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Natural-Killer-like B Cells Function as a Separate Subset of Innate B Cells
Immunity
Letter Natural-Killer-like B Cells Function as a Separate Subset of Innate B Cells Shuo Wang,1,* Pengyan Xia,1 and Zusen Fan1,2,* 1CAS Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 2University of Chinese Academy of Sciences, Beijing 100049, China *Correspondence: [email protected] (S.W.), [email protected] (Z.F.) http://dx.doi.org/10.1016/j.immuni.2017.07.023
We appreciate the comments made by Vivier and colleagues on our findings of natural-killer-like (NKB) cells. We identified a separate subpopulation of innate B cells (called NKB cells) that have CD19+NK1.1+ signature markers and mainly reside in the spleen and mesenteric lymph nodes (MLNs) (Wang et al., 2016). We found that NKB cells harbor a unique identity that is distinct from those of NK and B cells. NKB cells can produce large amounts of interleukin-18 (IL-18) and IL-12 at an early phase of infection and consequently activate type 1 innate lymphoid cells (ILC1s) and NK cells to initiate innate immunity against invading microorganisms. Of note, our results revealed the Lin CD122+CD19+NK1.1+ cell population in the spleen is the NKB precursor (NKBP) that gives rise to mature NKB cells. According to our findings, NKB cells, acting as a separate subset of innate B cells, play a critical role in the early stage of innate immune responses. In their letter, Vivier and colleagues assume that the acquisition of CD19+ NK1.1+ NKB cells might be caused by non-specific binding of NK1.1 and NKp46 antibodies. In order to determine the specificities of these antibodies, we utilized an isotype immunoglobulin (Ig) (APC-conjugated mouse IgG2a, kappa) for the NK1.1 antibody (PK136) as a staining control. We observed that the PK136 antibody detected its respective NK1.1 antigen on CD19+NK1.1+ NKB cells of mouse spleens and MLNs, and these NKB cells were positively stained with both antibodies against NKp46 and DX5 molecules (Figure S1A). However, the control isotype IgG2a was not detectable on CD19+ cells (Figure S1A). This isotype IgG2a control addressed the possibility that the Fc domain of PK136 was nonspecifically binding to CD19+ cells. But
it was also possible that the non-specific binding could be contributed by the Fab’2 domain. To further verify whether NK1.1 staining was non-specific on NKB cells, we conduced similar staining on splenic NKB cells from BALB/c and C3H mouse strains that do not express NK1.1 antigens. As expected, PK136 antibody staining was virtually negative in BALB/ c and C3H mice (Figure S1B). In contrast to Vivier and colleagues’ comments, the PK136 antibody could detect NK1.1 antigen in BALB/c mice in a manner similar to that in C57BL/6 mice, possibly as a result of different batches of PK136 antibody. In addition, Vivier’s group also demonstrated that CD19+NK1.1+ cells were detectable in primary and secondary organs other than mouse spleens. They showed that CD19+NK1.1+ cells were mostly detected in the spleen and blood of C57BL/6 mice, whereas we found that CD19+NK1.1+ NKB cells were mainly detected in the spleens and MLNs of C57BL/6 mice (Wang et al., 2016). In addition, we isolated NKB cells from the spleens of C57BL/6 mice and analyzed the mRNA expression levels of Klrb1c (encoding NK1.1 antigen) and Ncr1 (encoding NKp46 molecule). We noticed that, similarly to NK cells, NKB cells substantially expressed high amounts of Klrb1c and Ncr1, whereas these two genes were undetectable in conventional B cells (Figure S1C). Combined with our above staining results, these data suggest that NKB cells constitutively express Klrb1c and Ncr1 and present their respective proteins on the cell surface. Vivier and colleagues used Ncr1driven Cre models to test the expression of Ncr1. They found that CD19+ NK1.1+ NKB cells were negative for NKp46 expression. The non-expression
of NKp46 might be due to the inefficiency of Cre expression in NKB cells. The same concern of Cre efficiency could also be applicable to other genetic mouse models. In addition, Mcl1 is required for the development and maintenance of NKp46+ NKs. However, it might not be the case for NKB cells. In fact, unlike other cell lineages, NKB cells harbor unique fate-decision transcription factors that could specifically drive their progenitors to differentiate into mature NKB cells. Indeed, we found that NKB cells are derived from proB cells of the bone marrow and that NKBPs are the precursors to NKB cells. What and how transcription factors drive proB cells to differentiate into NKBPs and mature NKB cells still need to be further determined. We observed that NKB cells are unable to produce IgM with liposaccharide (LPS) stimulation via ELISA. Using