*2.2. Viruses*

The laboratory-adapted HIV-1 strain used in the experiments was the R5 tropic HIV-1BaL (courtesy of Drs. S. Gartner, M. Popovic, and R. Gallo; NIH AIDS Research and Reference Reagent Program) provided through the EU program EVA Centre for AIDS Reagents (NIBSC, Potter Bars, UK). The virus was inactivated with AT-2, which is able to change the zinc finger nucleocapsid proteins of HIV-1, therefore deactivating the viral infectivity as previously described [21].

SARS-CoV-2 (Virus Human 2019-nCoV strain 2019-nCoV/Italy-INMI1, Rome, Italy) was expanded on Calu-3 cells (ATCC ® HTB-55 ™) and TCID50 was calculated as previously reported [22]. SARS-CoV-2 inactivation (i-SARS-CoV-2) was obtained by incubation at 65 ◦C for 30 min [23].

### *2.3. ERAP2 Genotyping Analyses*

Whole blood was collected by all the subjects enrolled in the study by venipuncture in Vacutainer tubes containing EDTA (BD Vacutainer, San Diego, CA, USA). Total DNA was extracted by DNA purification Maxwell ® RSC Instrument (Promega, Fitchburg, WI, USA) and quantified using the Nanodrop 2000 Instrument (Thermo Scientific, Waltham, MA, USA). Two-hundred ng of DNA were used to perform an SNP genotyping assay for ERAP2 rs2549782 (G/T) (TaqMan SNP Genotyping Assay; Applied Biosystems, Foster City, CA, USA), which is in linkage disequilibrium with the non-coding rs2248374 (A/G). Analyses were performed on Peripheral blood mononuclear cells (PBMCs) from all the subjects recruited in the study as well as on lung adenocarcinoma cells (Calu3; ATCC ® HTB-55 ™). Allelic discrimination real-time PCR method was used to analyze the results.

### *2.4. Isolation of PBMCs and Monocyte-Derived Macrophages (MDMs) Di*ff*erentiation*

PBMCs, obtained from density gradient centrifugation on Ficoll (Cedarlane Laboratories Limited, Hornby, ON, Canada), were counted by automated cell counter ADAM-MC (NanoEnTek Inc., Seoul, Korea), which distinguishes viable from non-viable cells.

Flow cytometer analyses was used to quantify the percentage of CD14+ monocytes in PBMCs isolated from 3 HeteroAB HC. MDMs were obtained as previously described [24]. Briefly, 5 × 10<sup>5</sup> adherent monocytes were incubated for 5 days in RPMI with 20% of fetal bovine serum (FBS) (Euroclone, Milan, Italy) and 100 ng/mL macrophage-colony stimulating factor (M-CSF) (R&D Systems, Minneapolis, MN, USA). Optical microscope (ZOE ™ Fluorescent Cell Imager, Bio-Rad, Hercules, CA, USA) observation allows to verify MDM di fferentiation.

### *2.5. Cell Cultures for Microbial Antigen Stimulation*

PBMCs were resuspended at the concentration of 1 × 10<sup>6</sup> PBMCs/mL in RPMI 1640 medium (Euroclone, Milan, Italy) containing 10% fetal bovine serum (FBS), 1% levo-glutammin LG and 2% penstreptomicin. Subsequently, PBMCs and MDMs from HC were stimulated with antigens from di fferent pathogens: 32 μg/mL of CMV grade 2 Antigen (Microbix Biosystem, Mississauga, ON, Canada), 1 μg/mL of LPS, 1 ng/mL of HIV-AT-2 equivalents and 5 multiplicity of infection (MOI) of i-SARS-CoV-2 inactivated virus. Two live UV-inactivated influenza viruses (flu) were used as well: an influenza A virus (A/RX73 and A/Puerto Rico/8/34 strains; 1:800) and the 1998–1999 formula of flu vaccine (1:5000; Wyeth Laboratories Inc., Marietta, PA, USA). Cells were stimulated even with non-microbial stimuli: 100U of IFN α and 1 μg/mL of IL-1β (Sigma, Saint Louis, MO, USA). Unstimulated PBMCs were cultured as control as well. Cells were harvested 10 (PBMCs) and 36 (MDMs) h post-treatment for RNA and protein analyses, respectively.

### *2.6. In Vitro Infection of PBMCs and Calu3 Cells with SARS-CoV-2*

2.5 × 10<sup>5</sup> Calu3 cells (ATCC ® HTB-55 ™) were cultured in DMEM medium (Euroclone, Milan, Italy) supplemented with 2% FBS in a 24-well plate. DMEM containing 100 U/mL penicillin and 100 μg/mL streptomycin was used as inoculum in the mock-infected cells. Cell cultures were incubated with serial dilutions of virus supernatant in duplicate, (1000 MOI, 5 MOI, 0.5 MOI) for three h at 37 ◦C and 5% CO2. Cells were washed two times with lukewarm PBS and refilled with the proper growth medium (10%FBS). Optical microscope observation (ZOE ™ Fluorescent Cell Imager, Bio-Rad, Hercules, CA, USA) was performed daily to investigate the cytopathic e ffect. The infected cells were harvested for SARS-CoV-2 detection in the supernatant and mRNA collection at 48 h. Each culture condition was run in triplicate. All the procedures were performed in agreemen<sup>t</sup> with the GLP guidelines adopted in our laboratory.

