key: cord-0755720-45slgs9s
authors: Winheim, Elena; Rinke, Linus; Lutz, Konstantin; Reischer, Anna; Leutbecher, Alexandra; Wolfram, Lina; Rausch, Lisa; Kranich, Jan; Wratil, Paul R.; Huber, Johanna E.; Baumjohann, Dirk; Rothenfußer, Simon; Hellmuth, Johannes C.; Scherer, Clemens; Muenchhoff, Maximilian; Bergwelt-Baildon, Michael von; Stark, Konstantin; Straub, Tobias; Brocker, Thomas; Keppler, Oliver T.; Subklewe, Marion; Krug, Anne B.
title: Impaired function and delayed regeneration of dendritic cells in COVID-19
date: 2021-05-26
journal: bioRxiv
DOI: 10.1101/2021.05.26.445809
sha: fdbd55a001550084f17bac14693e3bf204a1bc59
doc_id: 755720
cord_uid: 45slgs9s

Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DC) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute disease to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage- HLADR+ cells lacking DC markers. Increased frequency of the recently discovered CD163+ CD14+ DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of PD-L1 in conventional DC (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate naïve CD4+ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients.

Coronavirus disease 2019 (COVID-19), caused by novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), has emerged in December 2019 (1) and is currently causing a global health emergency. COVID-19 is characterized by diverse clinical manifestations ranging from asymptomatic, mild, moderate, to severe disease, including pneumonia which may progress to acute respiratory distress syndrome and multi-organ failure (2) . Exacerbated systemic inflammatory responses and thrombophilia frequently leading to cardiovascular complications are hallmarks of the severe form of the disease (3) . Several contributors to a more severe disease outcome have been identified so far, such as age, male sex, comorbidities, immunosuppression, autoantibodies against type I IFN and genetic variants. The disease course is strongly influenced by the dynamic interaction of the virus with the immune system (4, 5) . Disease severity was shown to correlate strongly with reduced lymphocyte and increased neutrophil counts in the blood as well as high concentrations of inflammatory cytokines such as IL-6, TNF-a, IL-1b and chemokines (e.g. CXCL10 and CCL2) (2, 6, 7) . Antibody and T cell responses were found in over 90 % of convalescent individuals (5, (8) (9) (10) including T follicular helper cell activation and plasma cell expansion (10) (11) (12) .

Immunological memory develops after natural infection lasting at least 6-8 months (13, 14) .

As highly efficient antigen-presenting cells (APCs), DCs are essential in recognizing pathogens, orchestrating innate and adaptive immune responses and secreting inflammatory mediators. Each DC subpopulations has specific functions in the antiviral immune response. Conventional DC (cDC) are highly efficient in presenting antigens and stimulating naïve T cells to expand and differentiate. While cDC1 are specially equipped for cross-presentation of antigens to CD8 + T cells, cDC2 shape Th cell responses (15). DC3 in human blood share characteristics of both cDC2 and monocytes but are distinct in ontogeny and may exert specific functions, but their roles in peripheral tissues and during immune responses are still unclear. In COVID-19 patients, an overall reduction of cDC subsets in the blood was observed in several studies (16) (17) (18) (19) and activated cDC2 were found to accumulate in the lungs of critically ill COVID-19 patients (18). Plasmacytoid DC (pDC), which rapidly produce antiviral type I interferons and inflammatory chemokines are reduced in numbers and functionally impaired in COVID-19 patients (7, 16, 17, 20, 21) .

Monocytes are quickly recruited to inflammation sites and can differentiate into macrophages and monocyte-derived DCs (22). In COVID-19, the recruitment of monocytes into the inflamed lung and subsequent production of proinflammatory cytokines could contribute to disease progression and tissue damage (18, 23-25). However, in patients with severe COVID-19 monocytes and DCs in the blood were found to express lower levels of HLADR and CD86 (16, 17, 19, 20, (26) (27) (28) (29) (30) .

