Absence of severe COVID-19 in patients with clonal mast cells activation disorders: effective anti-SARS-CoV-2 immune response

Mast cells are key actors of innate immunity and Th2 adaptive immune response which counterbalance Th1 response, critical for anti-viral immunity. Clonal Mast Cells Activation Disorders (cMCADs) such as mastocytosis and clonal mast cells activation syndrome are characterized by an abnormal mast cells accumulation and/or activation. No data have been published on the anti-viral immune response of patients with cMCADs. The aims of the study were to collected, in a comprehensive way, outcomes of cMCADs patients who experienced a biologically-proven COVID-19 and to characterize both anti-endemic coronaviruses and specific anti-SARS-CoV-2 immune responses in these patients. Clinical follow-up and outcome data were collected prospectively for one year within the French rare disease network CEREMAST encompassing patients from all over the country. Anti-SARS-CoV-2 and anti-endemic coronaviruses specific T-cells were assessed with an enzyme-linked immunospot assay (EliSpot) and anti-SARS-CoV-2 humoral response with dosage of circulating levels of specific IgG, IgA and neutralizing antibodies. Overall, 32 cMCADs patients were identified. None of them required non-invasive or mechanical ventilation; two patients were hospitalized to receive oxygen and steroid therapy. In 21 patients, a characterization of the SARS-CoV-2-specific immune response has been performed. A majority of patients showed a high proportion of circulating SARS-CoV-2-specific interferon (IFN)-γ producing T-cells and high levels of anti-Spike IgG antibodies with neutralizing activity. In addition, no defects in anti-endemic coronaviruses responses were found in patients with cMCADs compared to non-cMCADs controls. Patients with cMCADs frequently showed a spontaneous IFN-γ T-cell production in absence of any stimulation that correlated with circulating basal tryptase levels, a marker of mast cells burden. These findings underscore that patients with cMCADs might be not at risk of severe COVID-19 and the spontaneous IFN-γ production might explain this observation. Author Summary Mast cells are immune cells involved in many biological processes including the anti-microbial response. However, previous studies suggest that mast cells may have a detrimental role in the response against viruses such as SARS-CoV-2, responsible for COVID-19. When a mutation occurs in mast cells, it can lead to a group of diseases called clonal mast cells activation disorders (cMCADs), characterized by deregulated activation of these cells. Hence, patients with cMCADs might be more susceptible to severe COVID-19 than general population. We therefore conducted a 1-year study in France to collect data from all cMCADs patients included in the CEREMAST rare disease French network and who experienced COVID-19. Interestingly, we did not find any severe COVID-19 (i.e. requiring non-invasive or mechanical ventilation) in spite of well-known risk factors for severe COVID-19 in a part of cMCADs patients. We then have studied the immune response against SARS-CoV-2 and other endemic coronaviruses in these patients. We did not observe any abnormalities in the immune response either at the level of T and B lymphocytes. These findings underscore that these patients might not be at risk of severe COVID-19 as one might have feared.

with cMCADs might be more susceptible to severe COVID-19 than general population. 121 We therefore conducted a 1-year study in France to collect data from all cMCADs patients 122 included in the CEREMAST rare disease French network and who experienced COVID-19. 123 Interestingly, we did not find any severe COVID-19 (i.e. requiring non-invasive or mechanical 124 ventilation) in spite of well-known risk factors for severe COVID-19 in a part of cMCADs 125 patients. 6 126 We then have studied the immune response against SARS-CoV-2 and other endemic 127 coronaviruses in these patients. We did not observe any abnormalities in the immune response 128 either at the level of T and B lymphocytes. These findings underscore that these patients might 129 not be at risk of severe COVID-19 as one might have feared.   Well-established capacity of mast cells to drive Th2 responses (9,10), which counterbalance Th1 165 responses, could make one fears that it impairs anti-viral immunity in patients with cMCADs.

