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    S. de Biasi - Marked T Cell Activation, Senescence, Exhaustion and Skewing Towards TH17 in Patients With COVID-19 Pneumonia

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    Susanyi Ervin

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    OR COMMUNICATIONS ARTICLE om owstews OPEN Marked T cell activation, senescence, exhaustion and skewing towards TH17 in patients with COVID-19 pneumonia Sara De Biasi!®, Marianna Meschiari2®, Lara Gibellini!, Caterina Bellinazzi', Rebecca Borella Licia Gozzil, Anna lannone!, Domenico Lo Tartaro@ |, Marco Mattioli@ ', Annamaria Paolini’, Marianna Menozzi?, Jovana Milié?, Giacomo Franceschi2, Riccardo Fantini?, Roberto Tonelli3, Marco Sita’, Mario Sarti®, Tommaso Trenti®, Lucio Brugioni®, Luca Cicchetti’, Fabio Facchinetti® ', Antonello Pietrangelo® |, Enrico Clini® 3, Massimo Girardis*, Giovanni Guaraldi® 7, Cristina Mussini? & Andrea Cossarizza@ '8™ Lucia Fidanza’, The immune system of patients infected by SARS-CoV-2 is severely impaired. Detailed investigation of T cells and cytokine production in patients affected by COVID-19 pneumonia are urgently required. Here we show that, compared with healthy controls, COVID-19 patients’ T cell compartment displays several alterations involving naive, central memory, effector memory and terminally differentiated cells, as well as regulatory T cells and PDT *CDS7* exhausted T cells. Significant alterations exist also in several lineage-specifying transcription factors and chemokine receptors. Terminally differentiated T cells from patients proliferate less than those from healthy controls, whereas their mitochondria functionality is similar in CD4* T cells from both groups. Patients display significant increases of proin- flammatory or anti-nflammatory cytokines, including T helper type-1 and type-2 cytokines, chemokines and galectins; their lymphocytes produce more tumor necrosis factor (TNF), interferon-y interleukin IL)-2 and IL-17, with the last observation implying that blocking IL-17 could provide a novel therapeutic strategy for COVID-19, ‘Department of Medical and Supa Sclences for Chicken and Adults, University of Madena and Rego Emilia School of Medicine, Via Camp) 287, 41125 Madera, tay, Infectious Diseases Cincs, AOU Polcinco and University af Modena and Reggio Envi, via del Pozzo 7, 41124 Maden, Maly. °Respiratory Diseases Unt, AOU Polictnico and University of Modena and Raggio kil, va del Porzo 71 41124 Madena italy.“ Department of Anesthesia and Intensive Care ADU Palteca an Universty at Modena anc Reso Emilia via dl Pe220 /) 124 Maden, aly. "Cina! Mierobielgy Unit. AOU Pelielines, va det Pozzo 71 1124 Modena, aly. Emergency Department, MIAC, AOU Policinico, va de! Pozzo 7, 41124 Modena, tay, ?Labospace, via Alle 4, 20128 Milano, aly, ® National Institue fr Cardiovascular Research, via leneio 4, 40126 Bologna, tal, "These authens contributed equal. Sara De Biss Mansana Mazchae, Mamas sndrasorearzrauninare = NATURE COMMUNICATIONS 02031!3434|Intes//éo rg /10 1038/4467 020 17292 4| www nature comratueconmanitons 1 ARTICLE NATURE COMMUNICATIONS | hitps//doiorg/10:1038/s41467-020-17292-4 respiratory syndrome coronavirus (SARS-CoV-2) that causes Corona Virus Disease-2019 (COVID-19)', is a dramatic threat to our species that is changing daily habits and social behaviors all over the world. In Italy, the first patients with severe pneumonia of unknown origin were observed in Lom- bardy, first conficmed case dating to February 2lst, 2020°. From, that moment, we saw an unpredictable number of patients with severe pneumonia, whose treatment often required admission into intensive care units. Unfortunately, many patients filed in their fight against the vieus, and, as of June 18th, ltaly has counted, ‘more than 35,000 deaths amongst over 240,000 infections ‘The pathophysiology of such virus i not yet completely understood. Previous studies on severe diseases caused by other coronaviruses indicated that pulmonary inflammation is asso ciated with increased plasma levels of proinflammatory cytokines®®, A_ similar phenomenon has been described in patients with COVID-19, who can experience the so-called “cytokine storm”. However, litle is known about the cells that, are involved in the production of these mediators or about the specific immune response of patients with COVID- 19 Currently, we, and others, are observing that COVID-19 is ‘much more severe in elderly patients, especially in those with different comorbidities, diabetes, and obesity, while it appears much less severe in chiklren and pregnant women’, Immu- nological aging is associated with a chronic, subclinical inflam- matory state, defined “inflammaging” where T helper CTH) type | Iymphoeytes play a erucial role, whereas children and pregnant women have a tendency to develop TH2 responses, so producing less proinflammatory molecules". In order to better under stand how immune response and cytokine production are orchestrated, we have deeply characterized T cells, and have studied the functional differentiation of T cells towards TH, ‘TH, or THI7. For this purpose, we have employed an array of flow cytometric approaches combined with sophisticated tech- niques for unsupervised analysis of complex T cell phenotypes, and we have investigated the proliferative capability of diferent CD4! and CDS" T cell subsets, along with mitochondria func tionality in CD4" T cells, We have finally measured plasma levels, ‘of 31 cytokines and the poiyfunctionalty of T calls. A retrospective study performed by our group evidenced that a drug able to block the biological activities of 1-6, such as toci- Tizumab, can significantly reduce invasive mechanical ventilation, or death in severe COVID-19 pneumonia (defined as the con comitant presence of a respiratory rate 230 breaths per minute, blood oxygen saturation <93%, a PaQ,/FiO> ratio <300 mmHg in, oom air and lung infiltrates >50% ofthe lung, filed within 24—48 hy, Even if our results need to be confirmed by randomized, trials it is to note that, in comparison with the control group. COVID-18 patients treated with tocilizumab had an increased prevalence of severe infection. Here we show that COVID-19 patients have reduction in absolute numbers of CD" and CD8* T lymphocytes, which display markers related to activation or exhaustion/senescence, along with altered expression of master regulators and of several, chemokine receptors. Meanwhile, their CD4* cells have an altered cell proliferation but not mitochondria functionality, measured as_mitochondrial_ oxygen consumption and. extra cellular acidification rate (ECAR). In these patients, a massive cytokine storm detectable in plasma is accompanied by an increased in vitro production of tumor necrosis factor (TNE), interferon (IFN)-y, interleukin (IL)-2, as well as by a skewing toward the THI? or Tel? phenotype. This not only underlines, the efficacy of a drug that contrasts cytokine storm, but also suggests the clinical evaluation of novel strategies, based upon the inhibition of the TL-17 pathway. T: dramatic pandemic caused by the severe acute Results Characteristics of the patients. We have