In this study, we sought to gain an in-depth understanding of the dynamic changes in frequencies, activation status, and functionality of blood monocyte and DC subsets in correlation with adaptive immune responses and disease severity in COVID-19 patients. We observed a long-lasting reduction of DC subpopulations with an expansion of proliferating Lineage -HLADR + cells lacking DC markers and delayed regeneration. High-dimensional longitudinal flow cytometric analysis revealed an early type I IFN induced response and a longer-lasting PD-L1 hi CD86 lo phenotype in DC3 and classical monocytes. This dysregulated activation was associated with a reduced ability to stimulate T cells and correlated with disease severity. CD163 + CD14 + cells within DC3 increased in the patients with more severe disease and correlated with inflammation and subsequent activation of Tfh cells and B cells, but not antibody titers. Our results provide evidence for long-lasting aberrant activation and delayed regeneration of circulating APCs in COVID-19.

From 65 patients with PCR-confirmed SARS-CoV-2 infection, a total of 124 samples of PBMC were used for flow cytometric analysis. Patients with active COVID-19 (mild/moderate or severe) were compared with recovered patients and a control group including healthy donors and SARS-CoV-2-negative patients (see Fig. 1a and Table S1 for a detailed description of the cohorts).

COVID-19 severity was assessed using an ordinal scale from 1 to 8 adopted from the World Health Organization (31). The maximal value (WHO max) reached by the patients in our cohort correlated with laboratory markers of inflammation and altered peripheral blood leucocyte composition that are associated with disease severity (Fig. 1b) . We first characterized monocytes and DCs in PBMCs by multi-dimensional flow cytometry (Fig. 2a) . In line with published observations, we observed a relative reduction of monocytes in patients with mild/moderate disease and an increase of low-density neutrophils within the PBMC in a subgroup with more severe disease (Figure 2b, S1) (28).

The percentage of cells within the DC gate (Lin -HLADR + CD14 -CD88/89 -CD16 -) tended to be lower in patients than in controls. Within CD88/89 + monocytes we found a relative increase of classical CD14 + CD16monocytes (mo 1) and a decrease of CD14 lo CD16 + non-classical monocytes (mo 2) in patients with mild/moderate and severe disease in our cohort (Fig. S4 ), confirming published results (28, 29). Mo 2 were significantly reduced and mo 1 concomitantly increased compared to controls within the first 15 days after diagnosis recovering thereafter (Fig. S4) .

Focusing on DCs, we found a significant relative reduction of cDC1, cDC2, and pDC within the Lin -HLADR + CD14 -CD88/89 -CD16population in COVID-19 patients compared to controls. tDCs showed significantly lower frequency in severe patients (Fig. 2 c) . At the same time, a population of cells lacking typical DC markers such as CD1c, CD141, CD123, and CD11c but expressing HLADR and partially CD86 ( Fig. 2c and Fig. S1 ), after that called non-DCs, was found to be significantly expanded within the Lin -HLADR + CD14 -CD88/89 -CD16 -

showed that these cells cluster separately from differentiated DC subpopulations (Fig. S1 ). We analyzed the expression of several markers of known progenitor cells and found this population to be CD34 -CD127 -CD117 -CD115with varying expression of CD45RA and detection of proliferation marker Ki67 (Fig. S1 ). This proliferative HLADR + CD86 +/population, therefore, did not phenotypically overlap with a defined progenitor population. The increased frequency of this DC-like population was long-lasting and could still be observed even in recovered patients more than 60 days after primary diagnosis of COVID-19 (Fig. 2d) .

Changes in blood DC numbers and subset composition after bacterial or viral infection are highly dynamic (32) . We, therefore, analyzed the frequencies of DC subsets within total DCs (excluding the non-DC fraction) at different time points (Fig. 2e) . While the percentages of cDC2, tDC and DC3 within differentiated DCs (after exclusion of non-DCs) were not consistently altered in patients versus controls, the frequencies of cDC1 and pDCs were significantly reduced at the earliest time points (£ 3 days after diagnosis) and largely restored in recovered patients (Fig. 2e) . Our results show that all subpopulations of differentiated circulating DCs are relatively reduced with cDC1 and pDCs being most affected.

Shift towards CD163 + CD14 + cells within DC3 correlates with COVID-19 disease activity and inflammatory markers.