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In addition, in vitro studies found that histamine blocks the activity of human plasmacytoid 167 dendritic cells, thereby further impacting on anti-viral responses (11). Furthermore, mast cells 168 may contribute to COVID-19-induced inflammation by releasing pro-inflammatory cytokines 169 such as interleukin (IL-)1, IL-6 and tumor necrosis factor (TNF) and may also exacerbate the 170 lung lesions via degranulation (12,13). Hence, patients with cMCADs could have been more 171 susceptible to severe COVID-19.

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Over one year, we prospectively collected data from all patients with cMCADs (MMAS and 173 mastocytosis) included in the CEREMAST rare disease French network and who experienced  proteins. Six pools were tested: S1 for Spike glycoprotein N-terminal fragment, S2 for Spike   Table   206 1. Patients were predominantly females (59.4%) with a median age of 49.7 years (ranging from    (16). However, our study extends our knowledge on cMCADs and COVID-19 due to the exhaustive nature of the inclusion that concerned the CEREMAST rare disease network, which encompasses cMCADs in the entire French population. Indeed, when a patient with mastocytosis not referenced in the network was hospitalized for COVID-19 disease in an intensive care unit, the local or national reference centers were systematically contacted to obtain an expert opinion on potential drug contraindications due to the mandatory precautions needed for anaesthesia. The exhaustivity of the recruitment of patients with advanced mastocytosis and severe or critical COVID-19 was confirmed through consultation of the computerized registry of APHP that did not retrieve any inpatient unknown to the CEREMAST network. For obvious reasons, the only bias is that we cannot be fully exhaustive concerning patients with asymptomatic, mild or moderate forms of COVID-19 disease that did not require hospitalization nor special advice from their referent physicians.

Characterization of anti-SARS-CoV-2 and anti-endemic coronaviruses specific T-cells with an enzyme-linked immunospot assay in patients with cMCADs
The clinical progression scale as well as patient #30 who have received recent administration of 2CDA did not seem different from others patients. 13 To evaluate the global anti-coronavirus immune response in patients with cMCADs, we studied

T-cell specific response against Spike glycoprotein of Human alpha and beta-coronavirus
HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1. Two pools of peptide were tested (S1 and S2) as for SARS-CoV-2 Spike glycoprotein (S1 figure). No significant differences were found when comparing response in cMCADs patients and non-cMCADs controls. Our study did not find any defects in anti-endemic coronaviruses responses in patients with cMCADs with comparable reactivities in terms of frequency and intensity compared to non-cMCADs controls.
Same observation was found when comparing response in cMCADs and controls to the EliSpot positive control CEFX Ultra SuperStim Pool containing 176 known peptide epitopes derived from a broad range infectious agent: the IFN-γ production was similar in mastocytosis as in non-mastocytosis patients (S2 figure).

Characterization of anti-SARS-CoV-2 humoral response in patients with cMCADs
In parallel with EliSpot, SARS-CoV-2 specific IgG and IgA antibodies were studied with a very sensitive technique: The S-flow assay in 15 cMCADs patients. Fourteen of 15 were positive for IgG and 7 of 15 for IgA. The IgG negative patient was the one with negative EliSpot (#6). A viral pseudo-particle neutralization assay was used to determine if IgG were neutralizing. In 12/14 (86%) of IgG seropositive patients we detected neutralizing antibodies.
We report here a high prevalence of anti-SARS-CoV-2 seropositivity with high titter of neutralizing antibodies (S3 figure).
Taking all these observations into account, it is believed that patients with mastocytosis were

