Figure S7. A) Frequency of CD4+ Foxp3+ regulatory T cells in colon LP of DSS-treated Csf3r+/+ and Csf3r-/-. Unpaired Student’s t-Test. Data are mean ± SEM. ns: not significant.

Figure S3. Neutrophils drive the expression of IL-23 by BMDMs. Related to Figure 3. A) Total number of ulcers in the colon of DSS-treated Csfr3+/+ (n=6) and Csf3r-/- (n=7) mice. B) IL-23 levels detected by ELISA in supernatants of BMDMs, neutrophils (NΦ) and BMDM-NΦ co-cultures after stimulation with GM-CSF+CpG. In indicated conditions neutrophils were pre-treated with diphenyleneiodonium (DPI) (10µM) or BMDM-NΦ were cultured in transwells (for all conditions n=6). C) Percentage of viable neutrophils (AnnexinV-/PI-) cultured alone or after co-culture with BMDMs. D) Schematic representation of the treatment schedule for anti-IL-22 treatment and neutrophil adoptive cell transfer during acute colitis. Days of treatment are indicated by red arrows. E) Percentage of body weight loss during DSS-induced acute colitis in Csf3r+/+(n=4), Csf3r-/-(n=4) and Csf3r-/- mice upon adoptive transfer of neutrophils treated with anti-IL-22 or isotype control (50µg/mouse) via i.p. injection (Csf3r-/- mice+Isotype n=3; Csf3r-/-+anti-IL-22 n=3). A) Representative data of five independent experiments. B) Representative data of three independent experiments. C) Representative of two independent experiments. E) One experiment. A) Unpaired Student’s t-Test. B-C) Multiple Student’s t-Test. E) Wilcoxon matched-pairs signed rank test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.

Supplementary Table 1. Colitis grade score.

Hepatitis B surface antigen (HBsAg) loss and seroconversion represent the current therapeutic goal for achieving a functional cure of chronic HBV infection. Understanding the immunological mechanisms that enable the break of tolerance against HBsAg is therefore a key priority.The liver is a unique immunological environment where tolerance and immunity are tightly balanced. In HBV-replication–competent transgenic mice (HBV Tg), hepatocytes express the full HBV genome, and both T and B cells are traditionally considered tolerant to viral antigens. Unexpectedly, we observed that ∼60% of HBV Tg mice spontaneously develop anti-HBsAg antibodies, achieving HBsAg seroconversion and clearance of circulating virions and subviral particles.Seroconversion required CD4⁺ T cell help, as HBV Tg mice lacking MHC-II or CD40L failed to mount anti-HBsAg responses. Notably, seroconversion often occurred among animals sharing the same cage, and fecal microbiota transplantation (FMT) from seroconverted donors was sufficient to induce seroconversion in recipients. This effect was lost in CD40L⁻/⁻ HBV Tg mice, indicating that microbiota-driven immune activation relies on intact CD4⁺ T cell–B cell cooperation.Shotgun metagenomics revealed distinct microbial signatures enriched in taxa with lactate- and GABA-producing pathways in seroconverted mice, suggesting a metabolite-mediated modulation of immune activation.Our findings uncover a microbiota-dependent mechanism capable of breaking B cell tolerance to HBsAg in a tolerogenic hepatic environment. By linking microbial metabolites to CD4⁺ T cell help and anti-HBsAg seroconversion, this model offers a conceptual framework to explore microbiota-driven immune reactivation as a potential avenue toward functional HBV cure.

Figure S5. γδ T cell subsets activation in DSS-treated mice. Related to Figure 4-5 A) Representative gating strategy for γδ T cell subsets in colon LP. B) Frequency of γδ T cell subsets from colon tissue of DSS-treated mice in colon LP. C) AhR expression in γδ T cells subset from colon LP of DSS-treated mice. D-E) Expression of IL-22 by γδ T cell subsets stimulated 4 hours with IL-23 plus IL-1β (D) and PMA plus ionomycin (E) analyzed by flow cytometry. F-G) Expression of IL-17 by γδ T cell subsets stimulated 4 hours with IL-23 plus IL-1β (F) and PMA plus ionomycin (G) analyzed by flow cytometry. H) RorγT expression in γδ T cell subsets from colon LP of DSS-treated mice. I) Body weight loss during DSS-induced acute colitis in Csf3r+/+(n=4), Csf3r-/- (n=4) and Csf3r-/- mice upon adoptive transfer of neutrophils (NΦ) (n=4) with and without antibiotic (ABX) oral treatment (Csf3r+/++ABX n=4 and Csf3r-/-+ABX n=4, Csf3r-/-+NΦ+ABX n=4). Red arrows indicated days of neutrophils transfer. A-H) Representative data of two independent experiments, Csf3r+/+(n=4), Csf3r-/- (n=4). I) One experiments. B-H) Multiple t-test. I) Wilcoxon matched-pairs signed rank test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.

