key: cord-331208-bgh1a14p
authors: Garcia, M.; Kokkinou, E.; Carrasco Garcia, A.; Parrot, T.; Palma Medina, L. M.; Maleki, K. T.; Christ, W.; Varnaite, R.; Filipovic, I.; Ljunggren, H.-G.; Bjorkstrom, N. K.; Folkesson, E.; Rooyackers, O.; Eriksson, L. I.; Sonnerborg, A.; Aleman, S.; Stralin, K.; Gredmark-Russ, S.; Klingstrom, J.; Mjosberg, J.; Group, the Karolinska KIK COVID-19 Study
title: Innate lymphoid cell composition associates with COVID-19 disease severity
date: 2020-10-14
journal: nan
DOI: 10.1101/2020.10.13.20211367
sha: 
doc_id: 331208
cord_uid: bgh1a14p

Objectives: The role of innate lymphoid cells (ILCs) in coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is unknown. Understanding the immune response in COVID-19 could contribute to unravel the pathogenesis and identification of treatment targets. To describe the phenotypic landscape of circulating ILCs in COVID-19 patients and to identify ILC phenotypes correlated to serum biomarkers, clinical markers, and laboratory parameters relevant in COVID-19. Methods: Blood samples collected from moderately (n=11) and severely ill (n=12) COVID-19 patients as well as healthy control donors (n=16), were analyzed with 18-parameter flow cytometry. Using supervised and unsupervised approaches, we examined the ILC activation status and homing profile. Clinical and laboratory parameters were obtained from all COVID-19 patients and serum biomarkers were analyzed with multiplex immunoassays. Results: ILCs were largely depleted from the circulation of COVID-19 patients compared with healthy controls. Remaining circulating ILCs from patients revealed increased frequencies of ILC2 in moderate COVID-19, with a concomitant decrease of ILC precursors (ILCp), as compared with controls. ILC2 and ILCp showed an activated phenotype with increased CD69 expression, whereas expression levels of the chemokine receptors CXCR3 and CCR4 were significantly altered in ILC2 and ILCp, and ILC1, respectively. The activated ILC profile of COVID-19 patients was associated with soluble inflammatory markers, while frequencies of ILC subsets were correlated with laboratory parameters that reflect the disease severity. Conclusion: This study provides insights into the potential role of ILCs in immune responses against SARS-CoV-2, particularly linked to the severity of COVID-19.

chemokine receptor CXCR3 20 . Hence, CXCR3 serves as a marker to define the ILC1 129 subset in peripheral blood. well as in the WHO guidelines 25, 26 . 164

Peripheral blood sample from all COVID-19 patients was collected 5 to 24 days 165 (median 14 days; IQR 5-24 days) after symptom debut and 0 to 8 days after 166 hospitalization. 167

Sixteen donors (6 females and 10 males; median age 54 years; age range 168 between 34 -69 years), all SARS-CoV-2 IgG seronegative and symptom-free at the 169 day of sampling, were included as a control group for this study. PBMCs was carefully decanted into a new tube, followed by two washes with PBS 184 containing 10% FCS. Platelets were removed by centrifuging the samples at 400 g 185 for 10 min. Pellets were resuspended, cells counted and subsequently stained for flow 186 cytometry. 187

Vacutainer serum tubes with spray-coated silica (BD Biosciences). After coagulation 189 for up to 2 hours at room temperature (RT), serum was isolated by centrifugation at 190 2000 g for 10 min and immediately stored at -80 ºC for later analysis. Table S1 in the Online Repository) for 20 min at RT in the dark. Cells were 196 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. Phenograph plugin and including the same markers as for UMAP and parameters k = 228 30 and Run ID = auto. Finally, ClusterExplorer plugin was used to study the phenotype 229 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in to normalize data to minimize both intra-assay and inter-assay variation. 254