DC3, which share phenotypic and functional features of cDC2 and monocytes, represent the largest subpopulation of DCs in the blood. Differential expression of CD163 and CD14 marks different stages of maturation and activation in DC3 (33, 34) , and an increased frequency of CD163 + CD14 + blood DC3 with proinflammatory function exists in patients with active SLE. We, therefore, hypothesized that the CD163 + CD14 + fraction within DC3 is expanded also in COVID-19 patients. We observed a significantly increased frequency of CD163 + CD14 + cells and decreased frequency of CD163 + CD14cells in the DC3 subset in COVID-19 patients compared to controls. This shift was most pronounced in patients with severe disease (Fig. 3a-d) and in samples taken up to 20 days after diagnosis (Fig. 3c) . The percentage of CD163 + CD14 + cells within DC3 returned to the level of healthy controls in the majority of recovered patients ( Fig. 3b and c) . The frequency of CD163 + CD14 + DC3 correlated positively, and the frequency of CD163 + CD14 -DC3 correlated negatively with disease severity (WHO Max), maximal CRP, and maximal IL-6 values during hospitalization and with the actual CRP values at the time of sampling ( Fig. 3e and f). Thus, the shift towards more mature CD163 + CD14 + DC3 in COVID-19 patients was a persistent phenotype associated with inflammation and higher disease activity.

Early transient expression of Siglec-1 and persistent CD86 lo PD-L1 hi

In addition to the described dynamic changes in DC and monocyte subset composition after SARS-CoV-2 infection, we postulated that the expression of costimulatory molecules, activation markers and chemokine receptors in these cell types is altered in patients with active COVID-19. A high-dimensional spectral flow cytometry analysis was performed on PBMC of 20 patients with mild/moderate disease, 6 patients with severe disease and 11 healthy donors (see Table S1 for a description of this subcohort). Expression levels of the indicated markers were compared between these 3 groups for each DC and monocyte subpopulation (shown in the heatmap in Fig. 4a ). Costimulatory molecule CD86 was downregulated in cDC subsets, mo 1 and mo int populations in patients compared to controls. HLADR expression in mo 1 and DC3 was downregulated only in severe disease and upregulated in mild/moderate disease. At the same time, CD40 and programmed death-ligand 1 (PD-L1) expression in DC and monocyte subsets were increased in both patient groups indicating opposing expression of costimulatory and regulatory molecules (Figure 4a and b) . The PD-L1/CD86 ratio in DC3 was increased in patients until late time points (Fig. 4c ). It correlated with inflammatory markers and disease severity and segregated patients from controls and patients with mild disease from patients with more severe disease in principal component analysis ( Fig. 4h and i) . Investigating the whole cohort (65 patients), we detected a distinct CD86 lo PD-L1 hi DC3 subpopulation, which was also present in cDC2 but not in monocyte populations ( Fig. 4b and d) . These CD86 lo PD-L1 hi DC3 and cDC2 were significantly enriched in patients with severe COVID-19 ( Fig. 4d) . Considering all samples measured, a sizable population of CD86 lo PD-L1 hi DC3 (> 10%) was found in 7.8 % of samples of the mild/moderate group, 43.3. % of the severe group, 33.3% of the recovered group and 8.3 % of controls (data not shown). This subpopulation had expanded in COVID-19 patients with and without glucocorticoid therapy (Fig. S2) . None of the healthy donors in the control group, but 3 non-COVID-19 control patients had more than 10% of the CD86 lo PD-L1 hi DC3. Two control patients with a high percentage of this subset suffered from COPD and interstitial lung disease indicating that this subset can also be found in other pathologies associated with prolonged inflammatory responses.

Higher expression of the CD163 was detected in monocytes and DC3 of COVID-19 patients with severe disease. TREM-1 was most highly expressed in mo 1 and mo int and significantly upregulated in mo 2 of COVID-19 patients.

Expression of CD143 (angioconverting enzyme, ACE) was increased in monocyte subpopulations, cDC2, DC3, and tDCs in COVID-19 patients compared to healthy controls (Fig. 4a) , especially at early time points (Fig. S4) .

ACE2, the primary entry receptor for SARS-CoV-2 was barely detectable on the surface of peripheral blood monocytes and DCs and not induced in COVID-19 patients compared to controls (Fig. S2 ). CD33 was found to be downregulated in all APC populations of the patients especially in severe disease (Fig. 4a ). This may be due to older age, as CD33 was also reduced in older compared to younger healthy donors (Fig. S2 ). CD143 expression was also influenced by age, but the difference between COVID-19 and healthy controls was more significant than the difference between the age groups ( Fig.   S2 ). We did not detect significant differences in expression levels between young and old healthy donors in other markers (Fig. S2 ).