studied a total of 39 patients, the first 21 of which were consecutively admitted into the Infectious Diseases Clinics of the University Hospital in Modena over the period of March 12th-March 30th, 2020, whereas the remaining patients were studied between April 15th and May 10th, All patients had symptoms including sore throat, fever, cough, dyspnoea, and chest pain. At admission, SARS- CoV-2 was detected in a nasopharyngeal swab specimen by real- time reverse-transcriptase PCR, and ‘in all of them pneumonia ‘was subsequently conlirmed by X-ray. Supplementary Tables 1=4 show in details the individual clinical and biohumoral char- acteristics of the patients. Note that, in general, they were lym- phopenic (mean: 1150 cells/uL, range: 360-2334), and in some cases Iymphocyte count was very low. For the immunological analyses described below, they were compared with a total of 25, age- and sex-matched healthy controls. Bor those controls in Which we investigated T cell phenotypes, we could measure the lymphocyte absolute number, whose mean was 2320 cells, range: 1580-3130 (controls vs. patients: p<0.0001), ‘Characterization and count of CD4! I cell subsets. We studied M1 and CDS" T calls in patents and controls using 18 pars- rcter flow cytometry. The analysis of the data was then! per formed by using two complementary methodologies. In the fst, wwe used the classical approach based upon the two-dimensional recognition ofa given cell type, followed by a fist gate that was required to identify the population of interest (:e, CDA" or CDs" lymphocytes), followed by sequential gates to idently markers of activation, differentiation, senescence, exhaustion, regulatory CD41 T eels, and memory stem T cells (Tye). Data obtained with this gating strategy were then analyzed by appeo- prise, non-parametric statistical tests, and represented in the Figures as seater plots with means and standard errors of the mean, In the second, we performed an unsupervised analysis that considers the entie, complex scenario presented by CD1" 07 CD8'T cells, This analytical technique uses the multi dimensional information obtained by FlowSOM meta-clustering coupled to & dimension-reduction method such as the Uniform Manifold. Approximation and Projection (UMAP). Heat maps finaly seport statistical analysis (see Methods for details). ‘The same approach was used to study CDS! T lymphocytes. Figure La shows the alorementioned gating strategy that we used to compare CD41" T cals of heathy donors and COVID-19 patients. A. first gate was set on the parameters “time” and "ability, then a second gate was set on CD3 CD45! cells (.e, T lymphocytes), among. which we identified CD4* or CD8t Iymphocytes. Within the CD1* population, we analyed markers commonly related to T cell avation (HLA-DR and CD38), Senescence and exhaustion (CD57 and PDI), liferentiation (CDISRA, CCRI, CD28, and CD27), regulatory T cells (Treg, considered as CD23 and CD127%"), among which we could ‘entity diverse differentiation stages, and finally Tye (se, CD9S* calls within CDASRA'CCR7 CD27 CD28" naive lymphocytes). Figure 1b shows that paticnts had a similar percentage of CDA! T calls to conteels, but the absolute number ofthese cells was significantly lower. A similar phenomenon was observed a8 Taras nave, central memory, and elector memory CD1" T cells, were concerned, whereas the percentage but not the absolute number of terminally diferentiated (TE) cells was higher in patients Figure lc reports that patients also expressed higher percentages, but not absolute numbers, of activated cell (co- fxpressing HLA-DR and CD38), of senescentlexhausted cells (PDI'CD37!) and of regulatory" cells (Treg) 2 NATURE COMMUNICATIONS] 202079434 Iitp=/deg/101038/=41467 02017292 4 wnt somtureconmnstons NATURE COMMUNICATIONS | hitps://doi ong/10.1038/s41467-020-17292-4 ARTICLE a me anette oot nin 3 e Cc 4 = 8 2 20 = G15 . & E10 $ gs z Zo 350 x $40 g B 30 = Ee & £10 8 BO * 100 %%cDs7+ PDI SG TREG ‘CONTROL COVID. CONTROL COVID ° CONTROL COVID’ ° CONTROL COVID: We then used a more sophisticated approach to detect fine analysis using FlowSOM'; this performs multivariate clustering changes occurring within different subpopulations of CDt! of cells based on the self-organized map (defined "SOM") T cells. For each patient and control, data from 5000 CD45' algorithm, categorizing cells into relevant meta-clusters based CD3'CD4" T calls were exported and concatenated in a unique on their surface markers. We first clustered all individual matrix. We explored the T helper cell panel by unsupervised cells into 25 distinct clusters based on the surface expression NATURE COMMUNICATIONS] c@o203m9434|Iitp=//doog/10 1038/2446? 02017292 4 wnt omtnecmmnitons 3 ARTICLE NATURE COMMUNICATIONS | hitps//doiorg/10:1038/s41467-020-17292-4 Fig, 1 Differentiation, activation, and exhaustion of CD4' T cells. a Gating strsteay used to anahyze makers related to cierentiaton, activation status senescence, and exhaustion together wit identification of Togs and TSCM within CDA T calls. Nave T cals are identfid as CCRT CD4SRA*CD28" 027+ cells: TSCM are CCR7*CDASRA*COZB*CD27"CD9St; central memory (CM) are CCRI-CD4SRA*CD28"CD27¥/~; effector memory (EM) a CCR7-CDASRA-CD281/-CO27!7-; terminal effector (TE) are CCRT” CD45RATCD2S-COI7'/-. Activated calls 2re CD38*HLA.DR Treg are C0127" CD25"; exhausted/senescent are PDI*CDS7*. b, € Percentages and absolte numbers of diferent CD4* T cell subpopulations in controls (n— 12) and patients (020, obtained by manual gating, Data represent individual values, mean (centre bar) SEN (upper and lawer bars). Statistical analyse by two-sided Mann-Whitney nonparametric test: if no indested, p valu is not significant. Source data ae provided as a Source Data fe marker proteins. Then, to reduce complexity, we merged the clusters that were very close each other and further te. clustered cells into 15 meta-custers representing different T. cell types on the basis of activation, differentiation, and exhaustion. Doing so, we used a dimensionality reduction method, the “UMAP" 10” distinguish several CD4!T cell populations (ig. 2a), whose percentages are reported in the heat map shown, in Fig 2b. Tes possible to immediately recognize the high amount, of naive T cells (red dots), that were CDASRA CD28*CCR7! CD27 "D127 125 CD95-C38- HLA-DR, and that were similar between the two groups; then, we identified reeenly activated naive T cells expressing CD38, and those expressing HLA- DR. We also found a small percentage of T cells representing CDA! memory stem cells characterized by the expression of CD95 and, CD38! that was similar across the two groups. Central memory T cells are characterized by expression of CDIBRA, CD28, CD27, CDI27, and CD9S molecules. Within these, a population expressing only CD38 has been identilied, and a population of cells that were activated (HLA-DR CD38) and, also expressed PDI. In patients, these two populations were significantly more frequent than in controls. Regarding the effector memory compartment, part of the transitional effector memory T cells are characterized by the lack of expression of CDA5RA and CCR7, but express CD28, Effector memory cll