Additionally, several soluble analytes were also measured in serum or plasma 255 by use of custom made multiplex Magnetic Luminex Screening assays (R&D 256 Systems), according to the manufacturer's instructions. Serum and plasma were 257 diluted 1:2 prior to analysis in multiplex. 258

Analytes from Olink data and Magnetic Luminex Screening assays that had 259 more than 33% and 25% of missing values, respectively, were excluded from analysis. 260

Left-censored data from the multiplex analysis were imputed using GSimp package 27 261 in R (v. 3.6.0) 28 . 262 263 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

A total of twenty-three patients with either moderate (n=11) or severe (n=12) 292 COVID-19 and sixteen control donors were included in the study (Fig. 1A) . The 293 COVID-19 patients showed profound perturbations in inflammatory markers, 294 coagulation factors, organ/muscle damage markers as well as biochemical and 295 hematological parameters (Fig. S1) . A detailed summary of the patients' clinical and 296 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.13.20211367 doi: medRxiv preprint laboratory parameters is presented in Tables 1-2. A timeline summarizing the major 297 clinical events of the patients is shown in Fig. 1B . 298

For the identification and analysis of peripheral blood ILCs we used 18-299 parameter flow cytometry and a modification of a well-established gating strategy 36 300 (Fig. S2A ). We found that the relative frequencies and absolute counts of total CD127 + 301

ILCs (hereafter referred to as total ILCs), as well as the absolute counts of the specific 302 subsets ILC1, ILC2 and ILCp, were decreased in peripheral blood of COVID-19 303 patients as compared with controls ( Fig. 2A-D) . Further characterization of the 304 remaining circulating ILC compartment showed reduced ILCp frequencies in COVID-305 19 patients, while the frequencies of ILC1 remained unchanged ( Fig. 2C and E) . Of 306 note, the moderately ill group showed elevated frequencies of both total ILC2 (Fig. 2E ) 307

and CD117 -ILC2, as compared with the control group (Fig. 2F) . 308

Taken together, we observed a general ILC depletion as well as compositional 309 changes in the circulating ILC compartment of COVID-19 patients. Interestingly, 310 changes in the relative frequency of ILCs differed between the moderate and severe 311 group, prompting a more detailed phenotypical study of the ILCs. 312

To deepen our understanding of the differentiation, activation, and migration of 313

ILCs during COVID-19, we assessed differentiation (CD56 and NKp44) ( This approach revealed a higher relative frequency of CD69-expressing total 320

ILCs in patients as compared with controls (Fig. 3A) . Additionally, Ki-67, a marker of 321 cell proliferation, was increased in severe compared with moderate COVID-19 but 322 decreased in moderate COVID-19 compared with controls (Fig. 3A) . However, the 323 overall Ki-67 expression level was low in all ILCs regardless of the study group, 324 suggesting either a low proliferating capacity of ILCs in peripheral blood or migration 325 of proliferative ILCs to tissues at an earlier stage. In line with an increase in CD69 in 326 COVID-19 patients, we detected reduced frequencies of total ILCs expressing 327 CD45RA and CD62L, two markers associated with ILC naivety 18 , in severe COVID-19 328 patients (Fig. 3A) . Additionally, we observed alterations in chemokine receptor 329 expression between controls and moderate COVID-19 patients, revealing a reduction 330 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . in the percentage of CXCR3 + and an increase in CCR4 + ILCs. The latter likely reflects 331 the increase in ILC2 frequencies in these patients (Fig. 2E) , as CCR4 is particularly 332 highly expressed on ILC2 (Fig. 3E ). Despite these changes in ILC phenotypes, the 333 PCA analysis could not discriminate between COVID-19 patients and controls on the 334 basis of the total ILC data (Fig. 3B) , suggesting that the COVID-19 related changes in 335 total ILCs are ILC subset specific. 336

Indeed, we observed no changes in expression of markers associated with 337 differentiation and activation in ILC1. There was, however, a slightly increased 338 percentage of CCR4 + ILC1 cells in COVID-19 patients compared to controls (Fig. 3C) . 339

