key: cord-326881-3j2e92dj
authors: Vassallo, M.; Manni, S.; Pini, P.; Blanchouin, E.; Ticchioni, M.; Seitz-Polski, B.; Puchois, A.; Sindt, A.; Lotte, L.; Fauque, P.; Durant, J.
title: Patients with Covid-19 exhibit different immunological profiles according to their clinical presentation
date: 2020-09-28
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2020.09.1438
sha: 
doc_id: 326881
cord_uid: 3j2e92dj

Objectives A novel beta coronavirus has been identified as responsible for the 2019 coronavirus infection (Covid-19). Clinical presentations range from asymptomatic cases to acute respiratory distress syndrome with fatal outcome. Such a broad spectrum of disease expression calls for an investigation of immune response characteristics. Methods We identified subjects admitted for Covid-19 in whom a large panel of immunological markers were measured, including B- and T- and NK-lymphocyte phenotypes, T-lymphocyte subpopulation cells and plasma cytokines. Patients were divided according to symptom severity during hospitalisation, in those with uncomplicated and complicated infection. Differences between groups were analyzed. Results Seventeen patients were included (mean age: 83 years; 9 women; mean delay of symptoms onset: 4 days). Six had uncomplicated infection, while 11 developed complicated forms during the hospitalization. CD10 + B lymphocyte levels were inversely correlated with clinical severity (5.8% vs 2.0%, p = 0.04) and CD10+ levels above 3% were independently associated with uncomplicated forms [Odds Ratio 0.04 (CI 0.002-0.795, p = 0.034)]. TNF-alpha, IL-1, Il-6 and Il-8 measurements upon admission differed between patients who died and those who survived (p < 0.01 for all comparisons). Conclusions In a population of elderly patients recently infected with Covid-19, CD10 + B cell levels were inversely correlated with clinical severity. Cytokine values upon admission were highly predictive of fatal outcome during hospitalisation. These findings could explain differences in the clinical presentation and allow rapid identification of patients at risk for complications.

A cluster of pneumonia cases of unknown origin occurred in Wuhan Hubei Province, China, in December 2019 [1] . The pathogen was then identified as a novel beta coronavirus now named severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) and responsible for the so-called 2019 novel coronavirus disease (Covid-19) [2] . Human-to-human transmission of has been established and the virus has rapidly spread worldwide [3] [4] [5] .

The typical clinical presentation described was initially that of an acute respiratory syndrome, but several other clinical signs have since been reported, such as diarrhea, anosmia, confusion and vasculitis [7] [8] [9] [10] [11] [12] [13] [14] .

However, clinical expression of Covid-19 is extremely variable, with, in some cases, asymptomatic or mild forms [15] , while in other subjects severe forms culminating in acute respiratory distress syndrome (ARDS) have been described, requiring patients to be transferred to Intensive Care Units. Consequently, very high mortality rates have been reported worldwide [16] [17] .

Among clinical predictive factors for severe forms of Covid-19, older age, male gender and obesity are the most described, while correlations between abnormal coagulation and hematological parameters with poor prognosis have been observed [18] [19] .

Immunity is probably one of the major determinants of clinical outcome, as ARDS has been associated with a so-called cytokine storm, characterized by an exaggerated and uncontrolled inflammatory response to the virus, potentially resulting in lung injury [20] . This immune response to Covid-19 calls for indepth investigation. Indeed, lymphocytes subsets and immune response during Covid-19 have only been partially studied. Qin et al. found lower levels of helper T cells and suppressor T cells in severe patients, but demographic characteristics of severe and non-severe patients were significantly different [20] . Moreover, while the induction of a robust cellular immunity is likely essential for efficient virus control, dysregulated T cell and NK cell response may contribute to disease severity [21] . Therefore, it is urgent to better elucidate how different immune responses across patients affect the clinical presentation.

The aim of this study was to retrospectively analyze the immunological profile of patients at early stages of Covid-19 in order to identify potential risk factors for subsequent clinical deterioration.

We conducted an observational, retrospective cohort study on patients admitted to the Internal Medicine and Infectious Diseases Department in Cannes General Hospital, from March to May 2020, with confirmed Covid-19 infection. All patients enrolled in this study were diagnosed and treated according to national guidelines for Covid-19. Blood samples were collected in accordance with the clinical guidelines for inpatients with Covid-19. The study was submitted to the French National Institute for Health Data (Institut National de Données de Santé, reference CNIL MR004) and patients gave informed consent for retrospectively collecting data.

Demographic characteristics, underlying comorbidities, duration of symptoms, clinical signs prior to admission, upon admission and during hospitalisation, laboratory findings during hospital stay and clinical outcome were collected from patients' medical records.

Among laboratory findings, we focused particularly on immunological markers, which were mainly prescribed as a consequence of initial lymphopenia by the -Subjects with complicated illness, in case patients experienced severe forms, either already at the admission, or during the hospitalization.

Severity was defined by signs of pneumonia associated with at least one of the following additional criteria: respiratory rate > 30 breaths/minute, severe respiratory distress, or spO2 < 90% on ambient air.

In case of patient transfer to another clinical setting, files were reviewed and physicians interviewed in order to confirm that no significant changes in clinical presentation had been observed for each patient. [24] . The Treg gating strategy was based on the gating of CD3+CD4+ and CD3+CD8+ lymphocytes, the CD127wkCD25+ gate being set on CD3+CD4+ T cells using the CD3+CD8+ T cells as a negative control [25] . In addition to expression of CD10, CD20 kappa and lambda on B cells, at least 6 different B cell populations were identified in each sample, namely naive B cells, switched memory B cells, marginal zone-like memory B cells and CD27-negative memory B cells, transitional B cells and plasmablasts.