IFN-γ spontaneous production in EliSpot assays of patients with cMCADs.
Reading EliSpot's plates revealed an interesting observation: significantly higher backgrounds were found in cMCADs when comparing with non-cMCADs control group. In non-stimulated wells, containing PBMC in culture medium without any peptide pool, we accounted more than 10 small spots/2 10 5 CD3+ in 10/24 cMCADs patients (with history or not of COVID-19) versus 3/31 non-cMCADs controls (Fisher's Exact Test: p=0,009) and 2/11 in controls patients with idiopathic mast cell activation syndrome ( Figure 3A).
Of note, size and intensity of SARS-CoV-2 specific spots were much greater than background spots ( Figure 3B-E). Thus, adjusting settings of EliSpot Reader made it possible to count SARS-CoV-2 specific spots accurately and objectively.
The phenomenon observed resulted from a spontaneous IFN-γ release in the absence of any stimulation. As we tested total PBMC, our assay did not allow to identify the specific IFN-γ NK1 secrete IFN-γ and inhibits IgE synthesis in allergy (21). Level of total IgE could therefore be used as indirect marker of NK1 activation. Thus, we determined total IgE in 17 cMCADs sera. Low IgE levels were found in our cohort, the median value was 20 UI/ml  and no correlation between total IgE levels and spontaneous IFN-γ release was found.
Further investigations are needed to characterize NK cells in patients with mastocytosis.
We aimed then to correlate with patient characteristics. No correlation was found with age, current symptomatic treatments, history of anaphylaxis or the presence of KIT D816V mutation.
Interestingly, we found that basal serum tryptase level was correlated with spontaneous IFNγ release in patients with CM, MIS and ISM (Figure 4, R2=0.61, p<0.0001). Although it seems very unlikely that tryptase is directly involved in this phenotype (especially since patients with advanced mastocytosis have very high tryptase level without any known protection against infection) we believe that it reflects a link between clonal mast cells burden and IFN-γ release in patient with non-advanced mastocytosis.
To our knowledge, this result has never been reported in the literature and may suggest some degree of additional protection against severe patterns of viral infections. Further works in our 16 laboratory are currently performing to determine if this observation is related to a specific cytokine profile in patient plasma or due to a direct cellular mechanism between mast cells and T-cells. If confirmed, this specific phenotype in cMCADs patients might lead to therapeutic implications in the field of infectious diseases.
Overall, our results showed that cMCADs were able to develop effective and protective cellular and humoral response to SARS-CoV-2 but all of evaluable patients (4/4) with serial serology negated their serology after a median follow up of 33.0 weeks. Thus, anti-SARS-CoV-2 vaccination is strongly recommended, but its effectiveness remain to be confirmed in this specific population.

Conclusion
In conclusion, non-advanced mastocytosis and monoclonal mast cells activation syndrome most likely do not confer an increased risk for severe COVID-19. A spontaneous IFN-γ production in patients with cMCADs may be involved in this observation and must be confirmed by further clinical and biological studies. If confirmed, this specific immune profile may explain protection against SARS-CoV-2 virus.

Patients
We have prospectively collected data from patients with cMCADs and COVID-19 documented

Ethic Statements
All patients with cMCADs were followed up in the CEREMAST network centers (mastocytosis reference centers in France). Patients were enrolled in a prospective, national, multicenter study sponsored by the French association for initiative and research on mast cell and mastocytosis (AFIRMM). This study was approved by the ethics committee of Necker Hospital and was carried out in compliance with the Declaration of Helsinki Principles protocol. A written informed consent was obtained (Comité de Protection des Personnes N°93-00). Blood samples were obtained as part of routine care in the follow-up for their cMCADs. Control cohorts were prospectively collected and analyzed as part of the COVID-HOP study (APHP200609).

Immunological assays
The Spike protein were used. The viral pseudotypes were incubated with sera to be tested (at a 1:100 dilution), then added on transduced HEK 293T-cells expressing ACE2 and incubated for 48h at 37°c. The test measures the ability of anti-S antibodies to neutralize infection. Neutralization was calculated as described (24).
Control group including convalescent COVID-19 patients with mild to moderate and severe forms who were previously tested for SARS-CoV-2 EliSpots and serology in the Necker's immunology laboratory.
Comparison tests were performed using Student's t test, chi-square and Fisher's exact tests