CCR2 was found to be expressed at higher levels in COVID-19 patients than controls in all monocyte and DC subpopulations except pDC ( Fig. 4a and b) .

The CCR2-CCL2 axis is crucial for the recruitment of inflammatory monocytes to the site of inflammation or infection. It could similarly be involved in the recruitment of DC3, which expressed comparably high levels of CCR2 as classical and intermediary monocytes. CXCR3, which mediates chemotaxis in response to IFN-induced inflammatory chemokines (CXCL9, CXCL10, CXCL11), was also upregulated in COVID-19 patients' DC3, cDC2 and monocyte subsets, but downregulated in cDC1, tDC, and pDC. CXCR3 expression in DC3 was significantly higher in patients with severe than mild/moderate disease. CX3CR1, which is linked with patrolling ability and survival of monocytes, was downregulated in cDC2, DC3 and monocytes in our patient cohort (Fig. 4a, b) . These results show that circulating cDC and monocyte subpopulations in COVID-19 patients are poised to migrate in response to inflammatory chemokine ligands.

Type I IFN-induced Siglec-1 (CD169) was strongly upregulated predominantly in DC3 and mo 1 in the majority of patients sampled until 4 days after diagnosis and in a small subgroup of patients sampled until 15 days after diagnosis, indicating an early transient type I IFN response in most of the patients. We observed rapid downregulation of Siglec-1 expression in longitudinally sampled patients (Fig. 4e) . Unbiased mapping of the pooled high-dimensional dataset showed that DC3 were continuously distributed between cDC2 and CD14 + monocytes and contributed to a cluster of Siglec-1 hi cells, which also contained mo 1 and some mo int ( and mo 1 was persisting and more pronounced in severe disease.

Increased myelopoiesis has been described in COVID-19 patients (28, 35) .To understand if the altered phenotype of DCs and monocytes was caused by an enhanced recruitment of immature recently generated cells from the bone marrow, we analyzed Ki67 expression as a marker of ongoing or recent proliferation. Even though DCs were reduced in frequency, we found a sizable population of Ki67 + cells in all cDC subtypes which tended to be highest in the mild/moderate group (Fig. 5a, b) . tDCs and the HLADR + non-DC population had the highest frequencies of Ki67 + cells even in healthy/non-CoV controls which was further increased in COVID-19 patients consistent with their precursor/progenitor function (Fig. 5a) . The percentage of Ki67 + mo 1 was significantly increased in patients with active disease compared to controls (Fig.   5c , d). Increased Ki67 expression was detected in DCs and monocytes of recovered patients and even later than 60 days after diagnosis in some patients indicating enhanced cellular turnover until late timepoints ( Figure 5 a-d). The plasma concentrations of FLt3L and GM-CSF, growth factors, which can promote the generation and expansion of DCs and monocytes, were slightly higher in patients compared to heathly controls, especially in those with mild or moderate disease severity (Fig. S4 ).

We hypothesized that the unusual phenotype of cDCs with downregulated CD86 (and HLADR in severe cases), and upregulated PD-L1 is caused by enhanced recruitment of immature DCs from BM to blood and should hence be found in the Ki67 + fraction. Therefore, we compared the expression of these markers on the surface of Ki67 + and Ki67cells. Remarkably, we found higher expression of CD86 and HLADR and lower expression of PD-L1 in the Ki67 + fractions of cDC2 and DC3 (Fig. 5f, g) , suggesting that this phenotype alteration in DCs of COVID-19 patients was not caused by the recruitment of immature progenitors from the bone marrow, but could have been induced by external factors such as inflammatory mediators in the blood. We detected increased from COVID-19 patients, which had lower CD86 expression (see Fig. 6e ), induced significantly less proliferation and CD69 expression in T cells than DC3 from healthy donors irrespective of glucocorticoid therapy (Fig. 6a, b) . Reduced T cell proliferation was also observed in cocultures with monocytes from COVID-19 patients (Fig. 6c ). Proliferation and CD69 expression of CD4 + T cells in response to stimulation with anti-CD3/CD28 were comparable between patients and controls. Therefore, the reduced T cell response in cocultures with DC3 or monocytes was not due to impaired responsiveness of the patients' T cells but to the reduced costimulatory activity of DC3 and monocytes. We also detected lower concentrations of the cytokines IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17A, IFN-g, and TNF-a in cocultures of CD4 + T cells and DC3 from patients than from controls consistent with the reduced T cell activation. In response to CD3/CD28 stimulation, CD4 + T cells from patients produced similar amounts of most of these cytokines and even higher amounts of IL-5 and IL-10, indicating that their ability to differentiate into cytokine-producing Th cells was not generally impaired (Fig. 6d) . These results show that phenotypic changes are accompanied by functional impairment of circulating DC3 and monocytes in COVID-19 patients.

