key: cord-0964432-1y94p7e3 authors: Rausch, Lisa; Lutz, Konstantin; Schifferer, Martina; Winheim, Elena; Gruber, Rudi; Rinke, Linus; Hellmuth, Johannes C.; Scherer, Clemens; Muenchhoff, Maximilian; Mandel, Christopher; Bergwelt-Baildon, Michael; Simons, Mikael; Straub, Tobias; Krug, Anne B.; Kranich, Jan; Brocker, Thomas title: Binding of phosphatidylserine-positive microparticles by PBMCs classifies disease severity in COVID-19 patients date: 2021-06-18 journal: bioRxiv DOI: 10.1101/2021.06.18.448935 sha: ce1c801dc296fffe4f9348bd3ce192deeced1fac doc_id: 964432 cord_uid: 1y94p7e3 Infection with SARS-CoV-2 is associated with thromboinflammation, involving thrombotic and inflammatory responses, in many COVID-19 patients. In addition, immune dysfunction occurs in patients characterized by T cell exhaustion and severe lymphopenia. We investigated the distribution of phosphatidylserine (PS), a marker of dying cells, activated platelets, and platelet-derived microparticles (PMP), during the clinical course of COVID-19. We found an unexpectedly high amount of blood cells loaded with PS+ PMPs for weeks after the initial COVID-19 diagnosis. Elevated frequencies of PS+PMP+ PBMCs correlated strongly with increasing disease severity. As a marker, PS outperformed established laboratory markers for inflammation, leucocyte composition, and coagulation, currently used for COVID-19 clinical outcome prognosis. PS+ PMPs preferentially bound to CD8+ T cells with gene expression signatures of proliferating effector rather than memory T cells. As PS+ PMPs carried programmed death-ligand 1 (PD-L1), they may affect T cell expansion or function. Our data provide a novel marker for disease severity and show that PS, which can trigger the blood coagulation cascade, the complement system, and inflammation, resides on activated immune cells. Therefore, PS may serve as a beacon to attract thromboinflammatory processes toward lymphocytes and cause immune dysfunction in COVID-19. (WHO 4), and 'severe' (WHO 5-8) groups for the subsequent analyses. Additionally, we also 16 included a group of healthy donors (HD, n = 30) and recovered patients (n = 12, >69 days post 17 1 st SARS-CoV-2 + diagnosis by PCR, either never hospitalized or released from the hospital 18 with WHO score 1 -2). The frequencies of PS + PBMC increased with severity of 19 disease in the following order: healthy controls (WHO 0) < recovered patients < WHO 1-3 20 (mild) < WHO 4 (moderate) < WHO 5-8 (severe) (Fig. 1C) . In severely diseased patients, 30 -90% of all PBMC were PS + (Fig. 1C) . Accordingly, the individual WHO scores positively Moreover, when we analyzed the frequencies of PS + PBMC over time, we found elevated 25 levels for up to 10 weeks in some patients (Fig. 1E) , indicating that the presence of PS + PBMC 26 in COVID-19 patients might be long-lasting. However, in recovering patients, PS + PBMC 27 returned to the levels of healthy controls (Fig. 1E ). In summary, the number of PS + PBMC in 28 the blood of COVID-19 patients represents a new parameter that correlates with strongly with 29 disease severity. Table 5 shows the number, age and gender of the different study groups. (C) Grouped analysis of the 6 data from (A). (D) Same as (C), but plotted against the WHO ordinal scale (n = 38-79) (B). PS + PBMCs correlate with the severity of the disease. The plot shows the Spearman 8 correlation test and linear regression line with 95% confidence interval shading (A, C, D: HD, 9 n = 30; RD, n = 12; COVID, n = 49). (E) Analysis of PS + PBMC as shown in (A) plotted against 10 days after initial SARS-CoV-2 diagnosis. Lines connect the same donors. Significance was 11 determined by Mann-Whitney test: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Asterisks in brackets show statistically significant differences as compared to HD. recently shown that most PS + cells in the spleen of virus-infected mice are not apoptotic, but 16 cells carried PS + extracellular vesicles 33 . To determine whether PS + PBMC in COVID-19 17 patients were