The Transcription Factor Bach2 Negatively Regulates Natural Killer Cell Maturation and Function

BTB domain And CNC Homolog 2 (Bach2) is a transcription repressor that actively participates in T and B lymphocyte development, but it is unknown if Bach2 is also involved in the development of innate immune cells, such as natural killer (NK) cells. Here, we followed the expression of Bach2 during NK cell development, finding that it peaked in CD27+CD11b+ cells and decreased upon further maturation. Bach2 expression positively correlated with that of the transcription factor TCF1 and negatively correlated with genes encoding NK effector molecules as well as genes involved in the cell cycle. Bach2-deficient mice showed increased numbers of terminally differentiated NK cells with increased production of granzymes and cytokines. NK cell-mediated control of tumor metastasis was also augmented in the absence of Bach2. Therefore, Bach2 is a key checkpoint protein regulating NK terminal maturation.


Introduction 47
Natural killer (NK) cells are innate lymphoid cells that have spontaneous cytolytic activity 48

against tumor cells and virus-infected cells. The development of NK cells occurs in bone marrow 49
(BM) as well as in secondary lymphoid tissues in both humans and mice. Multipotent We confirmed the transcriptomics data by quantitative PCR (qPCR) analysis performed on 211 sorted splenic NK cells from the Bach2 cKO and control mice respectively (Fig. 2C). We detected 212 a significantly lower expression of genes for example Tcf7, Kit, and Sox6 in Bach2 deficient NK 213 cells. For other genes such as Cd69, Cd62l (Sell), and Ccr7, we did not observe significant 214 differences but they all had a trend of downregulation in NK cells lacking Bach2 expression. The 215 genes we picked with upregulation in RNA-seq data were confirmed to be increased by qPCR 216 analysis. These genes included Gzmb, Klrg1, Ccl5, Klrb1b, Cd39, etc. except for Cx3cr1 which 217 did not reach significance even though displaying a trend of upregulation. Consistent with their 218 transcription level, the protein encoded by the genes was also revealed to be changed caused by 219 Bach2-deficiency (Fig. 2D). TCF1, Kit, CD69, and CD62L were all shown to be decreased at the 220 protein level. Although Tcf7 had a dramatic decrease transcriptionally, its protein was only 221 slightly downregulated. On the other hand, CD39, KLRG1, CX3CR1, and Granzyme B were 222 elevated followed by their changes at the RNA level. Thus, we confirmed that our RNA-seq data 223 were reliable to reflect the impact of Bach2 in regulating the expression of various genes in NK 224

cells. 225 226
Next, we asked how Bach2 participated in NK cell biology. It was reported that the enforced 227 Bach2 expression in exhausted CD8 + T cells resulted in the cells becoming exclusively stem-like 228 precursor exhausted CD8 + T cells, preventing their further differentiation into terminal exhausted 229 CD8 + T cells (Yao et al., 2021). We used gene-set enrichment analysis (GSEA) to determine the 230 effect of Bach2-deficiency by comparing against the gene signatures of stem-like CD8 + T cells 231 and terminally differentiated effector-like CD8 + T cells. We found genes upregulated in NK cells 232 induced by Bach2-deficiency positively correlated with terminal differentiated effector-like gene 233 signatures (Fig. 2E) whereas the genes downregulated showed a stem-like signature (Fig. 2F). 234 Another comparison was performed using GSEA analysis and we found Bach2-sufficient NK 235 cells displayed a naïve CD8 + T cell signature (Fig. S2E) while Bach2-deficient NK cells 236 resembled activated effector CD8 + T cells (Fig. S2F). These data suggested that Bach2 237 expression suppressed terminal differentiation of NK cells by repressing many effector genes. 238 239

Bach2 restrained terminal maturation of NK cells 240
Based on the expression pattern of Bach2 in NK cells as well as RNA-seq and confirmatory 241 qPCR data in Bach2-deficient cells, we tested whether Bach2 indeed restricted the terminal 242 maturation of the immature NK cells. To address this, we analyzed the maturation profile of the 243 NK cells compared between Bach2 cKO and control mice. In the bone marrow, we did not observe 244 a significant difference between control and Bach2 cKO mice regarding the frequency of the 245 CD27 + cells or CD11b + cells (Fig. 3A). However, an altered maturation profile of NK cells was 246 detected in the spleens of Bach2 cKO mice, i.e., there were more cells with mature NK cell 247 phenotype (CD11b + ) and fewer NK cells at the immature DP stage as compared to control mice 248 Bach2 cKO mice, we found the frequency of CD27 + NK cells, specifically the DP (CD27 + CD11b + ) 255 NK cells, was significantly reduced while the frequency of CD11b + NK cells was markedly 256 increased in both bone marrow (Fig. 3D) and spleen (Fig. 3E) in Bach2 -/mice. In agreement, the 257 expression of KLRG1 was upregulated and we also found more cells (~80%) expressing KLRG1 258 in Bach2-deficient mice compared to Bach2-sufficient mice (~50%) (Fig. 3F). Taken together, 259 NK cells were skewed towards the most mature NK cells in Bach2-deficient mice as compared 260 to Bach2-sufficient mice, with a concomitant decrease in the immature NK cells. 261

B16 tumor growth and metastasis is controlled by NK cells with Bach2-deficiency 263
Since differential gene analysis showed many effector molecules, especially cytotoxic genes 264 were increased with Bach2-deficiency, we evaluated whether Bach2-deficiency resulted in 265 changes in the rejection of target cells. We asked whether the increased representation of more 266 mature NK cells due to Bach2-deficiency would result in better control of tumor metastases in 267 vivo. We assessed the role of Bach2 in B16F10 metastasis. In tumor metastasis studies, 2.5×10 5 268 B16F10 cells were intravenously injected into Bach2 cKO or control mice and tumor metastases 269 were evaluated two weeks later by counting the black colonies formed in lungs ( The genes we detected with differential expression patterns correlated very well with the gene 317 signatures observed in CD8 T cells, their cytotoxic counterparts in the adaptive immune system. 318 One of the genes, Tcf7, was particularly interesting and may be important for the mechanism of 319 the regulation of NK development by Bach2. Tcf7 (encoding TCF1) is highly expressed in naïve 320