were characterized by the lack of expression of CDISRA, CCR7, CD27, CD28, CD38, and HLA-DR and mid expression of PDI, CD127, and CD25; in addition, effector ‘memory expressing marker related to exhaustion (Like PD1) were significantly higher in the patients’ group. Terminally effector memory T cells that express CDASRA, CD25, CD127, and CD95, did not differ between the two groups, as well as TSCM. nally, we are well aware that T regulatory cells (Treg) can be beter identified by the expresion of Foxp3, which is not present in, ‘our panel, However, thanks to the expression of CD127 (low) and, CDA (high), we were able to identify three different putative ‘populations of T regulatory ces those nave (expressing CDASRA CCR7'), central memory (CDASRA CCR7") and. the most represented subset of effector memory (CD45RA~CCR7-). The comparison of iferent clusters between healthy donors and, COVID-19 patients is shown in Fig. 2c Tes to note that patients, wore characterized by higher percentage of nave Treg cells (p value ‘was lower than the lower limit of the sltistcal analysis provided the software, ie, <10-%), by more central memory Treg cells, and by increased activated central memory calls expressing, PDI. We were able to analyze in more details cells from seven Patients and six controls, measuring the expression of several, chemokine receptors and of molecules that are considered master regulators (i ineage-specifying transcription factors) Figure 2d, shows a. representative comparison of the cytometric analysis, within CD3' CD4" cells ina conteol donor (upper panels) and a COVID-19 patient. The gating strategy for the identification of CDA! T calls is reported in Supplementary Fig. 1. {As indicated in Fig, 2, patients displayed a lower percentage of cells expressing CCR6 of CXCRS, and of those co-expressing CRG, and CD161, but higher percentages of CXCR4* of CCR" cals: no differences were noted in the expresion of T-bet or GAAS, Characterization and count of CDS" T cell subsets. Figure 3a shows the manual gating strategy that we used for the identii- cation of different types of CDS cells, which was similar to the strategy used for CD4! T cells As shown in Fig, 3b, healthy donors and COVID-19 patients displayed a comparable percen- tage of total CDS T cells, even ifthe absolute number was lower in patients. Both the percentage and absolute number of naive and central memory cells were lower in patients, while the per- centage, but not the absolute number of TE cells was higher. Figure 3c reports that patients had a higher percentage and absolute number of activated cells (expressing HLA-DR and 38), and higher percentages of cells expressing PDI and/or CDS7, markers that are associated to cell senescence/exhaustion. No differences were present as far as stem cell memory cells were concerned, similarly with what observed among CD41 T lymphocytes (not shown). We then performed the same unsupervised analysis by using FlowSOM meta-clustering to investigate the phenotype of CD8" ‘T cells, represented by the UMAP in Fig. a and by the heat map in Fig. tb. In this case, 13 clusters were identified and analyzed. As reported in Fig. 4c, showing the statistical analysis of chemokine receptors and master regulators. in patients vs controls, COVID-I9 patients were characterized by higher percentages of terminal effector cells expressing CD38 alone or in combination with CD57, and by activated effector memory calls expressing PDI or CD57. They also displayed significantly lower percentages of naive and central memory T cells, which could suggest that patients displayed an exhausted CD8! T cell compartment. Figure td shows a representative comparison of the eylometric analysis of CD3'CD8" calls in a control donor (upper panels) and in a COVID-19 patient (lower panels). The galing strategy for the identification of CD8' T cell is reported in Supplemen- tary Fig, 1. We could” also identify C8! T lymphocytes expressing high levels of CD161 that, along with CCR6, are present in most mucosal associated invariant ‘T (MATT) ‘cells characterized by Va7-2T cell reeeptor!”. Concerning the expres- sion of chemokine receptors and of transcription factors among 8 Tce, we foul hat patients expressed ower percentages of CCR6', CCRS!, T-bet! oF CD161"& cells, and of CXCRS" ‘T-het! or CCR6'CD161* lymphocytes (Fig te): they also had higher percentages of eells expressing CCR4, CXCR4, oF GATAS. ‘T cell proliferation and mitochondrial bioenergetics. Figure 5a, related to CD4' cells, shows in the upper panels the gating strategy for the identification of cells that had undergone either call proliferation (giving origin to the so-called “proliferation index”, Pl, revealed by those events in the red rectangles) or that replicated different times (“percentage divided”, PD, related to the different Quorescence peaks). A representative example of a hhealthy control and a COVID patient is shown. Lower pancls compare five patients and five controls, and show that TE C4! T cals from patieats had a lower proliferation index. Figure sb shows the same analysis applied co CDS! T cells. In this case, patients showed both a significantly lower proliferation index and ‘higher percentage of dividing cells. The gating strategy for 4 NATURE COMMUNICATIONS] 202079434 Iitp=/deg/101038/=41467 02017292 4 wnt somtureconmnstons NATURE COMMUNICATIONS | hitps//doiorg/101038/s41467-020-17292-4 ARTICLE as casero shave b CONTROL fines i i 2m t a ™ . Ein i pees ae ‘ acm ala - wees bjs 8s wae a gt Pop a nave gl ree gl,” en He be om a d “ g. contrat ge 1 of . ig ee 8.4 : 8 (a Po Fee, & £2 Fite CXCR-AFAES couse = * CoRA-PECESON CONTROL COVID * CONTROL CovID Fig. 2 Unsupervised analysis of CD" T cell and their characterization. a Uniform Manifold Aporoximation and Projection (UMAP) representation of the CDA Teal landscape. b Heat map representing different CDA T cell liters identified by FwSOM, with lative identity and percentages in healthy controls and COVID-19 pationts. The coors in the heat map represent the median of the arcsinh, 0-1 transformed marker expression calculated over calls from all the samples, varying from blue for lower expression to ved for higher expression. The dendkogram on the lft represent the hierarchical simiaity betwoon the motaclustrs (metric: Euclidean distance; linkage: average). Each cluster has a unique colr assigned (baron the let). Baplt along the rows (clusters) and values on the right indicate the relative sies of clusters. €Dillerental analysis between control (bar color: salmon: n= 13) and COVID-19 (emerald = 21). The heat represents acsine-square-cttransormed cll equencies that were subsequertly nomalized per cluster (rows) to mean of zero and standard deviation of ane. The coor ofthe heat varies from be indicating relative under representation to orange incicaing relative over representation. Bar and numbers atthe right inccate sgniicantGferentaly abundant clusters (green) and adjusted p values. Clusters are sorted according to adjusted p values, so thatthe cluster tthe top shows the most significant abundance changes between the lwo conditions d Representative ot plots relate tothe expression of ciflerent chemokine receptors and ineage-specivng tvansription factors in gated CDA‘ T trom a contrl (upper) 2nd.a patient (lower panel). Numbers inccate the percentage in each quadrant. Two experiments (one forthe contro group, one for patients) out of 13 are shown. Numbers indicate the percentage m each quadrant. The gating strategy forthe identification af CDA* T cells i ported in Supplementary Fi 1. ‘eercentages of different CDA} T cell subpopulations in controls (n= 6) and pationts (n= 7), obtained by manual gating. Data represent individual values (dots), mean (centre bar) SEM (opper and lower brs), Statistical analysis is performed by two-sided Mana’ Whitney nonparametric tes not indicated Dale ie not senficant. Source data ae provided 2° 9 Source Data fe. studying cll proliferation in CD4* and in Supplementary Fig. 2. 