A mouse model of systemic lupus erythematosus responds better to soluble TACI than to soluble BAFFR, correlating with depletion of plasma cells

The TNF family cytokines B‐cell activating factor (BAFF) and a proliferation‐inducing ligand (APRIL) support plasma cell survival. It is known that inhibitors of BAFF only (BAFFR‐Fc) or BAFF and APRIL (TACI‐Fc) administered early enough in an NZB/NZW F1 mouse model of systemic lupus erythematosus (SLE) ameliorate clinical outcomes, pointing to a pathogenic role of BAFF. In the present study, TACI‐Fc administrated at a later stage of disease, after onset of autoimmunity, decreased the number of bone marrow plasma cells and slowed down further formation of autoantibodies. TACI‐Fc prevented renal damage during a 12‐week treatment period regardless of autoantibody levels, while BAFFR‐Fc did not despite a similar BAFF‐blocking activity in vivo. TACI‐Fc also decreased established plasma cells in a T‐dependent hapten/carrier immunization system better than single inhibitors of BAFF or APRIL, and sometimes better than combined single inhibitors with at least equivalent BAFF and APRIL inhibitory activities. These results indicate that TACI‐Fc can prevent symptoms of renal damage in a mouse model of SLE when BAFFR‐Fc cannot, and point to a plasticity of plasma cells for survival factors. Targeting plasma cells with TACI‐Fc might be beneficial to prevent autoantibody‐mediated damages in SLE.

2. The authors should go through all labels of x and y axes in their diagrams. For example, in Fig. 2B it is not clear whether they documented frequencies or absolute cell numbers. The latter one is absolutely necessary for analyzing the effect on a cell population. The authors should check legends and labeling of all other figures for clarity.

28-Jan-2017
Dear Executive Editor, Thank you for taking a look at our manuscript. We are pleased that you find it interesting. Please find below a point by point answer to your comments: Answer to comment 1 (Provide gating strategies): We have added a Suppl Figure 2 with the gating strategies that we have used to identify CD138+ cells in the spleen and in the bone marrow, and B220+/CD19+ cells in the spleen. We apologize for not having provided this figure in the first place.
Answer to comment 2 (Clarify legends to x-and y-axes): It was unclear to the executive Editor if the legend of the y-axis in Fig  documented frequencies or absolute cell numbers. The answer is that this axis documents absolute cell numbers. We have changed the legend of the axis to "CD138+ PC [millions/spleen]" and changed the figure legend "Cell numbers were determined..." to "Absolute cell numbers were determined...". A similar case applied to Fig. 2D, where we have changed the y-axis from "CD138+ PC in BM [thousands]" to "CD138+ PC [thousands/2 femurs]" and the figure legend to "Same as panel B, but for absolute numbers of CD138+ cells in the bone marrow of 2 femurs ....".
We have added in the figure legends comments to mention whenever we measured absolute cell numbers.
Finally, we have edited the figure legends to follow Journal standards as recommended.
Changes made are highlighted in yellow in the manuscript and in supplemental file (we uploaded them as "Supporting information not for review"). Clean versions were uploaded as "Submission file" and "Supporting information for review". Although the referees have recommended publication, some revisions to your manuscript have been requested. Therefore, I invite you to respond to the comments of the referee 2 and revise your manuscript accordingly.
You should also pay close attention to the editorial comments included below. **In particular, please edit your figure legends to follow Journal standards as outlined in the editorial comments. Failure to do this will result in delays in the re-review process.** If the revision of the paper is expected to take more than three months, please inform the editorial office.
Revisions taking longer than six months may be assessed by new referees to ensure the relevance and timeliness of the data.
Once again, thank you for submitting your manuscript to European Journal of Immunology. We look forward to receiving your revision. This paper address a relevant issue, modulating the BAFF-APRIL system as a therapy for autoimmune diseases. Their results are clear. In this model a soluble receptor blocking BAFF only is less effective than a soluble receptor that blocks both BAFF+APRIL. This is not really surprising. Nevertheless, such data are informative, since anti-BAFF, but atacicept (blocking BAFF+APRIL) is not yet approved in human SLE.
The authors have adequately addressed the issues raized by the reviewers of the original submission.

Reviewer: 2
Comments to the Author In general the study is very well performed and the experiments are clearly described and discussed including possible limitations of the study. The study is clearly of relevance and interest.
There are however a few minor points that should be addressed before publications. P5, line 55 and Figure 1D: How long after depletion was the FACS analysis performed?
In Figure 2E. ANA titres were assumed to stay constant if a mouse had to be euthanized before the conclusion of the experiment. Is that a reasonable assumption and leave out the points that were not actually measured from the graph 2E? Figure 4A/B: Dual lines for statistical significance is confusing. No stats shown on the hTACI-Fc group?
Is there a possible explanation for the production of autoantibodies in the NZB/NZW F1 mice but not in the C57BL/6 mice and would that potentially be relevant for a clinical application of the human equivalent to Apry-1-1?