In plasma, cytokine measurement included Il-6, Il-1, Il-8 and TNF-alpha, measured with custom-designed cartridges Ella (ProteinSimple), following the manufacturers' instructions.

Categorical variables were described as frequency rates and percentages, while continuous variables were detailed with mean, median and inter-quantile range (IQR).

Patients with asymptomatic or mild forms were compared to those with moderate-to-severe forms.

χ2-tests and Student's t-tests were performed to compare variables, and independent risk factors were identified by multivariate analysis.

Variables with P ≤ 0.20 in univariate analysis were initially selected for the multivariate model and only those with P ≤ 0.05 were retained in the final model. All analyses were performed using Statview© software.

From February to Mai 2020, 101 patients were admitted to our Department for Covid-19. 

In a population of elderly patients at a very early stage of Covid-19, we found that levels of CD10+ B lymphocyte cells were predictive of the clinical severity.

In humans, CD10 is expressed on B cell progenitors in the bone marrow and subsequently disappears gradually with maturation, except on activated germinal center founder B cells, co-expressed with CD27 [27] . These activated B cells are also detected in peripheral blood, the majority of them consisting in immature/transitional B cells [28] . As CD10 is a marker known to be associated with immature/transitional cells, we compared transitional B cells between the two groups, which only revealed a trend (data not shown). Our study, which was not intended to specifically analyze this cell population, does not allow to conclude on the origin of these cells and their significance in Covid-19 infection, as the difference in levels of transitional cells was not statistically significant between the two groups. However, several hypotheses can be put forward: if they are not immature/transitional cells, they could be activated precursor B cells with early Ig rearrangement in order to produce a broad repertoire of antibodies capable of responding to the viral infection [21, 29] .

Indeed, a robust B-cell response and plasmablasts expansion is generally detected early during SARS-CoV-2 infection [30] . For reasons that still require investigation, only subjects who produce higher levels of precursors B-cells appear to develop less severe forms of Covid-19. Besides, Ho et al. showed that in HIV-infected patients with advanced disease there is an increased production of CD10+ cells, which are highly susceptible to intrinsic apoptosis, due to overexpression of the anti-apoptotic BCL-2 and BCL-x L proteins [26] . Thus, increased levels of CD10+ in patients with mild forms of infection could also be explained by their better ability to eliminate the virus as a consequence of a proapoptotic profile of B lymphocytes and accelerated cell turnover. Unfortunately, the analysis did not include Il-7 which has been associated with the homeostatic compensation of overrepresented precursor B cells [31] . Finally, another explanation for lower levels of CD10+ cells in complicated forms could be a vigorous virally-induced immunosuppression, responsible for a blunted lymphocyte response [32] .

If confirmed by larger studies, this work could furnish new insights about the immunological mechanisms of response to the virus and the broad spectrum of clinical presentation in a population at high risk of complication.

Interestingly, no difference between groups was found upon admission for values of the main plasma cytokines involved in the so-called "cytokine storm".

However, although the majority of patients had a mild clinical presentation upon admission, initial cytokine values were highly predictive of fatal outcome during hospitalisation. These results could have another important consequence for future management of Covid-19, suggesting that cytokine levels at entry could allow rapid identification of subjects who, despite moderate clinical symptoms, J o u r n a l P r e -p r o o f could be at risk for developing severe complications, and could thus be anticipated to benefit from anti-inflammatory strategies [33] .

To our knowledge, this is the first study to evaluate the immunological profile of patients preceding their clinical deterioration. As previously cited, lymphocyte subsets during Covid-19 have been already studied by Qin et al. [20] , but demographic characteristics of severe and non-severe patients, in contrast with ours, were significantly different between groups. Moreover, although time since symptoms onset is not described, we presume that patients were in a later stage of infection than in our cohort, as the immunological analysis was performed when patients already had signs of severity.

The study was not designed to determine the role of hydroxycholoroquine regarding clinical response, as only subjects with severe forms received this compound. Only randomized prospective studies on recently infected patients can be expected to establish its role. This study is limited by its retrospective character. However, files were systematically reviewed in order to confirm all described clinical data. Duration of infection might be another limitation, as it cannot be coincident with symptoms onset. We therefore cannot exclude a difference in duration of viral replication between the two groups. Only through systematic and prospective nasal swabs in asymptomatic subjects would it be possible to exactly determine the time of onset of infection.

In conclusion, our work shows that levels of CD10+ B cells are higher in patients with uncomplicated Covid-19 and that levels of cytokines at the early phase of infection are predictive of a poor outcome, arguing in favor of rapid interventional strategies for patients at risk. 

This work did not require any funding

The study was submitted to the French National Institute for Health Data (Institut National de Données de Santé, reference CNIL MR004) and patients gave informed consent for retrospectively collecting data.

J o u r n a l P r e -p r o o f

There are no conflict of interest to declare 

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We wish to thank all the nurses of the Department of Internal Medicine/Infectious Diseases, without whom this work would not have been possible.A special thank also to Nathalie Doux and to Aurelie Leguillermic for the work organization and to Brigitte Dunais for reviewing this paper.J o u r n a l P r e -p r o o f