Reduced numbers, phenotypic alterations and impaired costimulatory function of circulating DC and monocyte subpopulations found in our patient cohort could affect adaptive immune responses. We, therefore, investigated the frequency of blood T and B cell subpopulations and their activation status.

Lymphocyte counts and percentages correlated inversely with disease severity in our patient cohort as expected (see Fig. 1 ) and the percentages of CD3 + T cells were reduced, especially in the group of patients with severe disease (Fig.   S5 ) consistent with T cell lymphopenia. We observed a shift from naïve (CD45RA + ) to non-naïve (CD45RA -) CD4 + T cells in the severe group, while the frequency of CXCR5 -Th and CXCR5 + PD-1 + Tfh-like cells within CD4+ T cells was not considerably altered in COVID-19 patients ( Fig. 7a and S5 ). As specific T cell activation in response to acute viral infection can be detected by increased HLADR and CD38 expression (36) we investigated the coexpression of these molecules. The percentage of activated Th and Tfh-like cells was higher in patients with active disease compared to controls and recovered patients ( Fig. 7b and c) . Increased activation was observed in samples taken until 30 days after diagnosis (Fig. 7d) . The CXCR3 -CCR6 -Th0/2 cell fraction was increased in patients with severe disease but contained only a low percentage of activated cells. Circulating Th and Tfh-like cells expressing CXCR3 and or CCR6 showed increased activation in patients with active COVID-19 (Fig. S5 ). In the CD8 + T cell compartment, we observed a reduction of CD45RA + CD27 + naïve CD8 + T cells with a concomitant increase in CD45RA -CD27 + CD8 + T cells containing TCM ( Fig. 7e and S5 ). CD8 + T cell activation, which was detected mainly in the TCM and TEM containing fractions was increased in patients with active COVID-19 vs controls and recovered patients ( Fig. 7e -h). The highest frequencies of activated CD8 + T cells were observed between 6 and 15 days after diagnosis (Fig. 7g) .

B cell frequencies were similar in patients and controls, but the percentage of CXCR5 + B cells was significantly reduced in active COVID-19 ( Fig. 8a and b) .

We detected decreased naïve and memory but increased class-switched memory B cells compared to controls in a subgroup of COVID-19 patients ( Fig.   8c and g). Antibody secreting cells (ASC) were expanded in some but not all of the patients and returned to the level of healthy controls in the recovered patients ( Fig. 8d and g) . The expansion of ASC was already seen at early time points and persisted until 20 days after diagnosis and even longer in some patients (Fig. 8e ). Anti-SARS-CoV-2 spike S1 IgG antibodies were detected in 70.9% and anti-SARS-CoV-2 nucleocapsid IgG antibodies in 77.8% of patients at the latest available timepoint (n=54-55) and in 90.3 % and 93.5 % of patients sampled later than 15 days after diagnosis (n=32) indicating specific antibody production in the majority of patients (data not shown). Antibody levels To better understand the connection between the innate and the adaptive immune response, we performed a correlation analysis of innate parameters in the early phase (day 0-10) and adaptive parameters in the later phase (day 10-25 after diagnosis) in longitudinally sampled patients (Fig. 8h ). The expression of CCR2, CXCR3, HLADR and CD40 in DC3 and monocytes correlated with subsequent CD8 + T cell activation and inversely with anti-S1 antibody levels indicating that this APC phenotype could be relevant for CD8 + T cell activation in response to SARS-CoV-2 infection. The frequency of CD163 + CD14 + cells within DC3 and the PD-L1/CD86 ratio in DC3 correlated positively with the frequency of activated Tfh cells and cs-mem B cells (Fig. 8i) and ASC, but not with antibody titers (Fig. 8h) . This DC3 phenotype, as well as Tfh and B cell activation, also correlated with increased inflammatory markers (CRP, IL-6), neutrophil/lymphocyte ratio and disease activity ( 