18 T cells is reported mitochondrial oxygen consumption rate (OCR), shows that basal respiration, maximal respiration, spare respiratory Cellular bioenergetics was studied in CD4* T cells purified by magnetic sorting and stimulated overnight or aot with anti- CD3 plus anti-CD28 mAbs. Figure 6a, related to the analysis of capacity (SRO), and the amount of oxygen used for ATP production were similar in stimulated (8) oF aon-stimulated (5) cells from controls and patients. Figure 6b, related to the NATURE COMMUNICATIONS] c@o203m9434|Iitp=//doog/10 1038/2446? 02017292 4 wnt omtnecmmnitons 5 ARTICLE NATURE COMMUNICATIONS | hitps://doiorg/10.1038/s41467-020-17292-4 Diterentiton Actvation = “onan ‘saa cla TE i o 1000; B00 « pcos = C20) groom 2 2 p00 2 600 8 15. = £2 400 = a 845 8 z z 8 200 ase = 51 Z 10 © 35 3'5 300) p-0005 a 33 “B 200 3 4,40 3150 R 100. = ~ 230 100 . . 0 & 20 & = 30) wan 150, p<0.005 8 10 B 6° S 2 20 zo So eS ® ° 100 ° £80 80 300) | 8 60 = 60 = = 40 © 40 3 ® 20 ® 30 F100] 2 0 0 60 100. + 60 rv 8 40 = B & 20 oe we ° n ole Control covin CONTROL COVID ‘CONTROL COvID CONTROL COvID analysis of ECAR, shows that stimulated (S) or NS cells from Detecting cytokine storm in patients’ plasma, We measured patients and controls had the same capacity 10 switch from plasma levels of 31 cytokines, chemokines, and immune-related respiration to glycolysis, and had similar basal and maximal molecules in 21 patients and 13 healthy controls (Fig. 7). We ECAR. found that galectin-1, galectin-3, galectin-9, C-C motif chemokine ‘ NATURE COMMUNICATIONS] 202079434 Iitp=/deg/101038/=41467 02017292 4 wnt somtureconmnstons NATURE COMMUNICATIONS | hitps//doiorg/101038/s41467-020-17292-4 ARTICLE Fig. 3 Diferentiation, activation, and exhaustion of CDB* T-cell subsets. a Gating strateuy used to analyze markers of dileeniation, activation status senescence, and exhaustion together with identification of TSCM within CDB T cell. Nave T cols are identified as CCR7CD45RACD28'CD27" TSCM are CCR7"CD45RA#CD28 "CD27 CD95; central memory (CM) are CCR7-CDASRA*CD28"CD27-, efector memory (EM) CCR7-CD45RA (€028"/-CD271/-; termina effector (TE are CCRT” CDA5RA'CD28-CD2T!/~. Activated calls are CD38!HLA-DR'; exhausted/senescent are PDI" CD57. bye Percentages and absolute numbers of diferent CDB* T cel subpopulations in controls (n ~13) and patients (n—20, obtained by mana galing. Data represent individual values, mean (cenee bar) "SEM (upper and lower bats). Salitical analysis by two-sided Mann-Whitney nonparametric test if not incited vale is nt significant. Source data are provided as a Source Data fie ligand-2 (CCL2, also known as MCP-1), CCL3 (also known as MIP- 1a), CCLA (also known as MIP-1b), C-X-C motif chemokine ligand-6 (CXCL6), CD27, CD40, CDAOL, TNF, IFN-y, IL-1a, IL If, TL-4, IL-6, IL-7, IL-8, IL-10, TL-13 major histocompatibility complex class I chain-related protein A (MICA), elucocorticoid: induced 'TNER-related protein (GITR, also known as receptor superfamily member 18), and programmed death-ligand 1 (PD- 1) were significantly higher in COVID-19 patients, as compared. with controls. No statistical differences were found as far as MIP- 2 (also known as CXCL2) was concerned. Likely due to technical limitations of the multiplex assay. CCL?, GITR, 1-2, IL-3, and IL-5 were not detected in most patients and controls, Also due to the fact that levels of IL-1270, TL-15, and TL-17 were undetectable in almost all of the healthy controls, we were unable to perform statistical comparisons, even, although inmost patients these cytokines were present at detectable levels In vitro production of proinflammatory cytokines, We studied six different functions of CD4" and CDS" T cells, including the proxluction of IEN-y, IL-17, IL-2, TNE and granzyme-B, along, With the expression of the degranulation marker CD1072"°, Figure 8 shows a representative example of the detection of intracellular cytokines and the presence of CD107a in CD4* T calls (Fig 8a) or CD8" T cell (Fig. 8b) from a healthy donor (CONTROL, upper quadrants) and s patient (lower quadrants). ‘The gating strategy for studying intracellular cytokines in CD4" and CD8" cells is reported in Supplementary Fig. 3. As summarized in (Fig. 8), in comparison with six controls, a significantly higher amount of CD4'T cells from eight COVID- 19 patients was able to produce TNF, CD107a, TFN-y, TL-2, and particularly I-17 (that showed the highest difference). ‘The production of granzyme-B was similar in the two groups. A. similar trend was present among CDS! T cells (Fig. Sd), whose stimulation with anti-CD3/CD28 resulted in significantly higher production of CD107a, 1-17, and especially T-2. 4 trend, even if not reaching a. statistical significance, was present as far as the proxiuction of TNF and IEN-y is concerned. We then investigated CD4" and CDS T cell polyfunctionality by analyzing the simultaneous production of TNF, CD107a, [EN- y, IL-2 and I1-17 (Fig. 9). It isto note that in this case we indicate the percentage of cells exerting one or more functions (consider ing the entite set of cell, that is taken as. 100%), without considering the power of the global response", Based on the use of five markers, we could discriminate 31 different populations of CDA! of COB! T calls able to produce one or more molecules: the population that had no activity, ie» that was five times negative for all five markers, was excluded from the analysis. Figure 9a shows that CD4" and CD8' T cells from patients and controls had very global patterns of polyfunctionality. However, different functional types of CD4* cells producing TNF were present at higher proportions in patients as compared with controls (Fig. 9). Moreover, patients also showed more cells simultaneously producing IL-2 and IL-17 than controls, con- firming what was reported above. Discussion In this study we describe the main changes in the T cell com- partment of patients with COVID-19 pneumonia. Most were Iymphopenic, and most did not require noninvasive ventilation, indicating that at the time of blood collection the disease was severe, but not too advanced to require intubation and mechan- ical ventilation. Our data thus indicate that several immune alterations are present when the infection becomes clinically relevant. These data are also in agreement with the immunolo- gical changes recently described in a