Different immunomodulatory role of Ly6Ghi and Ly6Gint neutrophils during antiviral antibody therapy

Antiviral monoclonal antibodies (mAbs) can generate protective immunity through immune complexes (IC)-Fc{gamma}Rs interactions. We have shown the essential role of neutrophils in mAb-induced immunity of retrovirus-infected mice. Using this model, here we addressed how viral infection, with or without mAb therapy, affects neutrophils functional activation. We found that neutrophils activated by viral ICs secreted high levels of chemokines able to recruit monocytes and neutrophils themselves. Moreover, inflammatory cytokines potentiated chemokines and cytokines release by IC-activated cells and induced Fc{gamma}Rs upregulation. Similarly, infection and mAb-treatment upregulated Fc{gamma}Rs expression on neutrophils and enhanced their cytokines and chemokines secretion. Fc{gamma}Rs upregulation allowed to identify in vivo two splenic neutrophils subpopulations with distinct phenotypic and functional properties that differentially and sequentially collaborate with inflammatory monocytes to induce Th1-type responses in mAb-treated mice. Our work provides novel findings on the heterogeneity and the immunomodulatory role of neutrophils in the enhancement of immune responses upon antiviral mAb therapy.

The copyright holder for this preprint this version posted April 24, 2020.     activation was evaluated by measuring the CD86 co-stimulatory molecule expression on the cell (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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Virus-and ICs induced a weak secretion of most of the 12 cytokines analyzed, except for IL6 and 173 TNFa. However, no significant differences between unstimulated monocytes versus virus-or IC-174 stimulated monocytes were detected (Supplemental Figure 2A). This contrasted with high level 175 secretion of IL-6, TNFa and IFNg observed upon LPS stimulation (Supplemental Figures 2B-2D).

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The quantification of the chemokine release by activated monocytes showed some interesting 177 similarities with neutrophils. Following virus or ICs stimulation, monocytes produced CXCL5, 178 CXCL1 and CCL2, with no significant differences between virus versus ICs ( Figure 2C). As

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IFNg and IFN-I enhanced the secretion of CXCL10 by IC-stimulated monocytes and IFN-I 209 significantly increased the production of TNFa. Priming conditions also modulated the 210 cytokine/chemokine secretion profile of virus-activated monocytes, but in a more restricted way than 211 in IC-activated cells. Thus, CXCL1 secretion was enhanced by TNFa and IFNg and CCL2 by IFN-I.

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Both types of IFN also increased the release of CXCL10 by virus-activated monocytes ( Figure 3D).    238 239 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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In steady-state conditions, FcgRIV was highly expressed on CD11b + Ly6G hi and CD11b + Ly6G int   FcgRIV on CD11b + Ly6G int ( Figure 5C and 5D) suggested different and specific properties of this 287 neutrophil subpopulation. We thus characterized it further. We observed neutrophils Ly6G int to be 288 smaller than neutrophils Ly6G hi and displaying a lower granulosity ( Figure 5D). To determine the 289 specificities of this population, we studied the expression pattern of neutrophils and monocytes cell-290 surface markers in these cells at steady-state conditions ( Figure 5E). Similar to CD11b + Ly6G hi 291 neutrophils, CD11b + Ly6G int cells expressed low levels of the monocyte cell surface markers Ly6C and 292 CCR2 ( Figure 5E). They also expressed the CXCR4 chemokine receptor although at intermediate 293 levels as compared to CD11b + Ly6G hi neutrophils and inflammatory monocytes. However, they weakly 294 express the chemokine receptor CXCR2 which is highly expressed on CD11b + Ly6G hi neutrophils.

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These results revealed that Ly6G int cells display a neutrophil-like phenotype but with specific 297 characteristics as compared to classical Ly6G hi neutrophils, notably (i) smaller size and granulosity,

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The copyright holder for this preprint this version posted April 24, 2020. ; https://doi.org/10.1101/2020.04.22.055533 doi: bioRxiv preprint show that the activation state of the cells evolved over time. At day 8 p.i., ( Figure 6A) CD11b was 312 upregulated on neutrophils and monocytes upon viral infection either in the absence or in the presence 313 of immunotherapy. However, infected/treated mice showed a significantly higher CD86 upregulation 314 in CD11b + Ly6G hi and CD11b + Ly6G int neutrophils than infected/non-treated mice. MHC-II molecule 315 was also upregulated on CD11b + Ly6G int neutrophils and monocytes upon viral infection. At day 14 p.i.

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CD86 expression on CD11b + Ly6G int neutrophils was significantly increased in infected/treated mice, 319 suggesting a mAb-mediated upregulation of these costimulatory molecules on this neutrophil 320 subpopulation. Likewise, inflammatory monocytes from infected/treated mice also showed a 321 significantly increased expression of CD86 co-stimulatory molecules as compared to infected/non-

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We found that the secretion profile evolved over time and was cell type-and stimulus specific, with 338 globally enhanced cytokine/chemokines secretion in cells isolated from infected/treated mice ( Figure   339 7A and 7B), in particular at day 14 p.i.

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At day 8 p.i., the CD11b + Ly6G hi neutrophils subpopulation from both infected/non-treated and 342 infected/treated mice showed a broad chemokine and cytokine secretion profile as deduced from an 343 increased secretion of most of the 13 chemokines and 12 cytokines assessed, although at weak levels 344 ( Figure 7B and 7C). CD11b + Ly6G int neutrophils showed enhanced expression of several chemokines 345 mostly in cells isolated from infected/non-treated mice notably with a strong enhanced secretion of 346 CCL3, CCL5 and CXCL5. In addition, CD11b + Ly6G int neutrophils also showed a slight secretion of 347 multiples cytokines in infected/treated mice, but to a lesser extent than CD11b + Ly6G hi neutrophils (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted April 24, 2020. ; https://doi.org/10.1101/2020.04.22.055533 doi: bioRxiv preprint (lower fold-increase) ( Figure 7B and 7C). In contrast, infection and mAb-treatment hardly affected the 349 chemokine/cytokine secretion of Ly6C hi monocytes. They only showed a weak increase in CXCL1 350 secretion in infected/treated mice as well as a slight increase in cytokines secretion (both in terms of 351 diversity and fold increase) mainly in infected/non-treated mice ( Figure 7B).

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Interestingly, at day 14 p.i., the functional activation of the three different cell types assessed 354 completely differed from that observed at day 8 p.i. (Figure 7B and 7C). We found a more restricted

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We have previously shown that neutrophils have a key immunomodulatory role in the induction of

Phenotypical and functional activation of FcgRIV-expressing cell from spleen ex vivo: 521
Single-cell suspensions of splenocytes were obtained from naive, infected/non-treated and

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The copyright holder for this preprint this version posted April 24, 2020. ; https://doi.org/10.1101/2020.04.22.055533 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted April 24, 2020. ; https://doi.org/10.1101/2020.04.22.055533 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted April 24, 2020.

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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted April 24, 2020.

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The data represent 6 independent experiments and are expressed as means +/-SEM. Statistical 929 significance was established using a parametric 1-way ANOVA test (*p < 0.05; **p < 0.01; ***p <   960 independent experiments at D14 p.i with at least 6-9 mice per group (I/NT and I/T) and 3-5 mice per 961 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.          I IT  I IT  I IT  I IT  I IT  I IT  I IT  I IT  I IT  I IT  I IT  I I  IT  IT  IT  I  I   I  IT  IT  IT  I  I   I  IT  IT  IT  I  I   I  IT  IT  IT  I  I   >500  100  50