Peer review of: Cosmc controls B cell homing (2020)

Reviewers' comments: 

Reviewer #1 (B cell biology, microscopy) (Remarks to the Author): 

The manuscript describes a role for the T-synthase folding chaperone Cosmc in regulating B cell 
homing, in particular the entry of B cells into secondary lymphoid organs. While this observation is 
interesting, the manuscript fails to provide mechanistic insights beyond this initial observation and 
lacks stringency, as well as a proper discussion of the findings. For now, it seems somehow unfinished. 
Major concerns: 
The authors do not observe differences in rolling/adhesion behavior, but reduced accumulation of B 
cells in SLOs, so they conclude that Cosmc affects the later phases of endothelial transmigration. This 
should be analyzed more in detail, and molecular mechanisms should be analyzed or at the very least 
discussed. 
A more detailed analysis of the Cosmc deficient mice would be required, and data on homing behavior 
of the main hematopoietic subsets should be included. The paper focuses on the homing effect on B 
cells, but other important functions of are not mentioned, it is not clear whether they were just not 
analyzed. For example, in addition to homing, glycosylation plays an important role for other 
processes related to B cell biology, such as antibody glycosylation. It should be tested whether Cosmc 
is involved in those processes, i.e. is antibody glycosylation changed in the Cosmic knockout mice? 
The glycosylation pattern of antibodies crucially determines their function, and O glycosylation defects 
have been associated with autoimmunity. In that respect it would be interesting to know whether the 
Cosmc deficient mice develop autoimmunity with age. 
The authors find that B cells have a homing defect and are reduced in the spleen, but on the other 
hand the spleen weight is increased. This seems contradictory and should be explained, and other 
main immune cell subsets in the spleen should be analyzed and compared to wt, and the results 
should be discussed. In general, the manuscript would benefit from a more elaborate discussion. 
Another example is in Line 165, where higher expression levels of chemokine receptors were observed, 
which seems counterintuitive given the homing defect. Again, this is not at all discussed. 

Minor issues: 
Line 106: which background factors? 
Line 115: unclear to me, what the previous study is? what is consistent? 
Line 126: It may make more sense to move this paragraph upstream 
Line 134: Why do they look for Thy1.2. in particular? Why do they look in the liver? 
Line 143: Why are there no movies supplied? 
Line 146: How did they determine the sticking and rolling fraction? 
Extended Data Fig. 1a – please invert for better visibility 
Fig. 1b/c description missing (WT/KO)! 
Had problems finding E 
Fig. 1 G in the text it says there is CD19 staining but it’s only written B cells in the legend. Scale bar is 
missing 
Fig. 2 – are the two time points really sufficient? How about (immunizing and) looking at later 
timepoints in the BM? 
Fig. 3 a+b legend explaining symbols of glycosylation would be helpful 
For Extended data figure 4 D very low cell numbers, it would need more cells to support that 
statement 


Reviewer #2 (Glycomics, mass-spec analysis) (Remarks to the Author): 
The manuscript by Cummings and co-workers convincingly shows that B-cell mucin-type elongated O-
glycosylation is important for B cell homing to peripheral lymphoid organs. The manuscript point to 
the fact that the glycosylation of chemokine receptors may play an important role in B cell homing. 
The findings are of major significance as they shed light on the function and organization of 
immunological responses. I have a few comments which the authors may want to consider: 
While the study focuses on B cell homing to peripheral organs, I wonder how the COSMC mutation 
affects B cell development, abundance, lifetime, clearance, as well as B cell function in a broader 
sense. 
Also, the data pointing towards the involvement of the chemokine axis in causing O-glycosylation 
dependent B cell homing appear not fully conclusive, and further experiments may be needed to 
substantiate this. 

Reviewer #3 (Chemotaxis, B cell migration) (Remarks to the Author): 

In the present manuscript Zeng et al. investigate the phenotype of a novel mouse that carries B cell-
restricted deletion of the Cosmc gene. The authors report that B cells show a different pattern in O-
glycosylation in B cells and that homing of these cells is strongly impaired to lymphoid and non-
lymphoid organs. Mechanistically they show that the different interaction steps of homing B cells to 
HEVs are not affected. Instead they provide some preliminary evidence that impaired glycosylation of 
chemokine receptors might be responsible for impaired homing. This study is of clear interest but the 
current version is rather preliminary. 

Major points: 
1) The authors postulate that impaired homing of B cells in due to impaired glycosylation of 
chemokine receptors, in particular CXCR4 and CCR7. However only circumstantial evidence has been 
provided that this is the case. They therefore must show that the glycosylation pattern of these 
chemokine receptors is actually different in Cosmc-deficient B cells but e.g. not in T cells. Further, 
they must show that either binding of the corresponding chemokine(s) to their receptor is affected or 
subsequent receptor signaling is impaired – or both! 
2) The authors should also create mice with specific deletion of Cosmc in T cells and DCs and also 
characterize homing of these immune cell subsets: This is important since it has been shown that 
CCR7 is indispensable for homing of T cells and DCs while CXCR5 is more important for B cell homing. 
However, in the present study the authors demonstrate that Cosmc-/- B cells show unimpaired 
migration to CXCL13, the ligand for CXCR5. 
3) The authors describe a 50% reduction in splenic B cells, but also report splenomegaly in Cosmc-
deficient mice. Which cells contribute to the observed splenomegaly? Furthermore does Cosmc-
deficiency in B cells affect the architecture of the splenic white pulp? (GC-formation, follicle formation, 
MZ, marginal sinus, T cell positioning?) 
4) The authors report that the N-glycosylation pattern is not affected in Cosmc-deficient B cells. 
However, at least to this reviewer, ext. figs 3a and 3b show different patterns of glycosylation. 
5) The authors report a 50% reduction of B cells in the bone marrow. This has to be further 
investigated. Is B cell maturation in the bone marrow affected, or recirculation of mature B cells? 
6) Plasma cells have been shown to take advantage of CXCR4. Is plasma cell/blast homing to bone 
marrow affected in the mice analyzed? 
7) Fig 1J indicates that T cells are missing in PP in Cosmc-deficient mice. Quantitative analysis is 
needed to address whether this is indeed the case. Also, the section plane used for PP analysis is 
unusual. Sections showing FAE in the dome region as well as mucosa and serosa should be provided 

Minor: 
1) Give reference for the statement the the TN antigen is abnormally expressed 
2) Give reference for the Cosmc fl/fl mouse used in this study 
3) More information about COSMC must be provided in the introduction 
4) The authors frequently refer to reduced cell numbers as ‘fold reduction’. This phrase is scientifically 
incorrect. Therefore use e.g. ‘reduced to 5%’ instead of ‘20-fold reduction’