Figure legends
Fig 1. Activation Induced Marker (AIM) assay of circulating
memory Tfh revealed that cTfh cells of both infection naïve vaccinees
and convalescent vaccinees recognized spike proteins of both wild type
and delta strain. Representative
flow cytometry plot showing negative unstimulated control (A) .
Representative flow cytometry
plot showing cTfh response in infection naïve vaccinees to the Spike
protein of Wild type strain of SARS-CoV2 (SWT) (B) .
Representative flow cytometry plot showing cTfh response in infection
naïve vaccinees to the Spike protein of Delta strain of SARS-CoV2 (SDT)
(C) Representative flow cytometry plot showing cTfh response in
convalescent vaccinees to the Spike protein of Wild type strain of
SARS-CoV2 (SWT) (D) .
Representative flow cytometry plot showing cTfh response in infection
convalescent vaccinees to the Spike protein of Delta strain of SARS-CoV2
(SDT) (E) Representative flow cytometry plot showing stimulated
with α-CD3 and α-CD28 (F) . Cumulative graphical representation
of AIM assay indicated by CD25+OX40+cTfh cells responding to SWT and SDT in infection naïve vaccinees;
(n=20) (G ) Cumulative graphical representation of AIM assay
indicated by CD25+OX40+ cTfh cells
responding to SWT and SDT in convalescent vaccinees; (n=20)(H ). Cumulative
graphical representation of comparison of the AIM assay indicated by
CD25+OX40+ cTfh cell response to SWT
between infection naïve vaccinees (n=20) and convalescent vaccinees
(n=20) (I) Cumulative graphical representation of comparison of
AIM assay indicated by CD25+OX40+cTfh cell response to SDT between infection naïve vaccinees (n=20) and
convalescent vaccinees (n=20) (J,) . Representative flow
cytometry plot showing different T helper memory compartment response in
infection naïve vaccinees to the Spike protein of Wild type strain of
SARS-CoV2 (SWT) (K) , Representative flow cytometry plot showing
different T helper memory compartment response in infection naïve
vaccinees to the Spike protein of Delta strain of SARS-CoV2 (SDT)
(L). Representative flow cytometry plot showing different T
helper memory compartment response in convalescent vaccinees to the
Spike protein of Wild type strain of SARS-CoV2 (SWT) (M).Representative flow cytometry plot showing different T helper memory
compartment response in convalescent vaccinees to the Spike protein of
Delta strain of SARS-CoV2 (SDT) (N). Cumulative graphical
representation of T helper central memory cells indicated by
CCR7+CD45RA- responding to SWT and
SDT in naive vaccinees; (n=20) (O).Cumulative graphical
representation of T helper central memory cells indicated by
CCR7+CD45RA- responding to SWT and
SDT in convalescent vaccinees; (n=20) (P). Error bar indicates
SEM. Wilcoxon Test was used to
determine statistical significance of immunological response to the wild
type and delta spike proteins between same individuals of naïve
vaccinees and convalescent vaccinees groups. Mann-Whitney U test was
used to compare statistics between naïve vaccinees and convalescent
vaccinees. (*), p<0.01 was considered to be very significant
(**), P < 0.001 was considered to highly significant
(***), P < 0.0001 was considered extremely significant
(****) ns, not significant.
Fig 2. Spike specific cTfh cytokine response revealed that cTfh
response is damped in convalescent vaccinees as compared to infection
naïve vaccinees and spike specific IL-21 secretion from cTfh positive
correlated with neutralizing antibody in infection naïve vaccinees .Representative flow cytometry plot showing negative unstimulated control(A) . Representative flow cytometry plot showing cTfh cytokine
response in infection naïve vaccinees to the Spike protein of Wild type
strain of SARS-CoV2 (SWT) (B) . Representative flow cytometry
plot showing cTfh cytokine response in infection naïve vaccinees to the
Spike protein of Delta strain of SARS-CoV2 (SDT) (C).Representative flow cytometry plot showing cTfh cytokine response in
convalescent vaccinees to the Spike protein of Wild type strain of
SARS-CoV2 (SWT) (D) . Representative flow cytometry plot showing
cTfh cytokine response in infection convalescent vaccinees to the Spike
protein of Delta strain of SARS-CoV2 (SDT) (E). Representative
flow cytometry plot showing stimulated with PMA/Ionomycin (F) .
Cumulative graphical representation of cytokine assay indicated by
IL-21+ cTfh cells responding to SWT and SDT in
infection naïve vaccinees; (n=20) (G ).
Cumulative graphical
representation of cytokine assay indicated by
IL-21+ cTfh
cells responding to SWT and SDT in convalescent vaccinees; (n=20)(H ). Cumulative graphical representation of comparison of
cytokine assay indicated by IL-21+ cTfh cell response
to SWT between infection naïve vaccinees(n=20) and convalescent
vaccinees (n=20) (I). Cumulative graphical representation of
comparison of the cytokine assay indicated by IL-21+cTfh cell response to SDT between infection naïve vaccinees (n=20) and
convalescent vaccinees (n=20) (J) . Cumulative graphical
representation of % Neutralization between infection naïve vaccinees
and convalescent vaccinees (K). Graphical representation of
co-relation analysis between % IL-21+ spike specific
cTfh cells and % neutralizing activity in Infection naïve
vaccinees(L). Graphical representation of co-relation analysis
between % IL-21+ spike specific cTfh cells and %
neutralizing activity in Infection convalescent vaccinees (M).Representative flow cytometry plot showing different cTfh subsets based
on chemokine markers CXCR3 and CCR6 in infection naïve vaccinees(N) Representative flow cytometry plot showing different cTfh
subsets based on chemokine markers CXCR3 and CCR6 in convalescent
vaccinees (O). Cumulative graphical representation of T helper
central memory cells indicated by
CCR7+CD45RA- responding to SWT and
SDT in naive vaccinees; (n=20) (O). Error bars indicate SEM.
Wilcoxon Test was used to determine statistical significance of
immunological response to wild type and delta spike proteins between
same individuals of naïve vaccinees and convalescent vaccinees groups.
Mann-Whitney U test was used to compare statistics between naïve
vaccinees and convalescent vaccinees including the neutralisation assay.
The spearman’s rank correlation coefficient was used to measure the
correlation between two different variables (*), p<0.01 was
considered to be very significant (**), P < 0.001 was
considered to highly significant (***), P < 0.0001 was
considered extremely significant (****) and ns, not significant.
Fig 3. Cytokines analyses
from plasma of and healthy controls. Representative figures from T cell
cytokines analyzed in infection naïve vaccinees (n=20) and convalescent
vaccinees (n=20) are represented as dot plots. cTfh promoting cytokines
IL-21 (A), IL-10 (B), IL-4 (C). were elevated
in infection naïve vaccinees as compared to convalescent vaccinees.
Amongst anti-viral memory inducing cytokines IFN-γ (D) , IL-15(E), IL-12 (F) and IL-17 (G) were also
elevated in infection naïve vaccinees as compared to convalescent while
inflammatory cytokines IL-1β (H) and IL-6 (I) were
elevated in convalescent vaccinees as compared to infection naïve
vaccinees. Error bar indicates SEM. Mann-Whitney U Test was
performed to compare between the two groups, p<0.05 was
considered statistically significant(*), p<0.01 was considered
to be very significant (**), P < 0.001 was considered
to highly significant (***), P < 0.0001 was considered
extremely significant (****) ns, not significant.
Numbers between the brackets indicate range of the data set