Infection with SARS-CoV-2 is characterized by a broad spectrum of clinical syndromes, which may range from asymptomatic infection to mild symptoms to severe pneumonia and acute respiratory distress syndrome [2, 3].
According with the World Health Organization weekly epidemiological updates on COVID-19 (Edition 70), the current cumulative number of cases and deaths reported globally is almost 270 million and over 5.3 million respectively.
Immunological and clinical studies of acute and convalescent COVID-19 patients have observed that SARS-CoV-2-specific antibodies and T cell responses are strongly associated with milder disease and accelerated viral clearance [4-6] .
Varying approaches have been taken to quantify and characterize virus-specific T cell responses in acute, convalescent, and severe patients, in a quest to understand the nature of antigen specificity and function of the adaptive response to SARS-CoV- 2 [3, 6, 8, 9].
The response to the structural proteins, including spike (S), nucleocapsid (N), membrane (M), and non-structural proteins (nsp3, nsp4, ORF3a, and ORF8), has been the main targets for study. Using HLA predicted peptide megapools (MP) of the SARS-CoV-2 proteome, Grifoni et al.  demonstrated that SARS-CoV-2-specific CD4+ T cell responses were found in 100% of patients convalescing from COVID-19, with the majority of the CD4+ T cell reactivity directed to SARS-CoV-2 spike, M, and N proteins. On average, these antigens accounted for 27%, 21%, and 11% of the total CD4+ T cell response, respectively.
The CD4+ T cell responses to the SARS-CoV-2 S or N proteins have been shown to correlate with the magnitude of the anti-SARS-CoV-2 neutralizing antibodies in recovered patients [8, 10] , a finding suggesting a potential role for the S protein in triggering a protective response to COVID-19.
The SARS-CoV-2 M protein, in contrast, has been implicated in driving evasion of protective immune responses, a process felt to occur by the manipulation of innate antiviral immune responses ,most specifically by interfering with interferon (IFN) signaling pathways and by antagonizing the production of type I and III IFN production [11-14] . This M- driven antiviral immune suppression appears to favor SARS CoV-2 viral replication.
To understand in fine detail the molecular nature of the virus-specific CD4+ T cell response to the SARS CoV-2 structural proteins, here, we generated (in a non-biased manner) 12 human CD4+ T cell lines (TCLs) reactive to either S protein or M protein from naïve T cells obtained well prior to the COVID19 pandemic from 6 healthy donors with the aim of comparing their molecular properties and function. Our data suggest that SARS-CoV-2 S and M proteins each drive a strikingly distinct molecular signature in TCLs driven under neutral conditions.
Whereas the S-specific responses are virtually indistinguishable from those responses induced by other viruses (e.g. CMV), the M protein-specific CD4+ TCLs have a transcriptomic signature that demonstrates marked suppression of STAT1-IFRs-interferon pathway signaling, a signature that is virtually indistinguishable from the molecular signature seen associated with severe COVID-19.
There is a critical need for elucidating the nature of antigen specificity and function of the memory T cell responses to SARS-CoV-2. Understanding the contribution of adaptive immunity to a protective or pathogenic role in SARS-CoV-2 infection may lead the way to a fundamental knowledge that can possibly be used therapeutically in COVID-19 patients or as vaccine targets.
SARS-CoV-2 infected-patients develop specific antibodies, CD4+ T cells, and CD8+ T cells in response to the infection [3, 6, 8, 9] , although, CD4+ T cells had the strongest association with diminished COVID-19 disease severity compared with the other two arms (B cells, CD8+ T cells) of the adaptive immunity . Strikingly, the absence of SARS-CoV-2-specific CD4+ T cells was associated with severe or fatal COVID-19 .
Data from other another coronavirus (SARS-CoV-1) reported that SARS-CoV-1 spike protein was responsible for nearly two-thirds of the CD4+ T cell reactivity with limited reactivity for M and N proteins  . It seems, however, that the pattern of antigen predominance of SARS-Cov-2-driven immune responses is different from SARS-CoV-1 in that there is strong reactivity of CD4+ T cells to viral S, M and N structural proteins, as well as, to other non-structural proteins and open reading frames, including ORF3 and NSP3 [3, 10, 16-18].
The relationship between antigen-specific CD4+ T cell responses and COVID-19 severity remains unclear. First it has been demonstrated that mild COVID-19 patients, who typically recover without special treatment, showed broad SARS-CoV-2-specific CD4+ T cell responses to S and N proteins, responses that were highly correlated with specific antibody titers .