Elderly individuals affected by COVID-19 are vulnerable to severe respiratory failure for reasons that have remained poorly defined. Here, we show that platelet-released serotonin drives lung pathology in a mouse model of age-associated disease severity after SARS-CoV-2 infection. In middle-aged mice and aged human healthy donors, platelet serotonin release upon activation was enhanced, and increased morbidity with respiratory dysfunction was associated with activated platelets aggregating and promoting fibrin deposition in the lung microvasculature. Pharmacologic or genetic disruption of serotonin uptake, or blockade of serotonin-dependent signaling, attenuated platelet activation and protected against respiratory distress, independently of viral replication and immune responses. Inhibition of fibrin formation similarly reduced disease severity, implicating serotonin-driven platelet procoagulant activity as a key contributor to age-related lung dysfunction. Thus, serotonin-mediated platelet procoagulant activity is a major contributor to respiratory insufficiency during SARS-CoV-2 infection and a potential therapeutic target for preserving pulmonary function, particularly in the elderly.

Figure S2. Neutrophil deficiency is associated with intestinal dysbiosis. Related to Figure 2 A) Representative histological images of H&E-stained colon section from Csf3r+/+(left) and Csf3r-/- (right) mice, after one cycle of DSS, showing the increased inflammatory infiltrate and the presence of bacterial accretions (indicated by black arrow) in Csf3r-/-. B-C) Shannon Index representing the evenness in the overall number of bacterial species between wild-type and neutrophil deficient mice untreated (B) and DSS-treated mice (C). D-E) Taxonomic analysis of the phyla that compose the microbiota of feces of untreated (D) and DSS-treated (E) Csf3r+/+ (untreated n=4, DSS-treated n=3) and Csf3r-/- (untreated n=4, DSS-treated n=4) mice. F) Microbiota depletion efficiency measured by qPCR for 16S gene: complete depletion is achieved after 20 days of antibiotic treatment. G) Body weight loss during DSS-induced acute colitis in Csf3r+/+(n=11) and Csf3r-/- (n=9) mice with and without cohousing (Csf3r+/+Cohoused n=5 and Csf3r-/-Cohoused n=5). H) Body weight loss of AOM/DSS treated Csf3r+/+(n=7) and Csf3r-/- (n=5) mice with and without cohousing (Csf3r+/+Cohoused n=5 and Csf3r-/-Cohoused n=5); I) Macroscopic polyp count at the experimental endpoint. A-I) One experiment. B-C) Unpaired Student’s t-Test. F, I) Multiple Student’s t-Test. G-H) Wilcoxon matched-pairs signed rank test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.

Figure S1. Neutrophil deficiency is associated with increased lymphoid aggregates. Related to Figure 1.A) Number of total lymphoid structures and B) number of lymphoid structures with follicular dendritic cells detected by RNAscope staining for the follicular dendritic cell marker Mfge8 in colon tissue sections from Csf3r+/+(n=8) and Csf3r-/- (n=5) mice after AOM/DSS-induced CRC. C) Representative images of RNAscope analysis for Mfge8 in colon tissue sections from Csf3r-/- mice after AOM/DSS-induced CRC: aggregates with follicular structures are visible as Mfge8+ (red spot): on the top is visible a lymphoid aggregate at two different magnifications, with diffuse Mfge8+ cells; on the bottom right lymphoid microaggregate with Mfge8+ cells; on the bottom left aggregate without follicular structures (Mfge8+). D) Representative dot-plot of neutrophils frequency in blood (top panels) and colon LP (bottom panels) of Csf3r-/- mice and Csf3r-/- mice 4hrs and 18hrs after adoptive transfer of neutrophils. E-F) Frequency of neutrophils in blood (D) and colon LP (E) of DSS-treated Csf3r-/- mice (n=5) and Csf3r-/- mice 4hrs (n=5) and 18hrs (n=5) after adoptive transfer of neutrophils. A-B) Representative data of three independent experiments. C-E) One experiment. A-B, D-E) Unpaired Student’s t-Test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.

