Single Cell Analysis of B Cell Responses to Malaria

Abstract

Every day, thousands of people from primarily developing nations are afflicted by diseases with no currently effective vaccines, such as Malaria and AIDs. Furthermore, the COVID19 pandemic has brought a stark reminder to the developed world of the impact that novel infectious diseases can have on our lives. There is therefore a vital need for new vaccination technologies and strategies to combat these ever-present threats. However, before these technologies can be designed, a more fundamental understanding of many aspects of the immune system is required. In particular, advances in technologies such as single cell RNA sequencing are allowing scientists to studying many of these aspects in ways never attempted before.

It is known that the generation of a functional germinal centre (GC) to produce affinity matured long lived plasma cells (LLPCS) and memory B cells (MBCS) is crucial for the formation of life-long immune memory. However, there is still relative controversy over what factors influences the generation of these different B cell fates. Utilising high throughput sequencing to identify B cell clones via their heavy chain complementarity-determining region 3 (CDR3), we observed that the same clone could be found in multiple cell fates. To test whether BCR affinity is important for these processes, we made use of a mouse model enriched for populations of B cells with varying affinities for the central repeat region of the Plasmodium circumsporozoite protein. Following immunisation, we saw no difference in the affinity of cells that become either PBs, GC B cells, or GC-independent MBCs. We identified a single mutation that led to a dramatic increase in BCR affinity. Using single cell RNA sequence (scRNAseq), we observed that this mutation was not enriched in precursors to long lived plasma cells found within the early GC. However, this mutation was underrepresented in a population of precursor memory cells. Thus, we show that BCR affinity does not appear to influence the development of either the early PBs, or affinity matured LLPCs within the GC. However lower affinities do appear to favour the development of MBCs at multiple stages of the immune response.

Furthermore, the full diversity of the circulating MBCs following activation is also relatively unknown, particularly in humans. We used scRNAseq approaches to examine the diversity of both antigen-specific B cells and total B cells in healthy subjects and malaria-exposed individuals. This analysis revealed two B cell lineages: a classical lineage of activated and resting memory B cells, and a separate alternative lineage which includes previously described atypical B cells. Surprisingly, given that atypical cells have previously been associated with disease states, the alternative lineage was common in both malaria-exposed individuals and healthy controls. Using barcoded antibodies in conjunction with our transcriptomic data, we found that alternative lineage cells are generally characterised by higher expression of CD11c, while some populations additionally express CXCR3. To examine the dynamics of alternative lineage cells after primary and recall immunization we tracked circumsporozoite protein specific B cells after plasmodium parasite vaccination in malaria naive volunteers. We found that alternative lineage cells were primed after primary vaccination and responded to booster vaccination. Finally, we showed that with repeated antigen exposure, alternative lineage cells adopted an increasingly atypical phenotype. The data highlight that alternative lineage cells are a normal component of vaccine responses but that continued antigen exposure drives these cells to adopt an atypical phenotype. Show more