However, T cell responses were imbalanced in critical ICU patients with a functionally impaired CD4+ T cell response showing reduced production of IFN-g and TNF-a .
Indeed, an inflammatory cytokine and chemokine signature (elevated CXCL10, IL-6, and IL-8) accompanied by ineffective interferon responses has been strongly associated with failure to control a primary SARS-CoV-2 infection and with a higher risk of fatal COVID-19 [20-22].
Moreover, impaired, and delayed type I and type III IFN responses have been associated with a higher risk of severe COVID-19 . Interferons (IFNs), including type I (IFN-α and IFN-β) and type III (IFN-λ) are central to both combating virus infection and modulating the antiviral immune response [24, 25]. While type I IFNs are widely expressed and can result in immune- mediated pathology during viral infections, type III IFN (IFN-λ) responses are primarily restricted to mucosal surfaces and are associated with protection to viruses without driving damaging proinflammatory responses .
Interestingly, coronaviruses develop efficient immune evasion mechanisms by manipulating immune responses and by interfering with the IFN-related pathways . Indeed, several structural (M and N) and non-structural (NSP1 and NSP3) proteins from SARS-CoV and MERS-CoV can act as interferon antagonists . Notwithstanding, SARS-CoV-2 M protein has also been implicated to antagonize type I and III IFN production by affecting the formation of the RIG-I/MDA-5–MAVS–TRAF3–TBK1 signalosome that has been shown to attenuate antiviral immunity and enhance viral replication .
Here, using single-cell transcriptomes of human CD4+ TCLs reactive to either S protein or M protein, we have shown that SARS-CoV-2 M protein-reactive CD4+ TCLs in comparison with S protein, expressed higher levels of the inflammatory genes FOS, JUNB and lower levels of ISGs, including ISG15, IFI6, IFI35, IFI44, IFIT3, IFITM1, STAT1, OAS1, and interferon regulatory factors, including (IRF7). Viral recognition elicits IFN production, which in turn triggers the transcription of IFN-stimulated genes (ISGs), which engage in various antiviral functions. ISGs have a central role to regulate
the type I interferon . Among these ISGs, ubiquitin-like protein ISG15 is one of the most strongly and rapidly induced, and recent work has shown that it can directly inhibit viral replication and modulate host immunity [30-32]. Similarly, we also observed that the molecular signature of SARS-CoV-2 M protein-reactive CD4+ TCLs, is characterized by suppression in the interferon pathways, genetically associates with the transcriptional profile of severe Covid-19.
Notably, through signal cell RNAseq analysis of T-cell dysregulation in severe COVID- 19 it has been demonstrated that CD4+ T cells from severe COVID-19 patients expressed higher levels of a set of inflammatory genes that include FOS, FOSB, JUN and others, gene expression
 not dissimilar to those found in our M-specific TCLs derived from SARS-CoV-2 unexposed individuals. In parallel, this same study showed that CD4+ T cells from patients with severe COVID-19 showed decreased expression of interferon-induced genes including IFIT1, IFIT2, IFIT3, and IFITM1 and those downstream from interferon signaling , again striking similar to the molecular signature seen in the M protein-reactive CD4+ TCLs in this present study. Finally, the mechanisms by how the peptide megapools of M and S proteins underlies different responses of naïve CD4+ T cells remains unclear. Future studies are needed to elucidate if the M- driven dysregulation of interferon signaling pathway in the adaptive immunity resemble to the mechanisms already described including the interaction with pattern recognition receptors (PRRs)-downstream molecules of innate cells, or if it is induced by the interaction of the class II MHC-peptide complex with the restricted TCR repertoire of naïve T cells.
In conclusion, although it has been poorly understood how CD4+ T cell dysregulation can contribute to the immunopathogenesis of severe COVID-19, our study suggests a potential link between the antigen specificity of the reactive CD4+ T cells to SARS-CoV-2 with the development of a functional and efficient adaptive immune response. The discordant response to
S compared to M proteins suggest that the balance between the T cells of different specificities may alter immune evasion mechanisms that may, in turn, drive disease severity. Therefore, one could envision therapeutic approaches that also targets the SARS-CoV-2 M protein may also be important for amelioration of severity of COVID-19.
reference link : The study findings were published on a preprint server and are currently being peer reviewed. https://www.medrxiv.org/content/10.1101/2022.01.20.22269491v1