In this study, we provide an in depth characterization of DC and monocytes subpopulations in the blood of hospitalized patients with mild, moderate or severe COVID-19 compared with healthy controls. Changes in DC/monocyte composition and phenotype were connected with parameters of inflammation and activation of adaptive immunity. We found that all DC subpopulations were profoundly and persistently depleted from the blood in COVID-19 patients while The long-lasting reduction of all DC subsets in the blood which occurred in patients with mild/moderate and severe disease, was accompanied by increased proliferation, which -although detectable for a long time after diagnosis -did not fully restore the circulating DC compartment. The reduction in DCs, which has also been observed in previous studies (16, 18, 19, 37) may be due to increased emigration from the blood and sequestration in tissues, auch as the inflamed lung or lymphoid tissues. We found that similar to monocytes CCR2 and CXCR3 were upregulated in DC3 of COVID-19 patients suggesting that DC3 together with monocytes may be recruited from the circulation to the infected lung in response to a gradient of CCL2 and CXCR3 ligands CXCL9/10/11 (38) . Indeed inflammatory chemokines CCL2, CCL3 and CCL4 have been found at higher concentrations in the airways compared to the plasma in patients with severe COVID-19 (24). cDC2 may follow a similar route, while cDC1 did not upregulate these receptors and pDCs even showed downregulation of CCR2 and CXCR3. This is consistent with preferential recruitment of cDC2 versus cDC1 to the lung (18) and low numbers of pDCs found in the airways and lungs of COVID-19 patients (38, 39) . cDC1 and pDC could be reduced due to sequestration in lymphoid tissues or enhanced cell death as shown for pDCs (40) . Reduction of circulating DCs due to productive infection by SARS-CoV-2 is unlikely. We did not detect expression of the major entry receptor ACE2 on blood DC and monocytes in accordance with previous reports (41, 42) .

While differentiated DC subsets were reduced, we found that Lineage -HLADR + CD86 +/-CD45RA +/proliferating cells lacking typical DC markers were greatly expanded in the blood of COVID-19 patients. Their phenotype did not overlap with that of previously described DC or monocyte/macrophage or lymphoid progenitor populations. Expression of HLADR and lack of CD33, CD14 and CD15 expression indicated that they are not typical myeloid derived suppressor cells. It is unlikely that these cells were activated proliferating innate lymphoid cells or precursors due to lack of CD127 expression. A similar immature HLADR + cell type with poor antigen-presenting capacity and a low response to TLR stimulation was found to be expanded at the expense of cDCs and pDC in the blood of patients with cancer or acute malaria (43, 44) . The long duration of cDC reduction and immature HLADR + cell expansion in the blood of COVID-19, even in convalescent patients, indicated delayed regeneration of the DC compartment. The "non-DCs" described in our study could be an immature DClike population appearing in COVID-19 due to hyperinflammation and increased myelopoiesis.

We observed a shift towards a more mature CD163 + CD14 + phenotype within the DC3 subset in acute COVID-19 correlating with disease severity and inflammatory markers. A similar change in DC3 phenotype has been observed in the blood of SLE patients and in melanoma patients coinciding with inflammation (33, 45) . CD163 + CD14 + DC3 have been shown to express higher levels of proinflammatory genes, secrete more proinflammatory mediators and induce Th17 polarization more efficiently than CD163 -CD14 -DC3 (33). It is still unclear, however, if the CD163 + CD14 + phenotype of DC3 in the blood of COVID-19 patients contributes to or is a byproduct of the inflammatory response.

Clusters of monocytes and DC3 with high expression of Siglec-1 appeared in the blood of COVID-19 patients at early timepoints and disappeared at later time points, indicating a robust but transient type I IFN response. Consistent with this dynamic expression pattern Siglec-1 was shown to serve as a negative feedback regulator of type I IFN production in response to viral infection (46).

