Several publications previously evidenced the efficient generation of monocyte-derived DCs (moDCs) upon stimulation with GM-CSF and IL-4 in FCS-supplemented media (19). However, recent reports demonstrated that different DC subsets can be generated without the addition of FCS, ensuring xeno-free cultivation conditions and facilitating clinically oriented studies (20, 21). Therefore, we first aimed to phenotypically compare primary generated DCs cultured under different serum-supplemented conditions, specifically in presence of 10% FCS (FCS), 5% hPL (hPL) or 1% FCS-pre-coated wells with 5% human platelet lysate (chPL) supplemented RPMI media (as previously published by Rauch et al. (22) ( Figure 1A ). Human peripheral blood CD14⁺ monocytes were purified by magnetic bead separation as previously described (19) and stimulated with GM-CSF and IL-4 to induce DC differentiation. On day 6, DCs were analyzed for their expression of moDC-affiliated markers, i.e. CD11c and CD209 ( Figure 1B ). Flow cytometric analysis across all tested donors confirmed that monocytes differentiated in hPL-supplemented media display similar FSC versus SSC identification and gain DC-characteristic CD11c and CD209 positivity as cells cultivated in FCS-containing or FCS-coated media ( Figure 1B ). In parallel, no cell morphologic differences could be observed, albeit moDCs in the presence of hPL showed an increased number of adherent cells maintaining DC-like morphological characteristics ( Figure 1C ).

Depending on microenvironmental factors and functional necessity, monocytes and DCs possess high plasticity and can rapidly adopt phenotypically and functionally. Hence, moDCs are crucially implicated in inflammation and play a pivotal role during infection, i.e. HIV infection (23–26). Thus, we next sought to evaluate whether hPL-generated DCs provide a suitable model to study infectious diseases. The expression pattern of characteristic DC surface markers (CD83, CD86, CD11c and DC-SIGN) on complement-opsonized HIV-1-exposed (HIV-C) DCs was studied by FACS analyses and compared with that of LPS-treated DCs (mature DCs, mDCs) or untreated DCs (immature DCs, iDCs) ( Figure 2A ). Interestingly, loss of CD11c expression in DCs upon HIV-C exposure was consistently observed in all culture conditions, resembling an abundantly described key characteristic in HIV-infected patients (23) ( Figure 2B ). Moreover, as observed by Date et al. (21), hPL-grown DCs showed consistent elevated survival post infection, when compared to FCS-stimulated DCs ( Figure 2C ). Following, activation markers were monitored on FCS-, hPL- and chPL DCs in unstimulated control DCs (iDCs). The gating strategy of DC activation for flow cytometry assay is shown in Figure 2D . As expected, stimulation with LPS led to a consistent transition into mature DCs evidenced by increased levels in co-stimulatory molecules CD83 and CD86 levels. Similarly, exposure to HIV-C or LPS induced upregulation of CD83 or CD86 in all cultivation conditions ( Figure 2E ), although a much stronger maturation effect could be observed in FCS-supplemented condition. Moreover, in line with Švajger et al. (20), CD83 expression of iDCs remained low in all tested conditions, while CD86 levels were consistently higher in conditions using hPL. Finally, DC maturation was analyzed by confocal microscopy. As shown in in Figure 2F , DCs differentiated from monocytes using FCS revealed higher cell density compared to hPL and chPL-DCs. However, immunofluorescence staining, and flow cytometric analysis revealed positivity of HLA-DR under all tested conditions ( Figures 2F, G ). Consistent with Švajger et al. (20), the expression of activation markers was slightly lower in hPL conditions ( Figure 2F ). Thus, all additives possess capacity to induce a full mature DC phenotype by activating iDCs (LPS, HIV-C). The loss of CD11c expression in infected hPL-DCs might reflect the DC functionality in vivo ( Figure 2F ).

DCs are crucially implicated in virus capture and transfer process, specifically representing a main potential target for HIV-1 transmission (23). Therefore, full functionality of the variously differentiated DC cultures is fundamental for studying infectious diseases in vitro.

In a next step, we further elucidated the HIV infection ability of DCs by use of confocal microcopy. As in the initial infection phase, transferred HIV virions are opsonized by complement proteins (i.e.C1q or C3), enhancing viral uptake (27, 28), we compared the infection potency and distribution of complement-opsonized HIV (HIV-C) and non-opsonized HIV-1 (HIV-1) in FCS- and hPL DCs.