We also detected a reduction of CD56 + ILC1 in COVID-19 patients (Fig. S3A) . PCA 340 analysis illustrated these differences well, showing segregation of the ILC1 data on 341 the basis of CCR4 for COVID-19 patients and CD56 for controls (Fig. 3D) . 342

In contrast, ILC2 displayed an activated phenotype in COVID-19 patients, 343

showing higher frequencies of CD69 + and a tendency towards reduced frequencies of 344 CD62L + cells, as compared with controls (Fig. 3E) . Interestingly, Ki-67, generally 345 expressed at very low levels, was decreased in relative frequency in ILC2 from severe 346 COVID-19 patients compared to the control group, possibly reflecting tissue 347 recruitment of such cells in severe COVID-19. Indeed, we identified dysregulated 348 chemokine receptor expression on ILC2 in COVID-19 patients, specifically CXCR3 349 which was reduced in the patients as compared with controls (Fig. 3E ). PCA analysis 350 provided an illustration of these findings by segregating the ILC2 data from patients 351 and controls based on CD69 expression for patients and chemokine receptors as well 352 as CD62L and CD45RA for the controls (Fig. 3F ). The latter two were, however, not 353 statistically different between the groups (Fig. 3E) . 354

ILCp showed an activated profile in COVID-19 patients, with increased CD69 355 and HLA-DR expression as compared with controls. Additionally, CXCR3 was 356 decreased on ILCp in COVID-19 patients as compared with controls (Fig. 3G) . 357

Although PCA analysis did not separate the ILCp phenotype from patients and controls 358 clearly, there was a tendency towards separation of ILCp from controls on the basis 359 of CD62L and CD45RA (Fig. 3H ). These two markers showed reduced tendency but 360

were not statistically significant in the manual gating (Fig. 3G) . 361

Overall, these findings suggest that the ILCs remaining in the circulation of 362 COVID-19 patients are activated and show an altered expression of chemokine 363 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. Altogether, the unsupervised analysis revealed clusters specifically 388 accumulated in COVID-19 patients compared to controls and the presence of specific 389 ILC phenotypes that associated to the disease severity. Importantly, several of the 390 findings agreed with those obtained by manual gating. Specifically, we confirmed an 391 increase in the relative frequency of CD69 + ILCp and a decrease in CXCR3 + ILCp in 392 both moderate and severe COVID-19, an increase in ILC2 percentage specifically in 393 moderate COVID-19 and an increased CCR4 expression among ILC1 in the patients. 394

To analyze for potential factors involved in the activation and potential 395 recruitment of ILCs to tissues in the COVID-19 patients, we examined correlations 396 between ILCs and a wide array of soluble serum factors ( Fig. S5A-B ). Based on our 397 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.13.20211367 doi: medRxiv preprint findings on altered frequencies of CXCR3 + and CD69 + ILCs in COVID-19 (Fig. 3A) , 398

we focused on selected chemokines (CCL20, CXCL10 and CXCL11) and factors 399 previously reported to be increased in COVID-19 or other viral respiratory disease: IL-400 6, IL-10, IL18R1 and PD-L1 7-9,37-42 . Interestingly, we found that the percentage of 401 activated (CD69 + ) total ILCs and activated ILCp positively correlated with serum IL-6 402 levels in the COVID-19 patients (Fig. 5A) . Additionally, the relative levels of the 403 cytokine IL-10 also positively correlated with the levels of CD69 + ILCs in the COVID-404 19 patients (Fig. 5B) . Moreover, the percentage of CD69 + total ILCs and ILCp 405 positively correlated with CXCL10 levels in the COVID-19 patients (Fig. 5C) , 406

suggesting that this chemokine is related to the increase in the percentage of activated 407