patient with mild-to- moderate COVID-19 patient that required hospitalization, and showed a significant increase in activated T cells. Studies om asymptomatic, infected individuals are crucial to better under- stand the immunopathogenesis of COVID-19, In COVID-18 patients with pneumonia, we found an increase capubility of CD4* or CD8* T cellsto produce in vitro 1-17, that, is able to strengthen the inflammation response and to activate neutrophils. In peripheral blood, paticnts also showed low per- centages of both CDt! and CDS! T cell expressing CCR6 and high levels of CDI61, which is typical of THI7 and of rmucosal associated invariant T (MATT) cals, respectively. The loss of cir- culating CD4'CD161"CCR6" cells contributes to disease pro- gression in macaques infected with the simian immunodeficiency Virus, and it has been shown that these eells accumulate in the rectal mucosa, enhancing inflammation’. In the same infection model, the percentage of TL-17 producing CD8'CD161* cells present in the lung can be fourfold higher than that in peripheral, blood? Furthermore, cells present in the lung were able to produce more IL-17 than those irom peripheral blood. Taken together, these findings underline the importance of IL-17 in OVID-19, and likely could pave the way to novel therapeutic approaches based upon IL-17 blockage by biological drugs that are already available ‘Very few data exist on the changes in the T cell compartment in patients affected by SARS-CoV-2 infection, Previous studies, hhave shown changes in the T cell family, which is characterized by signs of exhaustion?®=4, In most COVID-19 patients the proportions of T cells subsets can remain within the normal ‘ange, buta decrease can exist in CD4! and CDS ‘T cell counts or in CD4*/CD8* ratio®™*, Our data are in agreement with these observations, but are enhanced by our analyses of dif ferent subpopulations of both CD4* and CD8* T lymphocytes Indeed, whereas on the one hand no gross changer between patients and controls are detected simply using markers related to naive, memory of effector cells, on the other hand our more sophisticated and detailed analysis reveals significant differences. “Treg are crucial for regulating immune homeostasis and self- tolerance’’**. They express the forkhead box transcription factor Foxp3, but can also be identified by detecting the high expression of the'IL-2 receptor a-chain (CD25) and low/null expression of IL-7 receptor a-chain (CD127)™, along with other surface molecules such as CD39 and CD73"!. They suppress auto- immune phenomena, dampen allergic reactions, or block trans plant rejection, but they can also inhibit a protective immune NATURE COMMUNICATIONS 02031!3434|Intes//éo rg /10 1038/4467 020 17292 4| www nature comratueconmanitons 7 ARTICLE NATURE COMMUNICATIONS | hitps://doiorg/10.1038/s41467-020-17292-4 a Cluster identification —-B CONTROL 2 5 Rehsmpane wae z Ewroiecosre BM 3 mneosi vumart c CONTROL co. em coaee,c057~ ‘ACT. EM POS om ecos7 5+ . NAIVE 215 recost+ Bo ™ 30M .! ke oi om €7 emposcos7s gr Be reco27+ ge at com po1vcos7 aye 2 be d Le ad = contnoi ai: els ¥ 21 ait, 8 wows sy poms oe Be a oe) gh a3 CHCRDAPAES—“CERLECFSON——CCRGSWEES———CKCREPE CONTROL coviD. ~~ CONTROL cOVID Fig, 4 Unsupervised analysis of CDB* T cells and their characterization. a Uniform Manifold Approximation and Projection (UMAP) UMAP representation of COB} T coll landscape. b Heat map representing different clusters identified by FowSOM, with relative identity and percentages in controls and patients. The coor in the heat map represents the median of the arcsnh transformed marker expression calculated over calls rom all the ‘samples, varying om blue fo lower expression to red for higher expression. The dendrogram onthe ll represents the hierarchical similarity between the retacustrs (metric: Euclidean distance; inkage: average). Each cluster has a unique coor assigned (bar on the left) Barpots along the rows (clusters) 2d values on te right indicat the elatve sizes of dusters. Dilleretil analysis between CTR (bar color: salmon; =13) and COVIDAB (emeral 19), The heat represents arcsne-square-oot transformed cell frequencies that were subsequently normalized per Custer (rows) to mean of zero and Standard deviation of one. The color ofthe heat varies from blue indicating relative under-epresentation to orange indicating relative over- representation. Bar and numbers a the right nicatesignticantciferentaly abundant clusters (green) and adjusted p values. Clusters are sorted according to adusted p values, so thatthe cluster atthe top shows the most significant abundance changes between the two conditions. Representative dot plots related to the expression of cferent chemokine receptors and ineage-specityng transcription factors in gated CDS T trom a contol donor (upper) and a patient (lower panel), Two experiments (one forthe contrel group, one for patients) out of 13 are shown, Numbers incicate the percentage in each quadrant. The gating strategy forthe identification of COS* T cells is reported in Supplementary ig, I-e Percentages of ferent COB* T cel subpopulations in controls (n—6) 2nd patents (n=7), obtained by manual gating, Data repcesent individual values (dts), mean (centre har) + SEM (upper and lower bars). Statistical analysis is performed by two-sided Mann-Whitney nonparametric test: f not indicated, p value isnot signficant. Source data are provided as» Source Data fie 8 [NATURE COMMUNICATIONS] 202031!434|Intps//éaeg/10 1038/4467 020 17292 4 ww ature comratueconmnitons [NATURE COMMUNICATIONS | hitps//doiong/10 1038 s41467-020-17292-4 ARTICLE a ALLCD4 NAIVE CM EM TE 1 5 jrizan 238 6 CONTROL = poi oe ‘ ‘ Se ery ge s 1 8 = Calg COVID i 2 = froses foe CFSE-CellTrace ~ __ALLCD4 NAIVE cM £25 16 25 = 20) «, =" 20 ° 2 5 isle sgt t4 15 te Satis € 1 ww Bo £ 05) a 05, = 00 10 oo = 50 40 80, zg 2 = 30 = 60 Poe dot do, Sot Sp 8 10 eee 20 20 af, “= o 0 o o CONTROL COvIO” CONTROL COvID CONTROL COVID CONTROL CaviO’ CONTROL COvID b Aucos. NAIVE cm eM Te gms, pas che nae CONTROL ¥ sb fos oes Se) => |) eS ep reso a7 pr.7 (rise covip fo feaka | fb ro CFSE-CellTrace ALLCD8. NAIVE cM —M Te S%7,. 2, .25 26 ta, poor = at ay 20 20% = 16 ¢ g st 15 6 18 tal ts a4 He ea us i 05, os 10 2 0 0.0 0.0 OB © 50, 6 80; . 80 40) P<0.05 40 a” 60 60 = 30 . PSY Go™ Be Pow Le 6 10 20, 20 = 40 : § 10 oe ec CONTROL coviD CONTROL OVID CONTROL covID response against invading pathogens or tumors", Here we show thatthe percentages of dlerent types of Treg are increased in CONTROL COVID CONTROL cOvID Patients with COVID-19 had increased amounts of CD8* T cells expressing CDS7, which is considered a key marker of peripheral blood from COVID-19 patients, and that their plasma in vitro replicative senescence and is associated either to human. contains high amounts of the inhibitory cytokine TL-10 aging or prolonged chronic infections’. CD57 has been used to NATURE COMMUNICATIONS] c@o203m9434|Iitp=//doog/10 1038/2446? 