Siglec-1 + expression on monocytes was a promising biomarker for the early diagnosis of COVID-19 in the emergency room (47). In contrast to this study, we detected high Siglec-1 expression in monocytes and DC3 only in half of the patients analyzed, most likely due to later sampling timepoints. We also found ACE/CD143 to be upregulated at early timepoints in monocytes and DC3 of COVID-19 patients correlating with markers of inflammation. Increased expression of this carboxypeptidase has been observed in bacterial infections but not typically in viral infections (48). ACE/CD143 was found to promote TNFa and IL-6 production, adhesion and transmigration of myeloid cells in response to CCL2 (49) and could therefore be involved in tissue migration and cytokine response of monocytes and DC3 in COVID-19 patients.

The expression of costimulatory molecules was differentially regulated in different blood APC subsets. In pDCs, we observed increased expression of CD86, CD40 and PD-L1, but did not detect diversification into distinct activated pDC effector subsets as described by Onodi In summary, we provide evidence that the depletion of circulating DCs, delayed regeneration and phenotypic alteration are long-lasting effects of COVID-19

infection. The persistent phenotypic alteration and dysfunctionality of circulating DCs and monocytes was especially apparent in more severe disease and associated with the prolonged inflammatory response. The consequences of depletion and dysfunctionality of blood APCs are not known. While these changes may reflect a regulatory mechanism to reduce overactivation of the immune response in COVID-19, the described long-lasting alterations together with the profound lymphopenia could make patients more vulnerable to secondary infections, which were shown to be more prevalent in COVID-19 patients (55, 56). This needs to be taken into account in the clinical management of COVID-19. 

The following commercial CE in vitro diagnostics (IVD) marked assays were 

Statistical analysis was performed using GraphPad Prism 9. 

The study was conducted in the framework of the COVID-19 Registry of the LMU University Hospital Munich. Written informed consent was received from participants prior to inclusion in the study and patient data were anonymized for analysis. The study was approved by the local ethics committee (No. 20-245).

Additional approval was obtained for the analyses shown here (No. 592-16) and for the use of blood samples from healthy donors (No. 18-415).

Conceptualization 

(A) Gating strategy for DC and monocyte subtypes in the blood: Within HLADR + Lineage (CD3, CD15, CD19, CD20, CD56, CD66b), negative (Lin -) cells monocytes were gated as CD88/89 positive cells and separated into mo 1 (CD14 + CD16classical monocytes, mo int (CD14 + CD16 + intermediate monocytes, mo 2 (CD14 lo CD16 + non-classical monocytes). HLADR + Lin -CD88/89 -CD16cells were regated on HLADR positive cells (DC gate). Within the CD123 + DC fraction pDCs (Siglec1 -Axl -) and tDCs (Axl + and/or Siglec1 + ) were distinguished. Within the CD123 -DC fraction cDC1 (CD141+ CD1c lo ), cDC2 (CD141 -, CD1c + , CD5 + ), DC3 (CD141 -CD1c+ CD5 -DCs) and "non-DC" (CD141 -CD1c -) were identified. DC3 were further separated into CD163 -CD14 -, CD163 + CD14and CD163 + CD14 + DC3 subsets. (B) Percentage of neutrophils (Lin + CD88/89 + CD16 + ), monocytes (Lin -, HLADR + , CD88/CD89 + ) and DCs (Lin -, HLADR + , CD88/CD89 -) of living PBMC. Healthy donors (=H, black symbols, n=28), hospitalized COVID-19 negative patients (=white symbols, n=4), acute COVID-19 patients with mild/moderate (=M, red symbols, 

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Left: CD8 + naïve and memory subsets according to CD27 and CD45RA expression. Right: CD38 and HLADR expression in CD8 + T cells. Numbers indicate percentages. (F) Percentage of activated cells within CD8 + T cells in the indicated groups (as in C). (G) Percentage of activated cells within non-naïve CD8 + T cells at different time points after diagnosis (n = 105, shown as in D). (H) Percentages of activated T cells within naïve and memory CD8 + T cell subsets in the indicated groups (as in C)