ILCs that remain in the circulation of COVID-19 patients. 408 We searched for potentially relevant correlations between ILCs and the clinical 424 and laboratory parameters. The frequencies of ILC1 and ILC2 correlated with several 425 of the measured parameters, whereas no correlations were found for ILCp frequencies 426 (Fig. S3B) . The percentage of ILC1 positively correlated with LDH levels, which was 427 significantly increased in severe COVID-19 patients (Fig. 6C, Fig. S1 ). ILC1 428 frequencies also positively correlated with parameters that did not differ between the 429 two COVID-19 patient groups, i.e. platelet counts, serum SARS-CoV-2 IgG levels and 430 days post symptom debut (Fig. 6C) . The percentage of ILC2, on the other hand, 431 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.13.20211367 doi: medRxiv preprint negatively correlated with the leukocyte, neutrophil and platelet count (Fig. 6D) . 432

Interestingly, high neutrophil levels, which have been described as partial predictors 433 of disease severity 44 , also contributed heavily to the separation between patient 434 groups in the current study ( Fig. 6A-B) . Of note, neutrophils have been found to inhibit 435 ILC2 function, thus preventing allergic airway inflammation 45 . Additionally, the relative 436 frequency of ILC2 correlated negatively with levels of D-dimer, a coagulation factor 437 that has been suggested as a systemic biomarker of disease severity in COVID-19 438 ( Fig. 6D) 44, 46 . Finally, we observed a negative correlation between ILC2 frequencies 439 and organ/muscle damage markers (myoglobin, troponin T, LDH) and the number of 440 days since symptom debut (Fig. 6D) . These findings suggest that among COVID-19 441 perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.13.20211367 doi: medRxiv preprint in lung tissue repair in humans is unknown and deserves further exploration in COVID-466

In contrast to ILC2, ILCp frequencies were diminished in COVID-19 patients as 468 compared with controls, suggesting their migration to the site of infection or 469 differentiation into mature ILC subsets in the circulation. This has previously been 470 perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

The authors declare that the research was conducted in the absence of any 539 commercial or financial relationships that could be construed as a potential conflict of perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.13.20211367 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in within the mother gate. Bar graphs are shown as median ± IQR, *p < 0.05, ** p < 0.01, 775 *** p < 0.001. Patients with low cell numbers (less than 20 events) in the corresponding 776 gate were removed from the analysis. 777 778 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.13.20211367 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. Patients with less than 10 events per ILC subsets (defined by Phenograph-identified 832 clusters) were excluded from analysis. Statistical differences were tested using the 833 Kruskal-Wallis test followed by Dunn's multiple comparisons test. Bar graphs are 834

shown as median ± IQR, *p < 0.05, ** p < 0.01. ILC and CD69 + ILCp; (D) serum CXCL10 and CXCL11 levels (pg/ml) and the 842 percentage of CXCR3 + ILCs; (E) serum CCL20 levels (pg/ml) and the percentage of 843 ILC2 and CCR4 + ILCs. IL-6, CXCL10, CXCL11 and CCL20 serum absolute levels 844

were measured with Magnetic Luminex Screening assay, and IL-10 relative levels with 845 a proximity extension assay, where data is shown as normalized protein expression 846 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in All rights reserved. No reuse allowed without permission.

perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.13.20211367 doi: medRxiv preprint preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.13.20211367 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.13.20211367 doi: medRxiv preprint All rights reserved. No reuse allowed without permission.

perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted October 14, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . 

Interleukin-18 and IL-18 binding 687 protein

Mild versus severe COVID-19: Laboratory markers

Neutrophils restrain allergic airway 692 inflammation by limiting ILC2 function and monocyte-dendritic cell antigen 693 presentation

Immunologic perturbations in 698 severe COVID-19/SARS-CoV-2 infection. bioRxiv [Preprint

Dysregulation of Immune Response in Patients With 701

COVID-19) in Wuhan, China

Characteristics of Peripheral Lymphocyte Subset 704

Alteration in COVID-19 Pneumonia

Hepatitis C virus infection induces the expression of 707

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