apoptotic cells contributing to the described lymphopenia or EV + cells, we 18 analyzed the images of PS + PBMC acquired by IFC. Some cells showed almost entirely PS + 19 cell bodies with strongly labeled apoptotic blebs, typical for cell death by apoptosis ( Fig. 2A ). These cells still have an intact cell membrane since they did not stain with the live/dead dye 21 used to exclude necrotic cells from the analysis. However, we also detected many cells with Whitney test: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Asterisks in brackets 15 show statistically significant differences as compared to HD. We confirmed these correlations in our patient cohort ( Fig. 3B ). In general, CD8 + T cells were more strongly associated with PS + EVs 37 than CD4 + T cells (Fig. 3A ). However, both T cell subsets showed a similar tendency of 38 increased PS + EV binding in patients with a higher WHO score (Fig. 3A) . show the mean percentage ± SD of all cells depicted inside the dot plot that lie within the 9 respective gate, while the graphs show the average frequency ± SD of EV + cells within the 10 analyzed subpopulation. Significance was determined by Mann-Whitney test: *P < 0.05, **P < 11 0.01, ***P < 0.001, and ****P < 0.0001. Asterisks in brackets show statistically significant 12 differences as compared to HD. with PS + EVs in the patients. The frequency of PS + EV + CD8 + T cells best reflected the severity To better characterize the EVs associated with lymphocytes in COVID-19 patients, we isolated To test this hypothesis, we stained PBMC from COVID-19 patients for CD41, a platelet marker, 16 part of a fibrinogen-receptor, and present on platelet-derived PMPs 48 (Fig. Suppl. 3, Fig. 4G ). Analysis of flow cytometry data of PBMC confirmed our assumption and showed that many 18 PBMC were positive for the platelet marker CD41 (Fig. 4G) . However, CD41 could not The CD41 hi gate contained mainly cells with large CD41 + particles attached, which were also 31 visible in the brightfield (BF) channel -presumably whole platelets. In contrast, the cells in the 32 CD41 low gate were associated with small, more dimly stained spots that were too small to be 33 visible in the BF channel -presumably PMPs (Fig. 4J ). While T cells very clearly had a strong PS + CD41 low T cells. Statistical significance was determined by paired Wilcoxon test and is 5 indicated by asterisks (ns P > 0.5; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; two-tailed unpaired t-6 test). PBMCs from COVID-19 patients associate to a high degree with PS + platelets and PMPs, 9 while T cells rarely bind whole platelets, but rather PMPs. CD62P, and CD274 compared to their PScounterparts (Fig. 5A, B) . The frequency of T cells, 26 which were positive for these markers, was also increased in PS + EV + T cells (CD41, CD63, 27 CD62P; Fig. 5B ). This finding indicated that PMPs carried the surface molecules of their 28 activated 'parent' platelets to the surface of activated CD8 + T cells in COVID-19 patients. When 29 we analyzed the images of CD41, CD63, CD274, and CD62P stained T cells, we also 30 observed an EV-like staining pattern of these markers, similar to the PS staining (Fig. 5C ). This finding further confirms that T cells can acquire these markers through vesicles. To confirm that these markers originate from activated platelets or their PMPs, we quantified Statistical significance was determined by paired Wilcoxon test and is indicated by asterisks 10 (ns P > 0.5; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). scores ranging from 1.3 to 1.8 (Suppl Fig. 5) . These results indicate that most of the PMPs 13 detected on CD8 + T cells originate from activated platelets and carried platelet markers. 