02017292 4 wnt omtnecmmnitons ° ARTICLE NATURE COMMUNICATIONS | httpsi//doiorg/10.1038/s41467-020-17292-4 Fig. 5 Cell proliferation of CD4* and CD8 T lymphocytes. a Upper wo rows: representative dot plas elated to cell proliferation in leven pes of (€D4* T cals froma contral danor (upper panel) and a patient (COVID, lower panel). Lower two rows indicate the proliferation index and the percantage of vided cel n all CDA T cel, or in nave, central memory (CMD, efector memory CEM), oF terminally diflerentiated (TE) cells, Data represent individual values (dots) from five pationts and five contols, maan (centre bar)= SEM (upper and lower bars). Statistical analysis is performed by two-sided Mann-Whitney nongaramettc testi nt indeatd,p vale is not sinificant. The gating strategy forthe identification of CD-4* T cells s reported in Supplementary Fig 2b Uaper two rows: representative dot pots elated to cal poileratio in dilret types of COB T calls from 2 contra donor (CTR, Upper) and a patient (COVID, lower panel). Lower two rows indicate the proliferation index and the percentage of divided cel in all CDB* T cal, or in nave, central memory (CM), elector memory (EM), or terminally diferentiated (TE) calls. Data represent indvidual values (Cas) trom five patients and five contol, mean (contre bar)*SEM (upper and lower bars). Statistical analysis is performed by two-sided Mann-Whitney nonparametric tst if not inciated,p valve fs not significant. The gating strategy forthe identification of CDS T cells is reported in Supplementary Fig. 2 o a ‘Oligo FCCP Rot/AntA 2 25 tee ocrns Fs cece, OM FEEP Ravana az Sms Eg zo oon BS sow! Tos = Ge tee Sows ze SE ew "Smt EE owt a BOR 8S Eee Me ine) S sme (ints) iin . te) gaol spas y se~ i AG ee . e& EE sen! Eton . BS a8 at + + ~ 3 EE soa, += e i. UE git aie 3 Be, aes) 5 = 2 rome 0.08 an <2 p00 Z ww, Zw, ge ss : a + g noe Bie . SE sont . RE ow ‘ SE mw ' 23 gE - ze i zt Eso est pot >it ool a 5 mos OMS NS 5 NSS NS SNS Ss contnot covio CONTROL covID contRot covin Fig. 6 Mitochondria bioenergetics of CDA" T calls.» Rearesentative traces (out of 12 experiments) of oxygen consumption rate (OCR) of unstimulated (NS) and stimulated (5) CD4* T calls rom healthy contol (CTR; n=7) and COVID patients (95). OCR was measured in real tise, under basal condition and in response t indicated mitochondria inhibitors lgomycin (Oligo, 2M), cyanide-4-(Wrilluoromethony)phenylrydrazone (FCCP, 0:5 aN, nd antimycin A plus ratenane (ROL/AA, 0:5 uM), Histograms show the quantification of basal espration, maximal respration, spare respiratory capacty (GRC), ATP inked respiration (ATP), percentage of SRC, and area under the cuve (AUC) incall stimulated (S) with anti-CD3/28 or non stimulated (NS) from controls and patients. The percentage of SRC represents the ratio between the seventh and the third measurement, AUC was obtained by analyzing ‘the area under the curve fom the sch tothe eleventh measurement. Data represent individual values (dots trom seven patients and five controls, mean (centre ba) SEM (upper and lower bars). Statistical analysis by two-sided Mann-Whitney nonparametric test shows no stalstica ferences between controls and patients. 8 Representa traces (out of 12 experiments) of extracellular acifieaton rates ECAR) of unstimulated (NS) and stimulated (5) CD4* T cals rom one heathy contral (CTR) and one COVID patient. ECAR was measured under basal conditions and in response to FCCP. Data represent individual values from seven controls and ive patients, mean (centre bac) * SEM (upper and lower bars) concerning the quantification of basal ECAR and maximal ECAR incall stimulated (5) with ant-CD3/28 or non stimulated (NS) from controls and pationts. Statistical analysis by two-sided Mann-Whitney nongarametrc tet shows no statistical cifferences between controle and patients, 10 NATURE COMMUNICATIONS] 202079434 Iitp=/deg/101038/=41467 02017292 4 wnt somtureconmnstons NATURE COMMUNICATIONS | hitps://éoiorg/10:1038/41467-020-1729: a ARTICLE eee) Galectin 9 ‘p<0,0002, vom Galectind. » =) Gealectin 3 om] pe0,0001 0} 0.0001 * F ccLa p<0.0001 ae 027 p<0,0001 i pao. , p<0.0033* NF p<0.0001 pg/mL 4 <0.0001 7 p<0.0007° I-10 pc0.0001+ CONTROL COVID CONTROL cOvID CONTROL COVID CONTROL CovID Fig. 7 Plasma level of cytokines and chemokines from COVID-19 patients and controls. Quanicsion of cytokines and ether mediators in plasms btsined from COVID-19 patients (n—23) and heathy contol (n= 15). Oat bars), Statistical analysis by two-sided Manf-Whitney nonparametric test fn Data fie detect functional immune deficiency in patients with auto~ immune diseases, infectious diseases, and cancers. It has been. suggested that CD57" cells display a high susceptibility to activation-induced cell death, and are not able to undergo cell proliferation despite their preserved ability to secrete cytokines NATURE COMMUNICATIONS] C@20m:9434 Iitp=//doeg/10 1038/4467 0201729 ta represent indvical values, mean (centre bar) *SEM (upper and lower ot dicated p value isnot slgniant. Source data ae provided as a Source after activation, We have found a decreased proliferation index among TE CD41! and CDS! T lymphocytes. izom COVID-19 patients. T cells show phenotypic features of an exhausted state including the upregulated expression of the inhibitory receptors such as PDI. T cell exhaustion is " ARTICLE NATURE COMMUNICATIONS | hitps//doiorg/10:1038/s41467-020-17292-4 ‘TWF-8vE0S: War PECy7 Granayme-6-8V421 L2-aec 0.0826, % of co4 cel producing Fy producing TNF z ag és” ! st an 28 BE a 3E tp CONTROL Covi ‘CONTROL COVID Fig. 8 Cytokine production by CDA! and CD8!-T calle after in vitro stimulation with anli-CD3/28, b contRoL A7-PE-CY7 a Granzyme-8-BV421 WL2-aPe do.* » lew-pFrre fe 7 3B : ai” Be : BE Sia F g ) p=0.0083 Be ga . Bt He, § "| prooaes 2 bee ee gee a6 | Be» geo a Sg ate £ eZ + CONTROL COVID: & CONTROL CovID Representative dot plots (out of 18 experiments) related to the intracellar cytokine staining of CD4* Tells ofa contol donor (upper panes) and a COVID patient (lower panels) attri vio stimulation with ant-CD3/CD28. The gating strategy fr the identification of CDA" calls reported in Supplementary Fig. 3. b Representative dt plots (ut of 12 experiments) related tothe intracellular cytokine staining of CD* Tells of a control donor (upper panels) and a COVID patient (ower panels) after in vitro stiulation with ant-CD3/CO28. The gating strategy forthe identification of CD8™* T cells is reparted in Supplementary Fig. 3. € Comparison between the total production of IFN, TNF IL-7, Il-2, CDI07a and granzyme-B by CD4*T ells after in vito stimulation with ant-CD3/CD28, Data represent individual ales from six controls and eight patient, mean (centre bar) * SEM Copper and lower bars) Statistical analysis by two-sided Mann-Whitney nonparametric teen indeate,p value isnt igntiant. Campariean between te otal production of IFN, TNF, IL-7, 1-2, CDIO7S and granzyme-B by CD T cells ater in vitro stimulation with nt-CD3/CD28, Data represent individual values from six controls and eight patients, mean (cenire ba) = SENA (upper and lower bar), Statistical analysis by two-sided Mann-Whitney nonparametic est: fot indcated, p vale snot significant. characterized by functional unresponsiveness, which prevents massive immunoactivation and associated autoimmune tissue damage. Thus, it could be possible that in COVID-19 patients the activation of these