a mix of proliferative plasmablasts and immature plasma cells.

We agree that the heterogeneous nature of CD20^- cells complicates the interpretation. However, we would like to emphasize that all D7 CD20^- cells are Ki67⁺ whereas all D21 CD20^- cells are nearly all Ki67^-. Though we concede these could include recently proliferated PCs at D21, it is consistent with this population becoming quiescent. To better address this question in a future revised version, we will include direct measurements of Ki67 levels in CD138⁺ and CD138^- cells at D21.

In Figure 3a and thereafter, the authors claimed that IRF4 acted earlier than BLIMP1, but both deletions strongly affected differentiation at day 7. IRF4 might have a stronger effect, but it does not mean that it had an earlier effect. To substantiate their claim, the authors would need to demonstrate that, at an earlier time point, deletion of IRF4, but not BLIMP1, results in defective differentiation.

We agree that the current data do not by themselves prove that IRF4 acts earlier than BLIMP1 in developmental time. We will revise the text to state that the data are consistent with a temporally ordered model, rather than demonstrating strict sequential action. The latter interpretation is based on the distinct IRF4 KO stunted PC state observed by D9 scRNA-seq (Fig. 3C), together with the stronger early phenotypic effect of IRF4 loss (Fig. 3A). However, because both factors are mutually reinforcing and because perturbations were not performed across multiple time points, alternative explanations remain possible, including differences in editing efficiency, protein stability, and feedback-dependent TF decay. We will modify the text to better explain the rationale behind this interpretation while also acknowledging alternative interpretations that do not involve sequential IRF4-BLIMP1 functions (see response to Reviewer 1).

In Figure 3b, the authors stated that in each individual KO the expression of the other transcription factor was lower. Given that there were no cells in the gate, it is puzzling to figure out how these expressions were compared.

In Figure 3c, on the UMAP the bottom right part of the activated B cell cluster does not appear to be attributed to any condition. How can it be? Besides, it is highly surprising that at D9 we cannot see any plasmablast on these UMAP, even in the control. Based on the G1/S and G2/M scores, none of the ASC represented were proliferating. Could the authors explain this strong discrepancy with Figure 1?

Another discrepancy exists between Figure 3b and c: Figure 3b depicted no IRF4- or BLIMP1-expressing cells in either KO, so what were the stunted PC and the BLIMP-KO PC reported in Figure 3c?

We thank the reviewer for identifying these points of confusion. We will revise Fig. 3B to display outlier events and frequencies more clearly and revise the figure legend to clarify the donor origin of the displayed UMAPs and corresponding supplemental analyses. We will also clarify that Fig. 3B and Fig. 3C represent distinct readouts: flow cytometry measures IRF4 and BLIMP1 protein abundance, whereas scRNA-seq resolves transcriptional states after perturbation. Thus, the “stunted PC” state in IRF4 KO cells is defined at a transcriptional level as a population positioned between prePCs and PCs, not as a population retaining normal IRF4 or BLIMP1 protein expression. We further clarify that the apparent reduction in proliferative plasmablast-like cells at D9 likely reflects both the later timepoint relative to D7 and differences between transcriptional cell-cycle gene scores and Ki-67 protein persistence.

What are the signature genes defining pre-PC and the score depicted in Supplementary Figure 3d, as the materials and methods only state that they are intermediate between PC and B cells?

The signature genes defining the scores in Fig. S3D are listed in Table S2. We will clarify the source of these genes in the figure legend of a future revised version.

Could the authors show IRF4, BLIMP1 and some of their known target expression in these populations?

We thank the reviewer for this suggestion to highlight IRF4, BLIMP1 and exemplar target genes in the various populations. We will update Fig. S3 to show transcript levels of IRF4, PRDM1 and an example of one of each of their target genes in unperturbed cells to demarcate their normal expression pattern.

The authors claim that BLIMP1 is not needed to initiate the transition from pre-PC to PC, but in Figure 1, the intracellular staining showed that at day 7 the antibody secreting cells already expressed BLIMP1. This would rather suggest that BLIMP1, unlike IRF4, does not need to be maintained once the cell reaches a certain point.

We agree that the data do not exclude the possibility that BLIMP1 is required before the perturbation window but is less continuously required once cells have progressed beyond a defined prePC stage. Our statement that BLIMP1 is not required to initiate the prePC-to-PC transition is based on the observation that PRDM1 KO cells did not accumulate in the prePC or stunted PC intermediate states observed after IRF4 loss. We will revise the text to make this interpretation more precise by stating that BLIMP1 appears less important than IRF4 for progression into a PC-like transcriptional state but is required for efficient consolidation of the mature PC program. We will also acknowledge that differences in editing efficiency, protein persistence, and timing of BLIMP1 action could contribute to the observed differences in phenotypes.