Maxwell ® RSC Viral Total Nucleic Acid Purification Kit (Promega, Fitchburg, WI, USA) was used to extract RNA from Calu3 cell culture supernatants by the Maxwell ® RSC Instrument (Promega, Fitchburg, WI, USA). Viral RNA was quantified, by single-step RT PCR -time PCR (GoTaq ® 1-Step RT-qPCR) (Promega, Fitchburg, WI, USA) on a CFX96 (Bio-Rad, Hercules, CA, USA) by using TaqMan probes which target two portions of SARS-CoV-2 nucleocapsid (N) gene (N1 and N2). Specifically, we used the 2019-nCoV CDC qPCR Probe Assay emergency kit (IDT, Coralville, IA, USA), which allows also to amplify the human RNase P gene. Viral copy number quantification was performed by generating a standard curve from the quantified 2019-nCoV\_N positive Plasmid Control (IDT, Coralville, IA, USA).

### *2.7. In Vitro HIV-Infection Assay*

3 × 10<sup>6</sup> PBMCs from all the subjects included in the study were in vitro HIV-infected as previously described [25] with 10, 1, and 0.1 ng p24 HIV-1Bal/1 × 10<sup>6</sup> PBMCs. After 5 days, supernatants were collected for p24 antigen ELISA (Cell Biolabs, San Diego, CA, USA), whereas PBMCs collected at 2 days post-infection were used for RNA extraction and gene expression analyses.

### *2.8. Gene Expression Analysis*

RNA extracted from 1 × 10<sup>6</sup> PBMCs, and Calu3 cell lines were retrotranscribed as previously described [16]. cDNA quantification for ERAPs was performed on antigen-stimulated and HIV-infected PBMCs as well as on SARS-CoV-2 infected Calu3 cells through a real-time PCR (CFX96 connect, Bio-Rad, Hercules, CA, USA) and an SYBR Green PCR mix (Bio-Rad, Hercules, CA, USA); all the reactions were carried out in duplicate. Results are shown as the media of the relative expression units to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin housekeeping genes calculated by the 2−ΔΔCt equation. The following thermal protocol was used: initial denaturation (95 ◦C, 15 min) followed by 40 cycles of 15 s at 95 ◦C (denaturation), 20 s at 60 ◦C (annealing) and 20 s at 72 ◦C (extension). Furthermore, a melting curve analysis was assessed for amplicon characterization. Ct values of 35 or higher were let o ff the analyses.

### *2.9. Quantigene Plex Gene Expression Assay*

Gene expression of 8 × 10<sup>5</sup> PBMCs was performed by quantiGene Plex assay (Thermo Scientific, Waltham, MA, USA) which provides a fast and high-throughput solution for multiplexed gene expression quantitation, allowing the simultaneous measurement of 70 custom selected genes of interest in a single well of a 96-well plate. The QuantiGene Plex assay is hybridization-based and incorporates branched DNA (bDNA) technology, which uses signal amplification for direct measurement of RNA transcripts. The assay does not require RNA purification.

### *2.10. Western Blot Analyses*

Cultured MDMs were removed by non-enzymatic cell dissociation solution (Sigma, Saint Louis, Missouri, USA), counted by the automated cell counter ADAM-MC (Digital Bio) and used for protein extraction by RIPA bu ffer (Sigma, Saint Louis, MO, USA). Extracted proteins were stored at −80 ◦C for further analyses. For WB analyses, samples from 3 HeteroAB subjects were sub-pooled (50 μg per pool). Equal amounts of proteins were separated by 4–20% SDS-polyacrylamide gel electrophoresis (Criterion TGX Stain-free precast gels and Criterion Cell system; Bio-Rad) and transferred onto nitrocellulose membrane using a Bio-Rad Trans-Blot Turbo System. Membranes were probed using a 1:1000 dilution of primary antibody goa<sup>t</sup> anti-ERAP1 (AF2334; R&D Systems, Minneapolis, MN, USA) goa<sup>t</sup> anti-ERAP2 polyclonal antibody (AF3830; R&D Systems, Minneapolis, MN, USA), rabbit anti-GAPDH polyclonal (VPA00187); Bio-Rad, Hercules, CA, USA] and a 1:10,000 dilution of secondary antibody conjugated with alkaline phosphatase anti-goat IgG (A4187; Sigma; goa<sup>t</sup> anti-rabbit (STAR208P); Bio-Rad]. Membranes were incubated with the appropriate antibody and, after being excited using the Clarity Western ECL substrate, bands were visualized with a ChemiDoc MP imaging system (Bio-Rad) and quantified for densitometry with the Bio-Rad Image Lab software.