cells is followed not only by the lack of clonal expansion (as revealed by the decreased proliferation) but also by the production of molecules that cause inflammation Furthermore, we have observed that cells from patients dis: played higher percentages of diferent cell types expressing of PDI, a crucial molecule for the induction and maintenance of peripheral tolerance, and for maintaining T cells stability and integrity". The PD1/PD-LI axis also mediates potent inhibitory signals to block proliferation and function of T elfector cells, causing inimical effects on antiviral immunity. We have found significantly high plasma levels of PD-L1 in our patients, and studies are needed to understand whether and how the infection uses this pathway for inhibition of an efficient antiviral immune response os NATURE COMMUNICATIONS] 202079434 Iitp=/deg/101038/=41467 02017292 4 wnt somtureconmnstons NATURE COMMUNICATIONS | hitps//éoiorg/101038/s4467-020-17282- ARTICLE a CONTROL (i ~ cos f ‘ ( o agg LOTINLD LATTES a07: 01 £ ps0.08 5) pxo.0s S — Bg LOMFNHLZILA7THFg gg, 1071 2s 3 20, 2 . ou 2 on ° 005, 00! a CONTROL coviD Fig. 9 Polyfunctionality of CD4! and CD8! T cals aftr in vitro stimulation ee Biya aanie gnc: a get: + Bae: : a : Bat of4 eit : SiG + alt : aot + | m-+ - moe + aos : :-: + TNF+ 045. W2HLa7+ poo oo. TNF* 0.05: CONTROL COVID a CONTROL coviD with ant-¢D3/28.a Fic charts representing the propertion of responding CD&* Cupper ies) and COB (lower pies) cells producing diferent combinations of CDOT 1-2, FN, and TNF ater anti-CD3/CD28 stimulation in control danas (let: n—7) and patients (ight;n—). Frequencies were corrected by background subtraction ac determined in non stimulated controls using SPICE software. Pie arches represent the total production of diferent cytokines. b Comparison between the production of diferent combinations of cytokines by CDA" T calls after in vito stimulation with ani-CD3/CD28. Dat ta present individual vale from 7 controls and 7 patent, sna (eentre bot) SEM (oper and lower bars). Statistical analysis by two-sided Mann-Whitney nonarametrc test: no indicated, p value is not significant. An excessive inflammatory response evidenced by elevated levels of proinflammatory cytokines and chemokines has been described in patients allected by the SARS-CoV epidemic in 200352", Moreover, in vitro experiments revealed that several NATURE COMMUNICATIONS] C@20m:9434 Iitp=//doeg/10 1038/4467 0201729 different cell types from those patients were able to produce high amounts of eytokines®*=°. High plasma levels of proinflammatory ‘molecules indicating a TIHL/THL? response were also reported in patients with MERS-CoV infection, with increased concentrations 2 ema re con strecemunietins 2 ARTICLE NATURE COMMUNICATIONS | hitps//doiorg/10:1038/s41467-020-17292-4 of IFN-y, TNF, I-15, and 11-17. Similar data, along with skewed in vitro cytokine production by T cells, have been described in patients infected by MERS coronavirus‘!. In our COVID-18 patients, plasma concentrations of many. proin- flammatory cytokines and chemokines were dramaticall incre which in accordance to al the forementioned reports describing the so-called “cytokine storm”, "The eflzcts of the prescace of high amounts of molecules that are produced by several ell types and act on innate immune cells, ‘must be considered, Amongst these molecules, IL-8 might be of a particular importance, since it recruits neutrophils from the blood, to infected oF injured tissu. IL-8 production can be induced by a wide range of stimuli, including TNF, TL-1, bacteria, viruses, and, cellular stress, and it can be synthesized by several cell types, including monocytes, macrophages, endothelial and. epithelial, cells, fibroblasts, T lymphocytes, hepatocytes, synovial cells, and keratinocytes" Its receptors, CXCRI and CXCR2, are expressed, fon neutrophils, monocytes, CDS! T and NK cells, mast cells, basophils, and myeloid-derived suppressor cells. In. neutrop receptor activation stimulates degranulation and the production, of reactive oxygen species”. When a respiratory virus, such as SARS-CoV-2, enters alveoli, it first encounters and infects, alveolar epithelial cell. which can then produce IL-8 that in turn attracts and activates neutrophils and macrophages. These star, damaging the organ, eventually triggering a much more complex, series of pathogenic events, including, amongst others, endothe- lial damage, platelet activation, and’ intravascular thrombosis. Although it was not possible to precisely determine the ste of IL: 8 production, even for our patients with pneumonia, it is rea sonable to hypothesize that alveolar cells are intimately involved, in this phenomenon. Surprisingly, we also observed a marked plasma increase of typical TH2 cytokines, including IL-4, I-10, and IL-13. This, could indicate that the activation of immune system is really ‘massive, indiscriminately involving all cells, and, similarly to what occurs during bacterial sepsis, could cause a form of immune paralysis Galectn-1,galectin-3, and galectin-9 were increased in patients as compared with controls Galectins represent a family of soluble Begalactoside-binding proteins widely expressed at sites of inflammation and infection that have emerged as a new class of damage-associated molecular patterns (DAMPs) oF resolution- associated molecular patterns, serving to magnify orto otherwise correct inflammatory responses'®"”. In particular, galectin-1 acts, typically as a proresolving mediator by repressing a number of innate and adaptive immune programs. Conversely, galectin-3 and -9 have been proposed to act as alarmins (or DAMPs) that amplify inflammatory responses during sepsis and several types, of infection. To our knowledge, these are the first data describing the presence of these types of soluble mediators in patients wit COVID-19, and further studies are needed to investigate their importance in the immunopathogenesis of the disease, or their Possible consideration as potential therapeutic targets. ‘The massive precuction and release of cytokinesis very similar to what occurs during polyclonal, superantigen-driven, T-cell, activation’, A fourfold increase ofthe levels of a THI molecule such as IFN-y was indeed observed in plasma from COVID-19, Patients as compared with controls. IFN-y activates macrophages. Which produce proinflammatory cytokines, which then over ‘helm the system. When cells from patients were stimulated with anti-CD3/28, an increased number of CD4* T cells producing, IFN-y, TNF, I-17, and IL-2 was observed as compared. with healthy controls. This indicates that T cells from COVID-19 patients have a higher ability over controls to respond in vitro to stimulatory challenges producing potentially dangerous mole cules. Studies are” underway to. investigate the functional phenotype of specific T cells ic. those responding to peptide antigens derived from SARS-CoV-2. Elevated levels of serum proinflammatory cytokines and che- smokines are known to contribute, as a cytokine storm, to the increased severity of disease caused by some strains of corona virus. Unique immunoregulatory system mediated by T cell exhaustion and suppressive