Interestingly, a higher cell number was displayed in the FCS supplemented media ( Figure 3A ). Immunohistology data of mCherry-tagged complement-opsonized HIV or HIV-1 (pink), HLA-DR (yellow) and CD11c (green) revealed successful viral entry in FCS and hPL culture conditions ( Figure 3A , left). Expectantly, increased virus particle areas were consistently observed in HIV-C infections compared to HIV-1 infections as depicted by ratios of HIV-C versus HIV-1 spot areas ( Figure 3A , right). When quantifying the virus particles normalized to the cell number, significantly higher virus spots upon HIV-C stimulation could be observed in all tested conditions ( Figure 3B , FCS–left, hPL–middle, chPL-right). To further confirm that these cells were productively infected and secrete infectious virus particles, supernatants were collected, and infectivity was measured. The HIV p24 antigen, as representing the most abundant HIV protein is clinically used to detect early HIV infection (29). Differentially cultivated DCs were infected and an ELISA setup targeting the HIV-1 capsid protein p24 was performed. Despite the generally higher p24 concentration levels in the FCS moDC culture, stimulation with HIV-C significantly upregulated p24 levels superseding HIV-1 in all DC conditions ( Figure 3C ). Finally, we assessed the difference in plasma membrane localization of virus particles between HIV-1 and HIV-C. Figure 3D represents a XYZ-analysis of HIV-1 and HIV-C upon uptake in DCs. Co-localization of HIV with CD11c was only observed when DCs were exposed to HIV-C independent on the culturing method (FCS, hPL) ( Figure 3D ), whereas non-opsonized HIV-1 did not accumulate in CD11c-rich fractions.

Human platelet lysate (hPL) used in this study was obtained from the Blood Bank of the University Hospital Innsbruck. The hPL was produced under standardized and GMP-compliant conditions as part of a long-standing collaborative effort involving the Innsbruck blood bank and the Gstraunthaler group. The production protocol ensures batch-to-batch consistency and includes stringent quality control procedures. Detailed compositional analyses of this hPL product—including levels of platelet-derived cytokines, coagulation factors, and growth factors such as TGF-β—have been previously published (44). In particular, key bioactive components were quantified and shown to support robust cell growth in serum-free culture systems (45). The same hPL formulation has also been referenced in international consensus efforts for GMP-grade hPL production and application in cell therapy and in vitro research (46). Based on this extensive prior characterization, no additional quantification of individual hPL components was performed within the present study.

Platelet concentrates (PLTs) used for hPL production were obtained either from pooled buffy coats or via apheresis from single donors. All procedures were carried out at the Central Institute for Blood Transfusion and Immunology of the Tirol Kliniken in Innsbruck under certified standard operating procedures and in accordance with Austrian national regulations. Pooled PLTs were prepared from six buffy coats using the CompoStop Flex Triple, T&B Set (Fresenius Kabi, REF: FT52600), centrifuged at 560 × g for 9 minutes and 10 seconds at 20°C, and leukocyte-depleted using the Macopress Smarter system (Fresenius Kabi). Apheresis PLTs were collected using either the Trima Accel (Terumo BCT, Zaventem, Belgium) or Amicus (Fresenius Kabi) cell separation platforms. All PLTs were suspended in 35% plasma and 65% platelet additive solution—either InterSol (PAS-C, REF: RGR8109B, Fresenius Kabi) or SSP+ (PAS-E, REF: SSP2130U, Macopharma, France). Pathogen inactivation was performed using the INTERCEPT blood system (Cerus Europe, Amersfoort, Netherlands) following the manufacturer’s protocol, as described previously (47). All products were tested for HIV, HCV, HBV, and a subset for bacterial contamination using the BACT/ALERT 3D system (bioMérieux, France). Only expired PLTs (stored for 7 days at 22°C) were used for the production of human platelet lysate (hPL). Platelets were lysed by three freeze (–80°C) and thaw (37°C) cycles. To minimize donor-to-donor variability, five platelet concentrates were pooled for each hPL batch. As each concentrate was derived from six buffy coats, this corresponds to a total donor pool of 30 individuals per batch. This approach follows published recommendations suggesting a minimum of four PLT units per batch to ensure reproducibility and reduce inter-individual variation (48). The pooled lysate was centrifuged at 5,500 × g for 15 minutes at 22°C, and the supernatant was sterile filtered using 0.2 µm filters (Nalgene Rapid-Flow, Cat. No. 565-0020, Thermo Fisher Scientific) under aseptic conditions in a class A laminar flow hood. Final hPL products were stored at –20°C until use. All PLT products were originally manufactured for clinical use under a license from the Austrian Agency for Health and Food Safety (AGES) and in compliance with ISO 9001:2015 quality standards.

