Functionally distinct neutrophils sequentially promote Th1-type responses upon antiviral antibody therapy

Antiviral monoclonal antibodies (mAbs) can generate protective immunity through immune complexes (IC)-FcγRs 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 the functional activation of neutrophils. 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 FcγRs upregulation. Similarly, infection and mAb-treatment upregulated FcγRs expression on neutrophils and enhanced their cytokines and chemokines secretion. FcγRs 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.


153
Inflammatory monocytes activated by viral determinants, free or in the form of ICs, secrete high levels 154 of chemokines able to recruit neutrophils and monocytes. Taking into account the high amounts of the 155 monocytes-recruiting CCL2 chemokine secreted by viral determinants-activated neutrophils, we next 156 assessed the phenotypical and functional activation of monocytes by these stimuli. Ly6C hi monocytes 6 Virus-and ICs induced a weak secretion of most of the 12 cytokines analyzed, except for IL6 and TNFa.

163
However, no significant differences between unstimulated monocytes versus virus-or IC-stimulated 164 monocytes were detected (Supplemental Figure 2A). This contrasted with high level secretion of IL-6, 165 TNFa and IFNg observed upon LPS stimulation (Supplemental Figures 2B-2D). The quantification of 166 the chemokine release by activated monocytes showed some interesting similarities with neutrophils.

167
Following virus or ICs stimulation, monocytes produced CXCL5, CXCL1 and CCL2, with no 168 significant differences between virus versus ICs ( Figure 2C). As compared to neutrophils, higher 169 amounts of the neutrophil-recruiting chemokine CXCL1 were detected as well as lower amounts of

198
(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 May 1, 2020. . https://doi.org/10.1101/2020.04.22.055533 doi: bioRxiv preprint the production of TNFa. Priming conditions also modulated the cytokine/chemokine secretion profile 199 of virus-activated monocytes, but in a more restricted way than in IC-activated cells. Thus, CXCL1   of this FcgR is all the more relevant to be studied in this experimental model as (i) it is a high affinity 232 receptor for IgG2a, (which is the isotype of the 667 mAb) and (ii) it is strongly expressed in neutrophils,

233
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

236
To this end, mice were infected and treated, or not, with the therapeutic mAb (infected/treated and 237 infected/non-treated, respectively). Then, the expression of FcgRIV on neutrophils and inflammatory

268
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

273
FcgRIV on CD11b + Ly6G int ( Figure 5C and 5D) suggested different and specific properties of this 274 neutrophil subpopulation. We thus characterized it further. We observed neutrophils Ly6G int to be 275 smaller than neutrophils Ly6G hi and displaying a lower granulosity ( Figure 5D). To determine the 276 specificities of this population, we studied the expression pattern of neutrophils and monocytes cell-277 surface markers in these cells at steady-state conditions ( Figure 5E). Similar to CD11b + Ly6G hi 278 neutrophils, CD11b + Ly6G int cells expressed low levels of the monocyte cell surface markers Ly6C and 279 CCR2 ( Figure 5E). They also expressed the CXCR4 chemokine receptor although at intermediate levels

280
as compared to CD11b + Ly6G hi neutrophils and inflammatory monocytes. However, they weakly 281 express the chemokine receptor CXCR2 which is highly expressed on CD11b + Ly6G hi neutrophils.

283
These results revealed that Ly6G int cells display a neutrophil-like phenotype but with specific 284 characteristics as compared to classical Ly6G hi neutrophils, notably (i) smaller size and granulosity, (ii)

302
( Figure 6B), CD11b + Ly6G hi splenic neutrophils were similarly activated in infected/treated-and 303 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

304
CD86 expression on CD11b + Ly6G int neutrophils was significantly increased in infected/treated mice, 305 suggesting a mAb-mediated upregulation of these costimulatory molecules on this neutrophil 306 subpopulation. Likewise, inflammatory monocytes from infected/treated mice also showed a 307 significantly increased expression of CD86 co-stimulatory molecules as compared to infected/non-308 treated mice. Notably, MHC-II expression was significantly increased in infected/treated mice in both 309 neutrophil populations as well as in monocytes as compared to infected/non-treated mice.

338
(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 May 1, 2020. . https://doi.org/10.1101/2020.04.22.055533 doi: bioRxiv preprint Interestingly, at day 14 p.i., the functional activation of the three different cell types assessed completely 339 differed from that observed at day 8 p.i. (Figure 7B and 7C). We found a more restricted but stronger 340 induction of chemokines/cytokines secretion, mostly in cells isolated from infected/treated mice. In 341 contrast to the broad cytokines and chemokines secretion profile observed at day 8 p.i., CD11b + Ly6G hi 342 neutrophils only secreted CCL4 and CCL5 chemokines and showed no cytokine secretion at day 14 p.i.

463
Our work show that mAb-mediated activation of neutrophils and monocytes might be essential to induce 464 strong Th1-polarized early immune responses that might be key to induce protective immunity by mAb.

465
In addition, the distinct secretion profile of Ly6G hi and Ly6G int neutrophils and Ly6C hi monocytes, in 466 (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 May 1, 2020.

Phenotypical and functional activation of FcgRIV-expressing cell from spleen ex vivo: 493
Single-cell suspensions of splenocytes were obtained from naive, infected/non-treated and

582
The authors declare no competing financial interests. 583 584 (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 May 1, 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 May 1, 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 May 1, 2020.   (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 May 1, 2020.

949
The data represent 5 independent experiments at D8 p.i. and 6 independent experiments at D14 p.i with at least 6-    (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 May 1, 2020.  (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 May 1, 2020.   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  (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.