cytokines such as IL-10 are respon- sible for limiting excessive inflammation and play an important role in homeostasis in the lungs. A balance in the levels of immunoactivation and immunosuppression may therefore be crucial in host defense against highly pathogenic corona virus infection ‘We are well aware ofthe limitations of our study. First of all, the fact that the relatively low number of patients, who were however chosen on the basis of similar clinical characteristics, does not allow us to divide them into those with a mild or severe course of the infection, having sufficient statistical power for further analyses. Second, due to fact that the study started when there was no treatment at all, and that inthe days following blood collection some patients would have been treated and others not, itis difficult to understand which immune modifications ean be considered predictive markers ofthe natural containment of the infection, or ofthe suecess ofthe therapy. Third, for this study we could not provide longitudinal data, but just compared the cohort with healthy donors, However, in COVID-19 patients with pneumonia, we have found the presence of: (i) increased markers Of T call exhaustions, activation, and senescence; (ii) an altered dierentiation of different cel subtypes: (ii) high plasma levels of a variety of cytokines, from those with proinflammatory action to those that are able to inhibit the immune response, from those that indicate a skewing towards THI to those that reveal a skewing towards TH2; (iv) massive in vitro production of several cytokines, with a potential skewing of activated cells towards ‘THI? phenotype; and (v) decrease of those circulating cells that are known to localize in the lung and other tissues, and recruit, neutrophils. Tn conclusion, in COVID-19 patients, concomitant aspects of| immune inhibition, activation, exhaustion, and complex altera~ tions within cells at different stage of differentiation exist, and a hhuge variety of cytokines are produced and released. The overall picture that emerges underlines the ability of SARS-CoV-2 to provoke, in avery short period of time but, fortunately, not in all infected individuals, a massive activation of immune responses. However, and thanks tothe considerable efforts that the scientific community is devoting to studies of the immunopathogenesis of COVID-19, the progression of the viral infection starts to show some weak points that may be therapeutically relevant. For example, a therapeutic approach now exists based onthe alministration of drugs that block IL-6 pathway, and is now significantly ameliorating the course of the disease. 1L-17 is crucial in recruiting and activating neutrophils, cells that can imigrate to the lang and are heavily involved in the pathogenesis of COVID-19, We show here that a significant skewing of T cell activation towards THI? functional phenotype exists in COVID- 19 patiens, and we therefore suggest that blocking IL-17 pathway by biological drugs that are already available ane used to treat, diferent pathologies could be a novel, additional strategy to treat patients infected by SARS-CoV-2. ‘Methods ‘tidy design ‘This i case-control cos sectional, sng-enre stay, approned bythe local Ebial Caries (Contato cy def Aes Vsta Emin Nod resco mum 17/200, March 1th, 2020) and by the Univrity pital Commitee (Deion Sanitaria dl'Acnda Ospedale Univestaia di Mox- 2, protecal muse 7531, March 1th, 220), Each pasta cud helay stl, provid informed conse! according lo Henk Declaration, spl ‘alban materi have been approved bythe sane Commits Atala 38 “ NATURE COMMUNICATIONS] 202079434 Iitp=/deg/101038/=41467 02017292 4 wnt somtureconmnstons NATURE COMMUNICATIONS | hitps//doiorg/101038/s41467-020-17292-4 ARTICLE ‘COVID-19 patents were ince in the stu they ha a median age of 6 years Iconge 3-94)? were females 3D men Patients were thes fr age and gender ‘witha tol of 25 Reathy donors (CTH median age aD years range 8-66 ea) 1We recorded demogaphe dats, model hetay, symptoms, sighs temperature. 2 iain aboratny Ring rom each patient ta were ced and cord a Fel 141.0 fr Moc ina detbane present in the Inectons Diese links and routine nse. The tl numer and typeof eakecytes in peripheral Hood was nse hy hemiyometer nthe Clin Laboratory ofthe Univers Hota ‘Sordi to route methods, lod cllction and isolation af mononuclear calls. po 200. of Mond wre ‘let fom exc pint in acter containing eleniamine trace {5 Blod was immediately proces sls of peripheral blood manonodear ‘ll (POMC) was performed wing fal hypaqu according to sandard proce ‘Sues PBMC wete then std in diferent squat at the concent of 310 rion. in lui nto in fel bovine serum supplemented with 10% Si al Pn hn cle eto 2 ed ‘Cunt ws Meisurements wre ken rm india poet in the cn of ‘plasma, each measurement was Performed in upbeat and ony the mean Wes onsen son 1 cll immunophenotype by polyehromate flow cytometry awed Ps Wwse washed ie wit RPM T600 supplemented with 1 fetal bonne serum and 1% each of -gutamin, sodium pyrite, nonesena amino acs, ant Peis, 0M HEPES, 55M merapethnol and 0.02 mg/ml DNAS or the dead analy of Teall phenotype, PBMC were counted and up to rmion PMC were sail wah the Duaclone IMT call pene (oun Beckman Couher Mim) added wis another five Muoresceat mAs apd 2 masher fel ‘ality. Aloo wit se and rad ater sgl signs were obtained from {ierentfuorechomelaeed mise, CDI conjugated th Krome Orange {CDS APC-A7S0, CDE APC, CS AF700, CD27 PCr, CDs? Pai Blue, CD29 (opi) ness, Chas HC, COR? PE, CDISRA FITC. HLA-DR BUYS) CD27 $880, CD25 BV78S, CD9s HUV395, CD38 BUVADG and Promolton #0 (Promekie, Pomel Heidelberg. Germany). A mins of 00.00 cal per Sample were acgured on a CWOFLEX LX fw tometer (Beckman Cale or the anlsisof Tcl skewing toward THI, TH o THI, and chemokine receptor exreson, thawed PBMC were washed tice with PES and tained th the nabiity marker AQUA LIVE DEAD (Uhermatahe) Then upto | ion (clk were washes tained af 3° with the felling mAb: anc CXCRS [AFI8§-CXCREPE Col were wate spin an sacl trounfemperatare ‘with ane-CDIGI-PC7,-CCRe-BVENb, -CCRE-PE-CHS94, -CDHATTOO -CDB- [APC-CY7. Cals were washed fae and permeabilized ung Foxp37Tanscrpion Factor Staining Baler Set (Uhermot ber). Finally cls mee said wath ant (GATAS-BVE2I apd antcTBET-APC, and yashed A muna of 0,00 PBSC nee acted by aig Ane NxT acoustic oes fom eyomeer Uhermatihe) ‘Supplementary Table 5 shows al th cloes an monotolantidisus in ths say Representation of high parameter flow cytometry tow Cytometry Standard (0S) 30 fer were pertain How sltware tern 9 (Sector Dickinson, San Tost CA), and analyzed by stance ating to limmate aggregates and dead cll an to ent CDS" CDA" T cals and CRT cll The, data fom 500 {CD#*T eal and 200 CD8HT cls pr sample were export for fre aaysis {by felling eit that makes of Rococo Mares an stata pcages (CATALYST L101). The srt i arable a tpg com! Fees C/CATALYSIY® The secon af eictor fr dat ransormaton wat heck on Cytobank premium version (Sos cyan). The plato How SOM (aval a hp icconductor ory packagesiclese/iocnlFowSOM. ‘ua was used perform the mctadstriag (K-20). Data were subsegueaiy

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