4. Discussion
The role of cellular immunity is actively evolving in case of SARS-CoV2. With vaccination the severity of SARS-CoV2 has come down5, but still due to evolution of different VOCs, there have been uptick of cases, leading to significant morbidity and mortality. Our study here aimed to understand the phenotype of Tfh cells in recovered individuals of breakthrough infections and how they are different from Tfh cells encountered in only vaccinated individuals Tfh are important cells for resolution of infections and are there to help B cells for affinity maturation and isotype switching6. Our results showed Tfh cells from both naïve vaccinees and convalescent vaccinees recognized the spike protein from both wild and delta strains showing that epitope is conserved for T cells to respond to VOCs. This is in line with previous study which showed conservation of epitopes in VOCs to which T cells can respond3. IL-21, is one of the signature cytokines of Tfh and is essential for B-cell maturation7, our study revealed that IL-21 expression from cTfh were higher in naïve vaccinees compared to convalescent vaccinees. This is in observation with another study which showed a decrease in spike specific cTfh after third booster dose with CoronaVac or breakthrough infection during early time points8. Interestingly, we did not observe the boosting of neutralization activity after breakthrough infection, although our data revealed a strong positive correlation between IL-21 secretion from cTfh and neutralization activity only in naïve vaccinees and not in convalescent vaccinees. This could be due the timing of sampling9. Most of the convalescent vaccinees showed higher levels of pro-inflammatory cytokines like IL-6 and IL-1 b compared to naïve vaccines. On the other hand, Tfh and memory response promoting cytokines like IL-21, IL-10, IL-4, and IL-15 were higher in naïve vaccinees compared to convalescent vaccinees7,10. Our data also revealed that most of the cTfh were of cTfh2 phenotype in both naïve vaccinees and convalescent vaccinees. The role of cTfh2 in physiology and pathology is still not clear but they can promote B-cell response better than cTfh1 but less than cTfh 17 11.
In summary our data showed that after breakthrough infections there are perturbations in memory cTfh cells and their response is dampened. This could be due to the pro- inflammatory microenvironment that existed in these individuals even after the infection is resolved or it may be the endophenotype (heterogenous immune response) in these individuals that may be the cause of these perturbations12,13. Since we did not do a longitudinal study, we cannot determine whether there cTfh response were already dampened before infection or it was the effect of infection and subsequent inflammation. It also may have been possible that the cTfh response may evolve with time in these individuals since the strength and duration of antigenic stimulation of TCR along with cytokine milieu in the microenvironment is important for differentiation of different T helper subsets14,15. Overall, our study shows that hybrid immunity may be time and dosage dependent as all our convalescent vaccinees had mild symptoms during infection and infection naïve vaccinees displayed stable immune response to both wild and delta strains indicating that further consideration to booster doses may be specific for individuals or population vulnerable to more severe infections.
Author Contributions: Experimental design and conceptualization- S.S and S.D. Sample collection, processing- P.K.B. Flow cytometry experiments— S.S, S.K.S. Cytokine multiplexing—S.S., and G.B Neutralisation Assay- SaC, S.S, S.K.S and S.C. Data analysis—S.S., S.K.S and S.D. All Authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by the core funding of Institute of Life Sciences, Bhubaneswar, Department of Biotechnology (DBT), Government of India. S.S. was funded by DBT fellowship, S.K.S., SaC, and G.B were funded by the CSIR fellowship.
Data Availability Statement: The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.