Dotted lines denote the 95% CI. Figure 1B shows that the 2 2 different assays detected a predominant spike-specific response in all the individuals and defined a matching profile of spike-specific T cell responses after the prime and boost vaccinations. analysis. The magnitude of these spike-specific T cell responses cannot be predicted from the neutralizing antibody levels. Hence, both humoral and cellular spikeCspecific immunity should be tested after vaccination to define the correlates of protection necessary to evaluate current vaccine strategies. 6; 48 samples). Dotted lines denote the 95% CI. Figure 1B shows that the 2 2 different assays detected a predominant spike-specific response in all the individuals and defined a matching profile of spike-specific T cell responses after the prime and boost vaccinations. The number of IFN- spots detected after boost vaccination matched that observed in the phase I/II trial involving individuals vaccinated with BNT162b2 (10) and with a similar preparation consisting of the trimerized secreted version of the spike receptorCbinding domain (BNT162b1; ref. 13), including a trial conducted in Chinese individuals vaccinated with BNT162b1 (21). In our study, although stimulation with the NP-specific peptide pool remained largely negative, the levels of IFN- in the blood and the number of IFN- spots showed identical peak responses that occurred 7C10 days after the first dose in individuals V4 and V5, and 7C10 days after the second dose in individuals V1, V3, and V6. There were, however, some minor discrepancies. The CRA did not detect boosting of spike-specific T cells induced by the second vaccine dose in subjects V4 and V5, perhaps in relation to the transient lymphopenia induced by the mRNA vaccination (13). IL-2 cytokine measurement (Figure 1C) revealed a pattern of spike-specific T cell responses equivalent to that achieved through IFN- release. However, IL-2 levels exceeded those of IFN- in all individuals 21 days after the first and second vaccine doses. Overall, we found a very strong correlation between IL-2 and IFN- secretion and the number of IFN- spots (Figure 1D), which allowed a precise estimation of the quantity of IFN-Cproducing cells related to the quantity of cytokines detected in whole blood (Table 1). Table 1 Estimated IFN- SFU/106 PBMCs derived from IFN- and IL-2 concentrations in SpG peptide poolCstimulated whole blood, based on the linear regression Vitamin A analysis in Figure 1D Open in a separate window Assessment of the spike-specific T cell response directly from fresh whole blood yields results comparable to those obtained with classical T cell assays. Since T cell analysis is often performed in a single centralized laboratory using cryopreserved samples collected at different sites, we also analyzed the spike-specific T cell response after vaccination by performing ELISPOT activation-induced cellular marker (AIM) assays using cryopreserved samples stimulated with an SpG peptide pool. We then compared the results with those from the ELISPOT and CRA performed using the corresponding fresh whole blood. As already shown (22), the Rabbit polyclonal to TrkB quantity of spike-specific spots detected by ELISPOT in cryopreserved PBMCs was reduced in comparison with the quantity detected in freshly isolated PBMCs (Supplemental Figure 1A), but the dynamics of the spike-specific response remained consistent with new PBMCs (Supplemental Number 1A) as also evidenced from the high correlation between the ELISPOT results from the in a different way processed samples (Number 2A and Supplemental Number 1B). The AIM assay, in our case, was less precise at detecting the dynamic development and contraction pattern of the spike-specific T cell response (Supplemental Number 1, C and D), probably because of the bad effect of cryopreserving PBMCs. Nevertheless, the ability of the AIM assay to differentiate between CD4+ and CD8+ T cell reactions is an asset that should not be discounted. Open in a separate window Number 2 Correlation matrix of different assays used Vitamin A Vitamin A Vitamin A to quantify spike-specific T cells.(A) The top matrix shows the significance of the correlation, and the Spearmans correlation coefficient is definitely shown in the matrix below (6; 24 samples). (B) Linear regression analysis of the concentrations Vitamin A of IFN- and IL-2 in SpG peptide poolCstimulated whole blood and the corresponding rate of recurrence of SpG-reactive T cells in cryopreserved PBMCs quantified by either IFN- ELISPOT or Goal assay (6; 24 samples). Dotted lines denote the 95% CI. We then assessed whether whole-blood CRA results could reflect those acquired using additional assays (Number 2, A and B). We correlated the results obtained in all the different assays of spike-specific T cells in cryopreserved and new PBMC samples with the results from the whole-blood CRA. We found that cytokines in whole blood remained well.
