In summary, our study presents the longest follow up of B cell depleted patients, highlighting specific T-and B cell signatures that can be used in the clinical setting to advise patients on timing of SARS-CoV-2 vaccination. Study funding This work was supported by R01AI104870-S1 (L.I.R.E, E.M., T.A.T), Austin Public Health grant 4700 NI210000003 (E.M. in a cohort of COVID-19 B cell depleted neuroimmune patients (T cell stimulation assays with peptide pools evaluating the SARS-CoV-2 spike (S), nucleocapsid (N), and membrane (M) proteins. After stimulation for 24?h, activated CD4+ T cells were quantified by flow cytometry, based on increased expression of CD40L, CD69 and OX40 (Fig.?4 ). Activation in response to stimulation was Cytisine (Baphitoxine, Sophorine) primarily Cytisine (Baphitoxine, Sophorine) observed for CD4+ T cells (Fig.?4A-?-B),B), with less strong activation of CD8+ T cells assessed by 41-BB and CD69 (Fig.?4C). Both COVID-19 controls and B cell depleted groups followed a similar pattern of moderate reactivity at the early (8C13 days post symptoms onset) timepoint and higher reactivity MST1R at the late timepoint (52C225 days post COVID-19 symptom onset), consistent with prior studies (Dan?et?al., 2021). Envelope protein stimulation showed no reactivity at either timepoint, consistent with other studies (Grifoni?et?al., 2020) (Fig.?4ACC). Open in a separate windows Fig. 4 COVID-19 T cell stimulation in B cell depleted patients. Stimulation Index to cytomegalovirus (CMV), spike (S), nucleocapsid (N), membrane (M), and envelope (E) megapools for (A) OX40+ CD69+ CD4+ T cells, (B) CD40L+ CD69+ CD4+ T cells, (C) 41BB+CD69+ CD8+ T cells. Average percentages of Na?ve, TCM, TEM, and TEMRA for CMV, PMA, S, N, and M megapool stimulation in (D) OX40+ CD69+ CD4+ T cells and (E) CD40L+ CD69+ CD4+ T cells. Timepoints were grouped into early (days 8-13) and late (days 52-225) in days from symptom onset, or for asymptomatic patients, days from first SARS-CoV-2 PCR positive test. All patients on B cell depleted therapies developed detectable SARS-CoV-2 reactive T cells that differentiated into TEM, TCM, TEMRA subsets (Fig.?4D,?,E).E). Interestingly, there was a lower frequency of na?ve T cells across all timepoints in B cell depleted patients in response to cytomegalovirus (CMV), spike, nucleocapsid, and membrane protein stimulation compared to COVID-19 controls (Fig.?4D,?,EE). 3.5. Patient immune trajectory timelines In Fig.?5 , data from RBD antibody testing, PBMC flow cytometry, and SARS-CoV-2 T cell stimulation were compiled for B cell depleted patients demonstrating individualized trajectories of immune responses to both acute SARS-CoV-2 contamination and vaccination. All numbered events were scaled to post symptom onset of COVID-19 (day 0, PSO). Participant B1 did not Cytisine (Baphitoxine, Sophorine) develop Cytisine (Baphitoxine, Sophorine) a measurable RBD antibody response and had very low detectable CD20+ B cells. This patient’s COVID-19 symptom onset occurred 190 days (6.2 months) post B cell depletion and the patient was not vaccinated over the course of 364 days of follow up PSO. Participant B2 developed COVID-19 180 days (5.9 months) post B cell depletion. The patient mounted a detectable RBD antibody titer that increased in response to vaccination. This patient had detectable live B cells at the time of acute COVID-19 symptoms, with a majority of CD20+ B cells. Of note, this patient was seronegative on day 2 and seroconverted by day 9 post SARS-CoV-2 PCR, and antibodies persisted even after B cell depletion therapy administration on day 30 post SARS-CoV-2 PCR. Participant B3 developed COVID-19 149 days (4.9 months) post B cell depletion and displayed a detectable RBD antibody titer in response to COVID-19 infection, which peaked by day 100 PSO, with the patient found to be seronegative by day 351 PSO. The patient was seronegative at days 8 and 12 PSO but was positive by day 100 PSO, despite receiving B cell depletion therapy on day 36 PSO and no vaccination events occurring prior to day 100 PSO. The patient did not have a higher RBD antibody response post vaccinations administered on day 143 and 164 PSO. Participant B4 developed COVID-19 104 days (3.4 months) post B cell depletion,.