16-Mar-2017
Point-by-point answer to the comments of reviewers.

Reviewer: 1
Comments to the Author I have got the revised version of this paper for review. This paper addresses a relevant issue, modulating the BAFF-APRIL system as a therapy for autoimmune diseases. Their results are clear. In this model a soluble receptor blocking BAFF only is less effective than a soluble receptor that blocks both BAFF+APRIL. This is not really surprising. Nevertheless, such data are informative, since anti-BAFF, but atacicept (blocking BAFF+APRIL) is not yet approved in human SLE. The authors have adequately addressed the issues raised by the reviewers of the original submission.
Answer: We thank reviewer 1 for her/his positive comments.

Reviewer: 2
Comments to the Author In general the study is very well performed and the experiments are clearly described and discussed including possible limitations of the study. The study is clearly of relevance and interest.
There are however a few minor points that should be addressed before publications. Point 1: Figure 1B: Legend is missing. I assume same legend as in Figure 1C but no mTACI-Fc shown in this panel and not included in the legend.
Answer: The reviewer is right. We have now identified symbols in panel 1B, including mTACI-Fc.
Point 2: P5, line 55 and Figure 1D: How long after depletion was the FACS analysis performed?
Answer: Inhibitors were administered 3 times a week on a Monday/Wednesday/Friday schedule for 12 weeks. At the end of the experiment, mice were sacrificed two days after the last administration of inhibitors. In some cases, this time was reduced to 1 day (for some animal that had to be sacrificed before the end of the experiment). At those time points, BAFF inhibitory activity was still measurable in the circulation (for those inhibitors that inhibit BAFF) (Fig. 1E).
Point 3: In Figure 2E. ANA titres were assumed to stay constant if a mouse had to be euthanized before the conclusion of the experiment. Is that a reasonable assumption and leave out the points that were not actually measured from the graph 2E?
Answer: The reviewer is right: our graph was not optimal. In its updated version, the last measure is shown at the actual time point of measurement and sacrifice is marked by termination of the line with a black circle.
Point 4: Figure 4A/B: Dual lines for statistical significance is confusing. No stats shown on the hTACI-Fc group?
Answer: In a statistical comparison by one way ANOVA, a group very different from others obliterates differences between less dissimilar groups, even if real. We discuss in the manuscript the exceptional behavior of hTACI-Fc in vivo compared to other inhibitors tested (mTACI-Fc, mBAFFR-Fc and Apry1.1). When hTACI-Fc is included in the analysis, nothing else scores significant. However, differences are significant when hTACI-Fc is excluded from the analysis, i.e. when the control is compared only to inhibitors of comparable potency. Similarly, the weak APRIL-inhibitory activity of mTACI-Fc is significant when compared to controls (bottom line of statistical significance in panel B), but not when more potent inhibitors are included in the analysis. This is the reason why we showed dual lines for statistical analysis. We have added the following explanation in the legend: "For panels A and B, only hTACI-Fc is significantly different from all other groups by one way Anova (p<0.001). The line of statistical results that is shown in panel A compares controls to inhibitors of similar potency (i.e excluding the group of hTACI-Fc) by one way Anova. For panel B, inhibitors of medium activity (Apry-1-1, upper line of statistical results) and of weak activity (mTACI-Fc, lower line of statistical results) are independently compared to controls by one way Anova". Point 5: Is there a possible explanation for the production of autoantibodies in the NZB/NZW F1 mice but not in the C57BL/6 mice and would that potentially be relevant for a clinical application of the human equivalent to Apry-1-1?
Answer: The reviewer raises an interesting point. Apry-1-1 is a single chain human antibody fused to the Fc portion of mouse IgG2b. In the presence of adjuvant, this will certainly be immunogenic in non-autoimmune mice also. In NZB/NZW F1, B cells can recognize and neutralize Apry-1-1 in the absence of adjuvant because they are prone to activation. We think that a fully mouse anti-mAPRIL antibody should not be immunogenic, or much less, in NZB/NZW F1 mice, unless anti-idiotype responses would take place, which is possible. Whether a fully human anti-hAPRIL antibody in autoimmunity-prone humans will elicit an anti-drug response is not a trivial question. Maybe, may be not. Time will tell.
We thank reviewer 2 for her/his input.

20-Mar-2017
Dear Dr. Schneider, It is a pleasure to provisionally accept your manuscript entitled "A mouse model of systemic lupus erythematosus responds better to soluble TACI than to soluble BAFFR, correlating with depletion of plasma cells" for publication in the European Journal of Immunology. For final acceptance, please follow the instructions below and return the requested items as soon as possible as we cannot process your manuscript further until all items listed below are dealt with.
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