Splenic red pulp macrophages (RPM) degrade senescent erythrocytes and recycle heme-associated iron. The transcription factor SPI-C is selectively expressed by RPM and is required for their development, but the physiologic stimulus inducing Spic is unknown. Here, we report that Spic also regulated the development of F4/80(+)VCAM1(+) bone marrow macrophages (BMM) and that Spic expression in BMM and RPM development was induced by heme, a metabolite of erythrocyte degradation. Pathologic hemolysis induced loss of RPM and BMM due to excess heme but induced Spic in monocytes to generate new RPM and BMM. Spic expression in monocytes was constitutively inhibited by the transcriptional repressor BACH1. Heme induced proteasome-dependent BACH1 degradation and rapid Spic derepression. Furthermore, cysteine-proline dipeptide motifs in BACH1 that mediate heme-dependent degradation were necessary for Spic induction by heme. These findings are the first example of metabolite-driven differentiation of a tissue-resident macrophage subset and provide new insights into iron homeostasis.

Bcl11a is a transcription factor known to regulate lymphoid and erythroid development. Recent bioinformatic analysis of global gene expression patterns has suggested a role for Bcl11a in the development of dendritic cell (DC) lineages. We tested this hypothesis by analyzing the development of DC and other lineages in Bcl11a (-/-) mice. We found that Bcl11a was required for expression of IL-7 receptor (IL-7R) and Flt3 in early hematopoietic progenitor cells. In addition, we found severely decreased numbers of plasmacytoid dendritic cells (pDCs) in Bcl11a (-/-) fetal livers and in the bone marrow of Bcl11a (-/-) fetal liver chimeras. Moreover, Bcl11a (-/-) cells showed severely impaired in vitro development of Flt3L-derived pDCs and classical DCs (cDCs). In contrast, we found normal in vitro development of DCs from Bcl11a (-/-) fetal liver cells treated with GM-CSF. These results suggest that the persistent cDC development observed in Bcl11a (-/-) fetal liver chimeras reflects derivation from a Bcl11a- and Flt3-independent pathway in vivo.

Despite a clear role in protective immunity, the durability and quality of antibody and memory B cell responses induced by mRNA vaccination, particularly by a 3 rd dose of vaccine, remains unclear. Here, we examined antibody and memory B cell responses in a cohort of individuals sampled longitudinally for ∼9-10 months after the primary 2-dose mRNA vaccine series, as well as for ∼3 months after a 3 rd mRNA vaccine dose. Notably, antibody decay slowed significantly between 6- and 9-months post-primary vaccination, essentially stabilizing at the time of the 3 rd dose. Antibody quality also continued to improve for at least 9 months after primary 2-dose vaccination. Spike- and RBD-specific memory B cells were stable through 9 months post-vaccination with no evidence of decline over time, and ∼40-50% of RBD-specific memory B cells were capable of simultaneously recognizing the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells induced by the first 2 doses of mRNA vaccine were boosted significantly by a 3rd dose and the magnitude of this boosting was similar to memory B cells specific for other variants. Pre-3 rd dose memory B cell frequencies correlated with the increase in neutralizing antibody titers after the 3 rd dose. In contrast, pre-3 rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit reactivation of immunological memory and constrain further antibody boosting by mRNA vaccines. These data provide a deeper understanding of how the quantity and quality of antibody and memory B cell responses change over time and number of antigen exposures. These data also provide insight into potential immune dynamics following recall responses to additional vaccine doses or post-vaccination infections.

The transcription factors Batf3 and IRF8 are required for the development of CD8α(+) conventional dendritic cells (cDCs), but the basis for their actions has remained unclear. Here we identified two progenitor cells positive for the transcription factor Zbtb46 that separately generated CD8α(+) cDCs and CD4(+) cDCs and arose directly from the common DC progenitor (CDP). Irf8 expression in CDPs required prior autoactivation of Irf8 that was dependent on the transcription factor PU.1. Specification of the clonogenic progenitor of CD8α(+) cDCs (the pre-CD8 DC) required IRF8 but not Batf3. However, after specification of pre-CD8 DCs, autoactivation of Irf8 became Batf3 dependent at a CD8α(+) cDC-specific enhancer with multiple transcription factor AP1-IRF composite elements (AICEs) within the Irf8 superenhancer. CDPs from Batf3(-/-) mice that were specified toward development into pre-CD8 DCs failed to complete their development into CD8α(+) cDCs due to decay of Irf8 autoactivation and diverted to the CD4(+) cDC lineage.

Both classical DCs (cDCs) and monocyte-derived DCs (Mo-DCs) are capable of cross-priming CD8(+) T cells in response to cell-associated antigens. We found that Ly-6C(hi)TREML4(-) monocytes can differentiate into Zbtb46(+) Mo-DCs in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) but that Ly-6C(hi)TREML4(+) monocytes were committed to differentiate into Ly-6C(lo)TREML4(+) monocytes. Differentiation of Zbtb46(+) Mo-DCs capable of efficient cross-priming required both GM-CSF and IL-4 and was accompanied by the induction of Batf3 and Irf4. However, monocytes require IRF4, but not BATF3, to differentiate into Zbtb46(+) Mo-DCs capable of cross-priming CD8(+) T cells. Instead, Irf4(-/-) monocytes differentiate into macrophages in response to GM-CSF and IL-4. Thus, cDCs and Mo-DCs require distinct transcriptional programs of differentiation in acquiring the capacity to prime CD8(+) T cells. These differences may be of consideration in the use of therapeutic DC vaccines based on Mo-DCs.

Defense against attaching-and-effacing bacteria requires the sequential generation of interleukin 23 (IL-23) and IL-22 to induce protective mucosal responses. Although CD4(+) and NKp46(+) innate lymphoid cells (ILCs) are the critical source of IL-22 during infection, the precise source of IL-23 is unclear. We used genetic techniques to deplete mice of specific subsets of classical dendritic cells (cDCs) and analyzed immunity to the attaching-and-effacing pathogen Citrobacter rodentium. We found that the signaling receptor Notch2 controlled the terminal stage of cDC differentiation. Notch2-dependent intestinal CD11b(+) cDCs were an obligate source of IL-23 required for survival after infection with C. rodentium, but CD103(+) cDCs dependent on the transcription factor Batf3 were not. Our results demonstrate a nonredundant function for CD11b(+) cDCs in the response to pathogens in vivo.

We examined antibody and memory B cell responses longitudinally for ∼9-10 months after primary 2-dose SARS-CoV-2 mRNA vaccination and 3 months after a 3rd dose. Antibody decay stabilized between 6 and 9 months, and antibody quality continued to improve for at least 9 months after 2-dose vaccination. Spike- and RBD-specific memory B cells remained durable over time, and 40%-50% of RBD-specific memory B cells simultaneously bound the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells were efficiently reactivated by a 3rd dose of wild-type vaccine and correlated with the corresponding increase in neutralizing antibody titers. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit the added protection afforded by repeat short interval boosting. These data provide insight into the quantity and quality of mRNA-vaccine-induced immunity over time through 3 or more antigen exposures.