14 15 PS + CD41 + PMPs are preferentially bound to proliferating CD8 + T cells 1 Next, we wanted to assess whether there are functional differences between PMP + and PMP -2 CD8 + T cells. For this, we performed RNAseq analysis of FACS-sorted PS + and PSnon-naïve 3 CD8 + T cells from peripheral blood of 4 patients (Fig. 6A, Suppl. Fig. 1G ). Despite 4 heterogeneity between patients, we could identify gene sets that showed clear enrichment in 5 PS + (Fig. 6B) and PS -T cells (Fig. 6C) . Although PSand PS + CD8 + T cells had few apparent differences in gene expression due to 17 the high degree of variability between individual patients, it is striking that binding of PS + PMPs 18 was associated with increased proliferation. To confirm the RNAseq results, we stained CD8 + T cells with the proliferation marker Ki-67, which labels dividing and recently dividing cells in 20 G1, S, G2, and M phase, but is absent in resting cells 58 and compared the frequency of PS -21 and PS + EV + Ki-67 + cells. PS + EV + CD8 + T cells (Fig. 6D, E) and PS + EV + CD4 + T cells (Fig. 22 Suppl. 6) contained significantly more Ki-67 + proliferating cells than their PS -T cell 23 counterparts from the same patient. In both cases, Ki-67 staining localized to the nucleus and 24 PS + PMPs to the periphery of the same cells (Fig. 6E, Fig. Suppl. 6B ). Our results indicate 25 that PS + PMPs preferentially bind to proliferating T cells and may affect T cells in this cycling 26 stage. We were also interested to find out, whether CD8 + T cells binding PS + PMPs exhibit an effector Our results contribute to the complex clinical picture of thromboinflammation (reviewed in 4,36 ). One of our most surprising findings was the high association of PBMC with PS + PMPs and 6 platelets over the disease course, shown with a novel PS-detecting method. The degree of 7 this association correlated more strongly with disease severity than established laboratory 8 indices such as lymphopenia, IL-6, D-Dimer, fibrinogen, and others measured simultaneously. "Long Covid" is a phenomenon that occurs in around 10% of COVID-19 patients and seems 35 to be associated with persistent tissue damage in severe cases. However, also patients with 36 mild COVID-19 disease scores might be affected 66 . We identified PS + PMP + PBMCs of patients during many weeks post initial diagnosis with only minimal signs of returning to normal 1 levels. Therefore, prolonged adverse effects of PS + PMPs on the immune system could 2 contribute to "long COVID". In vitro, platelets can inhibit proliferation, cytokine production, and PD1 expression of T cells 10 69 . Since PMPs derive from platelets, they may also have similar functions as their 'parent'-11 platelets. We found that between 10-80% of CD8 + T cells were associated with PD- Two recent studies showed that in COVID-19 patients, hyperactivated platelets could form 24 aggregates with leukocytes and macrophages 51,75 . As these previous studies relied on 25 conventional flow cytometry, but not IFC, they could not differentiate between CD41 + platelets 26 and CD41 + PMPs. Also, they did not analyze PS + PMPs on the surface of live lymphocytes. The Ethics Committee approved the study of the LMU Munich (No: 20-308; 18-415), and 5 patients included (³ 18 years, mean age 63, Suppl. Table 1 ) consented to serial blood 6 sampling. Additional approval was obtained for the analysis shown here (No. 20-308) and for 7 the use of blood samples from healthy donors (No. 18-415) . For this study, patient data were 8 anonymized for analysis, and blood samples were collected between April 2020 and February Recovered donors (RD, n=12, mean age 40, Suppl. Table 2 ) were adults with a prior SARS- CoV-2 infection (³ 69 days post positive PCR test), who were either diagnosed in the 18 ambulance or released from the hospital with WHO score 1-2. Healthy donors (HDs, n=35, mean age 39, Suppl. Table 3) Imaging flow cytometry and data analysis 23 Data analysis was performed using the IDEAS software (Version 6.2, Luminex). Compensation matrices were generated using single stained samples and applied to the raw 25 data, and data analysis files were created. Unfocused events were excluded from the analysis 26 based on gradient max feature values. PS + cells were gating using FMO controls. 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