flow cytometry, Vivier and colleagues found that CD19+NK1.1+ NKB cells could differentiate into CD138+Blimp+ plasmablasts after LPS treatment. These discrepancies might be due to different detection methods. In addition, Vivier’s group found that CD19+NK1.1+ and CD19+NKp46+ cells in MD4 transgenic mice were extremely rare in peripheral blood. Therefore, they proposed that CD19+NK1.1+ and CD19+NKp46+ cells are derived from the binding of these monoclonal antibodies to a subset of B cell receptors (BCRs) present in the normal polyclonal B cell repertoire. However, we noticed that NKB cells were undetectable in the peripheral blood of C57BL/6 mice. Innate B cells include B1 (B1a and B1b) cells and marginal-zone B cells, as well as some newly identified B cell subsets (Bendelac et al., 2001; Cerutti et al., 2013). They express a limited diversity of germline-encoded BCRs and are promptly
Immunity 47, August 15, 2017 ª 2017 Elsevier Inc. 201
Immunity
Letter activated upon challenge with innate stimuli. In addition, we observed that NKB cells exhibit a non-Gaussian distribution of CDR3 length, suggesting that NKB cells display a restricted BCR repertoire. Collectively, we conclude that NKB cells are a separate subset of innate B cells and are distinct from conventional B cells. In summary, we believe that our results indicate that the monoclonal antibodies we used to identify NKB cells are actually specific. Furthermore, NKB cells express high amounts of
202 Immunity 47, August 15, 2017
Klrb1c and Ncr1 and their respective proteins on the cell surface. In addition, NKB cells have their own signature features that are distinct from those of NK and conventional B cells. Upon microbial challenge, NKB cells rapidly secrete large amounts of cytokines to prime NK cells and ILCs, triggering innate immune responses against microbial infections. We are still focusing on the mechanisms through which NKB cells are developed and/or maintained in physiological and pathological settings.
SUPPLEMENTAL INFORMATION Supplemental Information includes one figure and can be found with this article online at http://dx. doi.org/10.1016/j.immuni.2017.07.023. REFERENCES Bendelac, A., Bonneville, M., and Kearney, J.F. (2001). Nat. Rev. Immunol. 1, 177–186. Cerutti, A., Cols, M., and Puga, I. (2013). Nat. Rev. Immunol. 13, 118–132. Wang, S., Xia, P., Chen, Y., Huang, G., Xiong, Z., Liu, J., Li, C., Ye, B., Du, Y., and Fan, Z. (2016). Immunity 45, 131–144.
Letter Natural-Killer-like B Cells Function as a Separate Subset of Innate B Cells Shuo Wang,1,* Pengyan Xia,1 and Zusen Fan1,2,* 1CAS Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 2University of Chinese Academy of Sciences, Beijing 100049, China *Correspondence: [email protected] (S.W.), [email protected] (Z.F.) http://dx.doi.org/10.1016/j.immuni.2017.07.023
We appreciate the comments made by Vivier and colleagues on our findings of natural-killer-like (NKB) cells. We identified a separate subpopulation of innate B cells (called NKB cells) that have CD19+NK1.1+ signature markers and mainly reside in the spleen and mesenteric lymph nodes (MLNs) (Wang et al., 2016). We found that NKB cells harbor a unique identity that is distinct from those of NK and B cells. NKB cells can produce large amounts of interleukin-18 (IL-18) and IL-12 at an early phase of infection and consequently activate type 1 innate lymphoid cells (ILC1s) and NK cells to initiate innate immunity against invading microorganisms. Of note, our results revealed the Lin CD122+CD19+NK1.1+ cell population in the spleen is the NKB precursor (NKBP) that gives rise to mature NKB cells. According to our findings, NKB cells, acting as a separate subset of innate B cells, play a critical role in the early stage of innate immune responses. In their letter, Vivier and colleagues assume that the acquisition of CD19+ NK1.1+ NKB cells might be caused by non-specific binding of NK1.1 and NKp46 antibodies. In order to determine the specificities of these antibodies, we utilized an isotype immunoglobulin (Ig) (APC-conjugated mouse IgG2a, kappa) for the NK1.1 antibody (PK136) as a staining control. We observed that the PK136 antibody detected its respective NK1.1 antigen on CD19+NK1.1+ NKB cells of mouse spleens and MLNs, and these NKB cells were positively stained with both antibodies against NKp46 and DX5 molecules (Figure S1A). However, the control isotype IgG2a was not detectable on CD19+ cells (Figure S1A). This isotype IgG2a control addressed the possibility that the Fc domain of PK136 was nonspecifically binding to CD19+ cells. But
