Analysis of T-cell responses directed against the spike and/or membrane and/or nucleocapsid proteins in patients with chilblain-like lesions during the COVID-19 pandemic.

Dear Editor, A range of cutaneous manifestations have been described in association with SARS‐CoV‐2 infection during the COVID‐19 pandemic. Among them, chilblain‐like lesions (CLL) occurred more frequently than expected. A direct link was demonstrated thanks to the visualization of viral particles in the skin endothelial cells by electron microscopy, which however was further questioned. An indirect link was highlighted with high prevalence of seropositivity in patients with CLL compared with the general population. However, numerous publications still question the link between CLL and SARS‐CoV‐2· Herein, we assessed this association in a cohort of 50 patients with CLL. The patients were aged 32 years (interquartile range 27–43), 29 (58%) had suggestive extracutaneous COVID‐19 symptoms and 20 (40%) had been in close contact with people with confirmed COVID‐19. We performed SARS‐CoV‐2 reverse‐transcription polymerase chain reaction (RT‐PCR) for direct viral assessment, SARS‐CoV‐2 serology for humoral response, and interferon (IFN)‐γ release assay for cellular T‐cell response.

Firstly, at recruitment, real‐time RT‐PCR testing for SARS‐CoV‐2 was performed using a nasopharyngeal swab for all patients (n = 50), and in the skin (n = 6) and the stools (n = 3) of some patients. RT‐PCR was negative for all samples tested.

COVID‐19 serological tests were performed using three different techniques at recruitment and 14 days later: (i) IgG Abbott Architect COVID test (Abbott Laboratories, Libertyville, IL, USA); (ii) IgG and IgA enzyme‐linked immunosorbent assay (ELISA) nucleocapsid COVID test (EUROIMMUN, Lübeck, Germany); and (iii) flow spike IgA and IgG detection. The serological results were compared with those from a separate cohort of three patients with RT‐PCR‐confirmed mild COVID‐19. The serological tests were all negative in the CLL group, except for four positive and four doubtful IgA ELISA anti‐SARS‐CoV‐2 tests at the first visit. All three samples from the COVID‐19 group were positive.

IFN‐γ release assay was performed using ELISpot. Briefly, cryopreserved peripheral blood mononuclear cells were thawed and stimulated with pooled overlapping peptides spanning the SARS‐CoV‐2 spike, nucleocapsid and membrane protein (each at 2 mg mL−1; Miltenyi Biotec, Bisley, UK). The response was compared between patients with CLL, patients with RT‐PCR‐confirmed mild COVID‐19, and healthy control samples collected before the pandemic. We detected reactive T‐cell responses directed against the spike and/or membrane proteins and/or nucleocapsid in 33% of CLL, 31% of healthy control and 100% of COVID samples. The CLL samples had higher levels of spot‐forming unit than healthy controls, although not significantly (median 23·3, interquartile range 3·3–98·3 vs. −12·2, interquartile range −21–170, P = 0·37) (Figure 1).

Figure 1

SARS‐CoV‐2‐specific T‐cell response. Peripheral blood mononuclear cells (PBMCs) isolated from patients with chilblain‐like lesions (CLL; n = 27), patients with COVID (n = 5) and healthy controls (HC; n = 19) were stimulated ex vivo with overlapping peptides spanning the immunogenic domains of the SARS‐CoV‐2 S, M and N proteins in an interferon‐γ ELISpot assay. Negative control wells lacked peptides, and positive control wells included a CEF (Cmv–Ebv–inFluenzae virus) peptide pool. (a) Interferon‐γ‐producing spot‐forming units (SFU) per 106 PBMCs in response to the peptide mix. Responses were normalized to the positive control using background subtraction. Each dot represents one donor. The horizontal line represents the median, and the error bars represent the interquartile range. (b) Frequencies of donors responding to the peptide mix in each group. Antigen‐specific responses were considered positive when the number of SFU per 106 PBMCs was above 50 after background subtraction. *P < 0·05, Kruskal–Wallis rank‐sum test with Dunn’s post hoc test for multiple comparisons. NS, not significant.

Although SARS‐CoV‐2 is known to elicit a strong antibody response towards both surface and nucleocapsid peptides during systemic and pulmonary severe and mild disease, little is known about the humoral response in asymptomatic patients. Moreover, specific T‐cell response has been less studied, but it has recently been shown that when asymptomatic, patients more frequently display a T‐cell response than a humoral response.

