Plasmodium-specific atypical memory B cells upregulate markers of activation and co-stimulatory molecules indicative of cellular interaction with T cells in response to a febrile malaria infection in children and adults.

In naturally acquired immunity to malaria, antibodies that reliably protect are only acquired after years of repeated Plasmodium falciparum (Pf) infections. We have shown in Mali that this inefficiency in humoral immunity to malaria is associated with a large expansion of CD21loCD27- 'atypical' B cells (atBCs). The function of atBCs remains elusive, as they exhibit altered B cell receptor (BCR) signaling and when activated fail to secrete cytokines and antibodies. We set out to study the participation and function of malaria-specific atBCs during the immune response to a febrile malaria infection. Here, we show in a Mali cohort spanning infants to adults, that atBCs frequencies within the malaria and influenza-specific MBC compartment are comparable to the overall atBC frequency, showing that atBCs are not predominantly expanded within Pf-specific B cells. We performed a transcriptional analysis of Pf- and influenza-specific atBCs, actBC and MBCs and show that CXCL16, IL-6, IL-10, TNFa, IL12R and IL12R are uniquely upregulated in Pf-specific atBCs. Comparative transcriptomic and flow cytometric analysis before and after an acute malaria infection showed that after malaria, Pf-specific atBCs are transcriptionally most like the actBC subset, sharing many common features, such as activation of intracellular signaling pathways and expression of high levels of T-bet, FcRL5, Ki67 and CD86. Unique to Pf-specific atBCs was an upregulation of IL-5RA, CD38, CXCR3, TLR7, 9 and 10 after a febrile malaria episode. Our data shows that atBCs exhibit a surface marker profile that could facilitate cellular interaction with T cells in secondary lymphoid tissues. Overall design: Using B cell probes as previously described (Hopp et al., 2021), malaria- and influenza-specific B cells were detected in ex vivo flow cytometry of cryopreserved PBMCs obtained from children residing in the malaria endemic village of Kalifabougou, Mali, a study site that has previously been described in detail (Tran et al., 2013). Malaria transmission in Mali is highly seasonal and PBMCs were collected at the end of the 6-month dry season in May (healthy baseline) and one week after treatment (convalescence) of the first febrile malaria episode in the ensuing 6-month malaria season. To glean insight into the function of the atypical B cell lineage, we performed a transcriptomic analysis of antigen-specific B cell subsets. Pf- and HA- specific IgD- CD10- B cells were bulk sorted from 16 Malian malaria-exposed individuals, into atBC, actBC and MBC subsets at healthy baseline from malaria. Transcriptome libraries were generated from a total of 73 samples using the SMART-Seq v4 Ultra low Input RNA kit (Takara). To understand how atBCs functionally respond to an acute febrile malaria episode, we performed transcriptomic analysis of antigen-specific B cell subsets obtained from the same 16 donors after their first acute febrile malaria episode that followed the healthy baseline timepoint of the previous analysis. Transcriptome libraries were generated from 91 samples.