Germinal center B cells that acquire nuclear proteins are specifically suppressed by follicular regulatory T cells
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eLife assessment
It is well known that Tfr cells have the capacity to preferentially suppress autoimmune antibody responses, but it is not known why such specificity exists. This important work provides new information as to how self-reactive antibody responses are regulated and has significant implications to the fields of autoimmunity and vaccine design. The overall experimental designs and the data quality are largely convincing, but the authors should include more careful controls.
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Abstract
Follicular regulatory T cells (Tfr) restrict development of autoantibodies and autoimmunity while supporting high-affinity foreign antigen-specific humoral response. However, whether Tfr can directly repress germinal center (GC) B cells that acquire autoantigens is unclear. Moreover, TCR specificity of Tfr to self-antigens is not known. Our study suggests that nuclear proteins contain antigens specific to Tfr. Targeting of these proteins to antigen-specific B cells in mice triggers rapid accumulation of Tfr with immunosuppressive characteristics. Tfr then exert negative regulation of GC B cells with predominant inhibition of the nuclear protein-acquiring GC B cells, suggesting an important role of direct cognate Tfr-GC B cells interactions for the control of effector B cell response.
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Author Response
Reviewer #2 (Public Review):
In this study, The authors developed a mouse model to specifically investigate whether GC B cells that present nuclear protein (NucPr) could be specifically suppressed by Tfr cells. Most current mouse models that have been used in investigating Tfr functions are based on the overall readout of autoantibody production in the scenario of loss-of-function of Tfr cells. The proposed model of gain-of-function of Tfr cells is novel and valuable.
The authors mainly compared two boosting immunizations by Strepatividin (SA) alone or SA-conjugated with nuclear proteins (SA-NucPr) and demonstrated SA-NucPr boosting immunization was able to expand Tfr cells, suppress overall and SA-specific GC/memory/plasma cell responses. The results are mostly convincing.
One major concern is the conditions and …
Author Response
Reviewer #2 (Public Review):
In this study, The authors developed a mouse model to specifically investigate whether GC B cells that present nuclear protein (NucPr) could be specifically suppressed by Tfr cells. Most current mouse models that have been used in investigating Tfr functions are based on the overall readout of autoantibody production in the scenario of loss-of-function of Tfr cells. The proposed model of gain-of-function of Tfr cells is novel and valuable.
The authors mainly compared two boosting immunizations by Strepatividin (SA) alone or SA-conjugated with nuclear proteins (SA-NucPr) and demonstrated SA-NucPr boosting immunization was able to expand Tfr cells, suppress overall and SA-specific GC/memory/plasma cell responses. The results are mostly convincing.
One major concern is the conditions and controls used in the study. The control group (SA boosting immunization) would have enhanced T and B cell responses by this boosting. Unfortunately, there was no non-boosting control group so the level was unclear. It is therefore to strictly match such boosting condition in the SA-NucPr group. Notably, both SA and SA-NucPr were used at 10ug for boosting immunization. Considering NucPr were comparable or much larger (Nucleosome, about 200KDa) than SA (about 60KDa), the dose of SA in the SA-NucPr group was far less than that in the SA group. Due to this cavity, it is difficult to judge the difference between two groups was due to less SA boosting immunization or NucPr-induced Tfr function. This was a fundamental issue weakens the conclusion.
The single cell analyses clearly demonstrated the expansion of Tfr clones. It remains unclear why other Treg populations other than Tfr cells were not expanded? The Treg cells in the CXCR5intPD-1int population were recently activated and should be able to respond to the boosting immunization. On an alternative explanation, the changes in Tfr cells could be indirectly driven by the changes in Tfh cells. For example, Tfh can produce IL-21 and restrict Tfr expansion (Jandl C, et al.2017). This could be the case of the reduction in Tfr cells in the SA-OVA group as compared to the SA group.
As the reviewer, we were surprised not to detect significant increase in the levels of CXCR5intPD-1int Tregs in the original experiment after the boosting with SA-NucPrs(Fig.1). Our interpretation of this result was that the fraction of NucPr-specific CXCR5intPD-1int Tregs was small as compared to the total CXCR5intPD-1int Tregs and proliferation of this small fraction of cells would not be detectable by flow cytometry analysis of the total CXCR5intPD-1int Tregs numbers. Alternatively, the observed rapid accumulation of Tfrs was due to proliferation of the NucPr-specific Tfrs that may be abundant after a standard immunization with foreign antigen.
In single cell analysis we have used only presorted CXCR5highPD1high follicular T cells so majority of CXCR5intPD-1int Treg population was excluded from the analysis.
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eLife assessment
It is well known that Tfr cells have the capacity to preferentially suppress autoimmune antibody responses, but it is not known why such specificity exists. This important work provides new information as to how self-reactive antibody responses are regulated and has significant implications to the fields of autoimmunity and vaccine design. The overall experimental designs and the data quality are largely convincing, but the authors should include more careful controls.
