Ssion of PD-L2 at day 50 pi in both resistant C57BL/6 and susceptible BALB/c mice. PD-L2 expression was upregulated by all MF sub-populations of infected BALB/c mice in comparison with naive controls (Figure 4B) whereas infected C57BL/6 mice exhibited substantially lower frequencies of PD-L2+ MFs relative to BALB/c mice (Figure 4B). Indeed, there was negligible detection of PD-L2 expression by the F4/80hi population of C57BL/6 mice at day 50 pi (Figure 4B), regardless of being 80 bone marrow derived (Figure 3B). This gives more evidence that productive integration into the resident niche results in an inability to upregulate PD-L2 in response to helminth infection, constant with recent findings (Gundra et al., 2017). As a result, PD-L2+ cells within the F4/80hi population of infected BALB/c mice supports a model of inefficient integration of bmMF in to the resMF niche in this strain. PD-L2 has particular relevance to L. sigmodontis, exactly where its ligand PD-1 is very expressed on TH2 cells throughout chronic infection of susceptible BALB/c mice. In this setting, PD-1/PD-L2 interaction induces a state of TH2 cell-intrinsic hypo-responsiveness which is characterised by considerably diminished production of TH2 signature cytokines IL-4 and IL-5 (van der Werf et al., 2013). To assess if the enhanced recruitment and subsequent M(IL-4) activation of F4/80lo MF and monocytes was contributing to susceptibility by means of the induction of hypo-responsive TH2 cells, the strength with the immune response was analysed at day 50 pi in each resistant C57BL/6 and susceptible BALB/c mice. Expression of the TH2 master transcription factor GATA3 was substantially higher in CD4+ T cells isolated from the pleural cavity of C57BL/6 mice compared with BALB/c (Figure 4C) even though no substantial difference in expression of IL-4 or IL-5 by GATA3+CD4+ cells was detected (Figure 4C and D). Interestingly even so, there was a substantially greater proportion of IFN-g making CD4+ cells from infected BALB/c mice in comparison with C57BL/6 mice (Figure 4D). Notably, there was a strong good correlation between the percentage of PD-L2+ MF and worm recovery price (Figure 4E).F4/80lo MF and monocytes are detrimental to nematode killingThe information hence far recommended that in BALB/c mice recruitment of bmMF was detrimental to parasite killing. To test this hypothesis we used a monocyte depleting anti-CCR2 antibody to block monocyte recruitment in BALB/c mice prior to the peak of monocyte influx. Anti-CCR2 was administered dailyCampbell et al. eLife 2018;7:e30947. DOI: https://doi.org/10.7554/eLife.eight ofResearch articleImmunologyA***RELM+ (Freq of F4/80hi)80 60 40 20RELM+ (Freq of F4/80lo)80 60 40 20*Naive C57BL/6 Infected C57BL/6 Naive BALB/c Infected BALB/cBL/6 B/c BL/6 B/c Infected NaiveBL/6 B/c BL/6 B/c Naive InfectedB** *PD-L2+ (Freq.1345728-51-9 web of F4/80lo)100 80 60 40 20** **PD-L2+ (Freq.1020065-69-3 Chemscene of Monocytes)one hundred 80 60 40 20PD-L2+ (Freq.PMID:23443926 of F4/80hi)80 60 40 20***BL/6 B/c BL/6 B/c Naive InfectedBL/6 B/c BL/6 B/c Naive InfectedBL/6 B/c BL/6 B/c Naive InfectedCGATA3+ (Freq. of CD4+)**IL-4+ (Freq. of GATA3+CD4+)CD4+ T cells (1X105)***25 20 15 ten 5B/c BL/6 InfectedB/c BL/6 InfectedB/c BL/6 InfectedDIL-5+ (Freq. of GATA3+CD4+)E**PD-L2+ MBL/6 B/c Infected100 80 60 40 20 0 0 10 20 30 40Spearman R = 0.IFN+ (Freq. of CD4+)B/c BL/6 InfectedWorm Recovery RateFigure 4. PD-L2+ MF are associated with susceptibility. Expression of (A) RELMa or (B) PD-L2 by F4/80hi, F4/80lo and/or monocyte populations isolated from naive and L. si.