Given that chilblains are described as a later manifestation of COVID‐19, it is unsurprising that the patients had negative PCR results. Moreover, RT‐PCR can give false negatives if the amount of viral genome is insufficient or if the correct time window of viral replication is missed. Discrepancies between studies concerning positive serologies, some reporting seropositivity in up to 30% of patients with CLL, may be explained by many factors. Among them are different sensitivity, different timing between onset of disease and blood collection, and searching for only certain isotypes. Therefore, we used three different serology techniques to improve sensitivity, collected blood at two timepoints and searched for IgA anti‐SARS‐CoV‐2 antibodies, as they have been shown to be associated with vasculitis manifestations. The S‐Flow assay has the advantage of capturing all anti‐SARS‐CoV‐2 S protein antibodies and providing excellent sensitivity. Nevertheless, none had a positive serology, consistently with previous reports.

We and others have previously demonstrated that the cellular infiltrate plays a key role in the pathogenesis of CLL, and more particularly type I IFN, T helper 1 polarization and cytotoxic infiltration, highlighting the role of the cellular response. However, we demonstrate for the first time that patients with CLL have the same specific T‐cell response towards either the S, N or M protein as healthy controls. This could be explained by a pre‐existing cross‐reactive CD4 T‐cell memory towards common‐cold coronaviruses, as described previously in up to 20–50% of people, or by the absence of a link between CLL and SARS‐CoV‐2. Further studies should be conducted studying specific CD4 and CD8 responses towards different peptides separately. For example, cross‐reactivity with common‐cold coronaviruses seems more important in the context of nucleocapsid‐specific CD4 T cells. Our data therefore do not demonstrate the role of SARS‐CoV‐2 in the pathogenesis of CLL through specific T‐cell activation.

Acknowledgments

We thank all of the medical staff from the dermatology department of Saint‐Louis Hospital, and particularly Drs Marie Jachiet, Antoine Petit and Anne Saussine. Our thanks also go to Marie‐Hélène Durand, Elisabeth, Julie and Alain for their valuable assistance. We are also grateful to Drs Luc Sulimovic and Michel Rybojad and all members of the SNDV (French National Union of Dermatologists‐Venereologists) (listed in Appendix  S1; see Supporting Information). The authors gratefully acknowledge an equipment grant from Dormeur Investment Service Ltd, who provided funding to purchase the plate reader used here.

Author Contribution

Charles Cassius: Conceptualization (lead); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing‐original draft (equal); Writing‐review & editing (equal). Marine Merandet: Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal). Laure Frumholtz: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing‐original draft (equal); Writing‐review & editing (equal). David Bergerat: Investigation (equal); Methodology (equal); Software (equal); Writing‐review & editing (equal). Assia Samri: Investigation (equal); Methodology (equal); Software (equal); Validation (equal); Writing‐review & editing (equal). Chloé Grolleau: Investigation (equal); Methodology (equal); Writing‐review & editing (equal). Ludivine Grzelak: Data curation (equal); Investigation (equal); Methodology (equal); Writing‐review & editing (equal). Olivier Schwartz: Investigation (equal); Methodology (equal); Resources (equal); Software (equal); Writing‐review & editing (equal). Nader Yatim: Conceptualization (equal); Methodology (equal); Resources (equal); Software (equal); Writing‐review & editing (equal). Parna Moghadam: Investigation (equal); Writing‐review & editing (equal). Léa Jaume: Methodology (equal); Resources (equal); Writing‐review & editing (equal). Martine Bagot: Data curation (equal); Funding acquisition (equal); Resources (equal); Validation (equal); Writing‐review & editing (equal). Jérôme Le Goff: Funding acquisition (equal); Investigation (equal); Methodology (equal); Resources (equal); Writing‐review & editing (equal). Constance Delaugerre: Funding acquisition (equal); Investigation (equal); Resources (equal); Supervision (equal); Writing‐review & editing (equal). Jean David Bouaziz: Conceptualization (equal); Data curation (equal); Funding acquisition (equal); Writing‐original draft (equal); Writing‐review & editing (equal). Hélène Le Buanec: Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Supervision (equal); Writing‐original draft (equal); Writing‐review & editing (equal).

Appendix S1 Members of the French National Union of Dermatologists‐Venereologists (SNDV) and member of Saint‐Louis CORE.

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