The viruses used for the experiments in this project are produced in house in batches and listed in Supplementary Table 1 . Supernatants resulting from the transfection of HEK293 cells with plasmids, provided by Prof. Thomas J Hope from the Northwestern University in Chicago, are concentrated via ultracentrifugation at 20,000 rpm for 90 min at 4°C. An aliquot of each virus produced is inoculated with normal human serum that was stored at -80°C at maximum for 6 months to retain complement activity to generate complement-opsonized virus stocks. The concentrations of the viruses are analyzed via p24 ELISA. Their opsonization pattern is checked via virus capture assay and the infectivity (TCID50) is deduced by application of infection assays. The m-cherry tagged HIV-1/HIV-C are produced via calciumphosphate-mediated transfection of HEK293T/17 cells with R9BaL and mCherry proviral plasmids.

Ultracentrifuged, concentrated virus was opsonized in 200µl with medium alone (HIV), or normal human serum (NHS) as active complement source (HIV-C). Subsequent to opsonization, the different preparations were washed using 1 ml RPMI1640 without supplements (RPMI) and the virus was pelleted by ultracentrifugation. The virus was re-suspended in 200µl RPMI, aliquoted and the presence of C3 fragments or IgGs on the viral surface was confirmed by a virus capture assay (VCA) as described (34, 35).

Peripheral blood of healthy donors was obtained by the Central Institute for Blood Transfusion and Immunological Department, Innsbruck, Austria (EC1166/2018). Monocyte isolation was performed as previously described (19, 34). Briefly, PBMCs were isolated by use of a density gradient centrifugation using a Ficoll Paque Premium (GE Healthcare) gradient. Classical CD14+ monocytes were isolated using anti-human CD14 Magnetic Beads (BD). Only cells >98% purity were considered for further analysis. For efficient generation of immature DCs (iDCs), 1x10⁶ monocytes/mL were seeded and stimulated with IL-4 (200 U/ml) and GM-CSF (250 U/ml) for 5 days. Cells were either cultured in RPMI media containing 10% FCS or 5% hPL. For FCS-pre-coated human platelet lysate (chPL) DCs, wells were pre-incubated with 1% FCS (1h, 37°C) before cultivation in 5%hPL. L-glutamine was added in all culture conditions.

Dendritic cells were stimulated in U-bottom 96-well plates. The assay was performed in triplicates. Cells were either treated with 100 ng/mL LPS or 50 ng/100 µL HIV. RPMI media was used as a blank control. The cells were incubated for 24h at 37°C, 5% CO2. The supernatants were harvested for p24 ELISA analysis.

p24 ELISA was performed as described previously (49). All antibodies were kindly provided by Polymun Scientific, Vienna, Austria.

Cells are prepared as described above. The assay was performed on 96-well Perkin Elmer LLC CellCarrier-96 Ultra Microplates. To analyze infection, mCherry-tagged non- and complement-opsonized HIV-1 preparations were used (HIV, HIV-C). Prior to fixation, the cell surface was in additionally stained using CD11c Alexa 647 and HLA-DR Alexa 488. The next day, an intracellular staining was performed using an antibody mix of CD11c Alexa 647, HLA-DR Alexa 488 and Hoechst 33342. Plates were imaged on the Operetta^® CLS™ high content screening system (Perkin Elmer) and spots, XYZ and 3D analyses were calculated using the Harmony™ software.

For flow cytometry analysis, cells were stained and analyzed as previously described and are routinely tested in the laboratory using a viability dye, e.g. Ghost Dye Violet 540 Fixable Viability Dye (Cell Signaling Technology) or fixable Viability Dye (Thermo Fisher Scientific) (19, 50). In brief, cells were harvested and washed with FACS wash (0.5% BSA, 0.1% sodium azide in PBS). Prior to antibody or live/dead staining, Fc receptors were blocked, if indirect staining was performed. All samples were fixed in 4% FACS-fixation buffer, stained according to standard operating procedures and analyzed using BD FACS VERSE™ flow cytometer and the Flowlogic™ software. All antibodies used are listed in Supplementary Table 2 .

Differences between 2 groups were analyzed using GraphPad Prism software (Version 8.0.1) (GraphPad Software Inc.) with the unpaired Student t test (2-tailed). For multiparameter comparisons, values from the experiments were analyzed by use of One-way ANOVA, corrected with Tukey multiple comparison testing. Tests used are indicated in the Figure legends.