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Reviewer #1 (Public Review):
Autoantibodies to nuclear proteins are commonly associated with autoimmune conditions. Since their discovery, several reports have suggested that T-follicular regulatory cells (Tfr) Tfr cells have the capacity to preferentially suppress autoimmune antibody responses. Tfr have a TCR repertoire strongly skewed to self-antigens and in this report Ke et al. probe the idea that Tfr directly recognize nuclear proteins and inhibit nuclear protein specific B-cells. They find that vaccination of mice with an ongoing GC reaction to a foreign antigen using nuclear proteins causes expansion of Tfr and a Tfr dependent inhibition of the germinal center. Overall, this is a well written paper that significantly advances the idea that Tfr can control autoreactive B-cells in a selective manner. Most experiments are …
Reviewer #1 (Public Review):
Autoantibodies to nuclear proteins are commonly associated with autoimmune conditions. Since their discovery, several reports have suggested that T-follicular regulatory cells (Tfr) Tfr cells have the capacity to preferentially suppress autoimmune antibody responses. Tfr have a TCR repertoire strongly skewed to self-antigens and in this report Ke et al. probe the idea that Tfr directly recognize nuclear proteins and inhibit nuclear protein specific B-cells. They find that vaccination of mice with an ongoing GC reaction to a foreign antigen using nuclear proteins causes expansion of Tfr and a Tfr dependent inhibition of the germinal center. Overall, this is a well written paper that significantly advances the idea that Tfr can control autoreactive B-cells in a selective manner. Most experiments are convincing. Some of the novel methods regarding the use of nuclear proteins during sequential vaccinations in mice or Tfr-B-cell doublet formation will be of interest to members of the same fields.
A primary weakness of the paper is that despite detailed analysis of cells involved in antibody production, there is very little analysis of the antibodies themselves. Particularly when Tfr deficient mice are used in figure 5 analysis of both anti-SA and anti-NucPr antibodies between the Tfr cKO and other groups would significantly advance the findings.
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Reviewer #2 (Public Review):
In this study, the authors developed a mouse model to specifically investigate whether GC B cells that present nuclear protein (NucPr) could be specifically suppressed by Tfr cells. Most current mouse models that have been used in investigating Tfr functions are based on the overall readout of autoantibody production in the scenario of loss-of-function of Tfr cells. The proposed model of gain-of-function of Tfr cells is novel and valuable.
The authors mainly compared two boosting immunizations by Strepatividin (SA) alone or SA-conjugated with nuclear proteins (SA-NucPr) and demonstrated SA-NucPr boosting immunization was able to expand Tfr cells, suppress overall and SA-specific GC/memory/plasma cell responses. The results are mostly convincing.
One major concern is the conditions and controls used in the …
Reviewer #2 (Public Review):
In this study, the authors developed a mouse model to specifically investigate whether GC B cells that present nuclear protein (NucPr) could be specifically suppressed by Tfr cells. Most current mouse models that have been used in investigating Tfr functions are based on the overall readout of autoantibody production in the scenario of loss-of-function of Tfr cells. The proposed model of gain-of-function of Tfr cells is novel and valuable.
The authors mainly compared two boosting immunizations by Strepatividin (SA) alone or SA-conjugated with nuclear proteins (SA-NucPr) and demonstrated SA-NucPr boosting immunization was able to expand Tfr cells, suppress overall and SA-specific GC/memory/plasma cell responses. The results are mostly convincing.
One major concern is the conditions and controls used in the study. The control group (SA boosting immunization) would have enhanced T and B cell responses by this boosting. Unfortunately, there was no non-boosting control group so the level was unclear. It is therefore to strictly match such boosting condition in the SA-NucPr group. Notably, both SA and SA-NucPr were used at 10ug for boosting immunization. Considering NucPr were comparable or much larger (Nucleosome, about 200KDa) than SA (about 60KDa), the dose of SA in the SA-NucPr group was far less than that in the SA group. Due to this cavity, it is difficult to judge the difference between two groups was due to less SA boosting immunization or NucPr-induced Tfr function. This was a fundamental issue weakens the conclusion.
The single cell analyses clearly demonstrated the expansion of Tfr clones. It remains unclear why other Treg populations other than Tfr cells were not expanded? The Treg cells in the CXCR5intPD-1int population were recently activated and should be able to respond to the boosting immunization. On an alternative explanation, the changes in Tfr cells could be indirectly driven by the changes in Tfh cells. For example, Tfh can produce IL-21 and restrict Tfr expansion (Jandl C, et al.2017). This could be the case of the reduction in Tfr cells in the SA-OVA group as compared to the SA group.
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