Supplementary Table 2. List of critical reagents and antibodies used.

Figure S6. Neutrophil infiltration is associated with tissue repair gene signatures in patients with ulcerative colitis. Related to Figure 6. A-E) Enrichment plots for gene ontology term epithelial cell (GO:Epithelial Cell Proliferation (A, D); GO:Epithelial Cell Development (B, E); GO:Maintanance of Gastrointestinal Epithilium (C)) in CSF3Rhigh versus CSF3Rlow UC patients GSE109142 (A-C) GSE87473 (D-E). F-K) Enrichment plots for gene ontology term antimicrobial response (GO:Response to molecule of bacterial origin (F, I); GO:Defense response to bacterium; GO:Antimicrobial humoral immune response mediated by antimicrobial peptides) in CSF3Rhigh versus CSF3Rlow UC patients GSE109142 (F-H) and GSE87473 (I-K). CSF3R gene expression values were stratified by quartiles and patients belonging to the upper (GSE109142 n=51; GSE87473 n=21) and lower GSE109142 n=51; GSE97473 n=21) quartiles were considered for differential expression analysis. FDR, false discovery rate.

Figure S4. Neutrophil deficiency is associated with decreased expression of IL-22 by γδ T cells. Related to Figure 4-5. A-C) Representative gating strategy used to gate myeloid population (A), lymphoid population (B) and innate lymphoid cells (ILC) (C) in colon LP. UTC: unconventional T cells (TCRβ+, CD8-, CD4-). D) IL-22 expression in myeloid cells derived from DSS-treated Csf3r+/+(n=3), Csf3r-/-(n=3) stimulated 4 hours with IL-23 analyzed by FACS. E-F) Expression of IL-22 by lymphoid cells derived from DSS-treated Csf3r+/+(n=4), Csf3r-/- (n=4) stimulated 4 hours with IL-23 plus IL-1β (E) and PMA plus ionomycin (F) analyzed by FACS. A-F) Representative data of three independent experiments. D-F) Multiple t-test. Data are mean ± SEM. *** p < 0.001 ** p < 0.01 * p < 0.05.

This is an update to the Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition), Chapter 3: 12C, by Cossarizza et al. Administration of anti-ARTC2 nanobody(S+16a) prevents cell death during tissue processing. We demonstrate that the phenotype of CD44midTreg is significantly impacted, whereas the eTreg phenotype remains stable following S+16a treatment, outlining specific protocols for population recovery.

Chronic hepatitis B virus (HBV) infection is marked by dysfunctional HBV-specific CD8+ T cells, and restoring their effector activity is a major therapeutic goal. Here, we generated HBV-specific CD4+ T cell receptor transgenic mice to show that CD4+ effector T cells can prevent and reverse the CD8⁺ T cell dysfunction induced by hepatocellular priming. This rescue enhances antiviral CD8+ T cell function and suppresses viral replication. CD4+ T cell help occurs directly within the liver, independent of secondary lymphoid organs, and requires local antigen recognition. Kupffer cells, rather than dendritic cells, are the critical antigen-presenting platform. CD4+ T cells license Kupffer cells via CD40–CD40L interactions, triggering interleukin (IL)-12 and IL-27 production. IL-12 expands the CD4+ T cell pool, while IL-27 is essential for CD8+ T cell rescue. Exogenous IL-27 similarly restores HBV-specific CD8+ T cell function in mice and in T cells isolated from chronically infected patients. These findings identify IL-27 as a tractable immunotherapeutic target in chronic HBV infection. Here the authors show that CD4+ effector T cells prevent or reverse CD8+ T cell dysfunction by licensing Kupffer cells to trigger IL-27 production, defining a liver-specific immune circuit and a potential target for chronic HBV therapeutics.

Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.