it was also possible that the non-specific binding could be contributed by the Fab’2 domain. To further verify whether NK1.1 staining was non-specific on NKB cells, we conduced similar staining on splenic NKB cells from BALB/c and C3H mouse strains that do not express NK1.1 antigens. As expected, PK136 antibody staining was virtually negative in BALB/ c and C3H mice (Figure S1B). In contrast to Vivier and colleagues’ comments, the PK136 antibody could detect NK1.1 antigen in BALB/c mice in a manner similar to that in C57BL/6 mice, possibly as a result of different batches of PK136 antibody. In addition, Vivier’s group also demonstrated that CD19+NK1.1+ cells were detectable in primary and secondary organs other than mouse spleens. They showed that CD19+NK1.1+ cells were mostly detected in the spleen and blood of C57BL/6 mice, whereas we found that CD19+NK1.1+ NKB cells were mainly detected in the spleens and MLNs of C57BL/6 mice (Wang et al., 2016). In addition, we isolated NKB cells from the spleens of C57BL/6 mice and analyzed the mRNA expression levels of Klrb1c (encoding NK1.1 antigen) and Ncr1 (encoding NKp46 molecule). We noticed that, similarly to NK cells, NKB cells substantially expressed high amounts of Klrb1c and Ncr1, whereas these two genes were undetectable in conventional B cells (Figure S1C). Combined with our above staining results, these data suggest that NKB cells constitutively express Klrb1c and Ncr1 and present their respective proteins on the cell surface. Vivier and colleagues used Ncr1driven Cre models to test the expression of Ncr1. They found that CD19+ NK1.1+ NKB cells were negative for NKp46 expression. The non-expression
of NKp46 might be due to the inefficiency of Cre expression in NKB cells. The same concern of Cre efficiency could also be applicable to other genetic mouse models. In addition, Mcl1 is required for the development and maintenance of NKp46+ NKs. However, it might not be the case for NKB cells. In fact, unlike other cell lineages, NKB cells harbor unique fate-decision transcription factors that could specifically drive their progenitors to differentiate into mature NKB cells. Indeed, we found that NKB cells are derived from proB cells of the bone marrow and that NKBPs are the precursors to NKB cells. What and how transcription factors drive proB cells to differentiate into NKBPs and mature NKB cells still need to be further determined. We observed that NKB cells are unable to produce IgM with liposaccharide (LPS) stimulation via ELISA. Using flow cytometry, Vivier and colleagues found that CD19+NK1.1+ NKB cells could differentiate into CD138+Blimp+ plasmablasts after LPS treatment. These discrepancies might be due to different detection methods. In addition, Vivier’s group found that CD19+NK1.1+ and CD19+NKp46+ cells in MD4 transgenic mice were extremely rare in peripheral blood. Therefore, they proposed that CD19+NK1.1+ and CD19+NKp46+ cells are derived from the binding of these monoclonal antibodies to a subset of B cell receptors (BCRs) present in the normal polyclonal B cell repertoire. However, we noticed that NKB cells were undetectable in the peripheral blood of C57BL/6 mice. Innate B cells include B1 (B1a and B1b) cells and marginal-zone B cells, as well as some newly identified B cell subsets (Bendelac et al., 2001; Cerutti et al., 2013). They express a limited diversity of germline-encoded BCRs and are promptly
Immunity 47, August 15, 2017 ª 2017 Elsevier Inc. 201
Immunity
Letter activated upon challenge with innate stimuli. In addition, we observed that NKB cells exhibit a non-Gaussian distribution of CDR3 length, suggesting that NKB cells display a restricted BCR repertoire. Collectively, we conclude that NKB cells are a separate subset of innate B cells and are distinct from conventional B cells. In summary, we believe that our results indicate that the monoclonal antibodies we used to identify NKB cells are actually specific. Furthermore, NKB cells express high amounts of
202 Immunity 47, August 15, 2017
Klrb1c and Ncr1 and their respective proteins on the cell surface. In addition, NKB cells have their own signature features that are distinct from those of NK and conventional B cells. Upon microbial challenge, NKB cells rapidly secrete large amounts of cytokines to prime NK cells and ILCs, triggering innate immune responses against microbial infections. We are still focusing on the mechanisms through which NKB cells are developed and/or maintained in physiological and pathological settings.
SUPPLEMENTAL INFORMATION Supplemental Information includes one figure and can be found with this article online at http://dx. doi.org/10.1016/j.immuni.2017.07.023. REFERENCES Bendelac, A., Bonneville, M., and Kearney, J.F. (2001). Nat. Rev. Immunol. 1, 177–186. Cerutti, A., Cols, M., and Puga, I. (2013). Nat. Rev. Immunol. 13, 118–132. Wang, S., Xia, P., Chen, Y., Huang, G., Xiong, Z., Liu, J., Li, C., Ye, B., Du, Y., and Fan, Z. (2016). Immunity 45, 131–144.