IL-4 induces CD22 expression to restrain the effector program of self-reactive virtual memory T cells

Parasitic helminths induce the production of interleukin (IL)-4 which causes the expansion of virtual memory CD8+ T cells (TVM), a cell subset contributing to the control of viral coinfection. However, the mechanisms regulating IL-4-dependent TVM activation and expansion during worm infection remain ill defined. We used single-cell RNA sequencing of CD8+ T cells to investigate IL-4-dependent TVM responses upon helminth infection in mice. Gene signature analysis of CD8+ T cells identified a cell cluster marked by CD22, a canonical regulator of B cell activation, as a specific and selective surface marker of IL-4-induced TVM cells. CD22+ TVM were enriched for IFN-? and granzyme A and retained a diverse TCR repertoire, while enriched in CDR3 sequences with features of self-reactivity. Deletion of CD22 expression in CD8+ T cells enhanced TVM responses to helminth infection, indicating that this inhibitory receptor modulates TVM responses. Thus, helminth-induced IL-4 drives the expansion and activation of self-reactive TVM in the periphery that is counter-inhibited by CD22 Overall design: For 10X single cell RNA sequencing, CD8+ T cells were enriched from spleen samples from individual mice of one group, followed by hashtagging and pooling of 1 million cells per mouse sample. Groups consisted in WT or E8i-CreIl4ra-lox/lox BALB/c mice, receiving 3 different treatments (PBS ip injection at day -4 and day -2, IL-4c injection at day -4 and day -2, or infected with 200 L3 larvae of H. polygyrus). For BD Rhapsody sequencing, CD8+ T cells were enriched from mesenteric lymph nodes of 3 E8i-CreIl4ra-lox/lox BALB/c mice at day 15 after infection with 200 L3 larvae of H. polygyrus. After enrichment, CD8+ T cells were stained and FACsorted for CD124+ or CD124- cells. Then, each sorted population was Sample Tagged, and 500,000 cells of the tagged cells were pooled before cell capture. For bulk RNA-seq and TCR-seq, WT BALB/c mice were treated i.p. with PBS or with IL-4c (day -4 and day -2). At day 0, CD8+ T cells were enriched by bead based negative selection followed by immunostaining in order to sort by FACS CD44low naive T cells or CD44highCD49dlow Tvm from PBS-treated mice (n=4); and CD44low naive T cells or CD44highCD49dlowCD22- Tvm CD44highCD49dlowCD22+ Tvm from IL-4c-treated mice (n=4). 200,000 cells were sorted before cDNA library preparation and sequencing.