T-cell responses after COVID-19 vaccination in anti-CD20-treated lymphoma patients. (A) T-cell response rates in vaccinated patients (n = 50) and vaccinated healthy controls (n = 7) both after V2. A response was considered positive if mean spot-forming units (SFU) in peptide stimulated wells was >3 SFU above background, defined as mean SFU + 2 standard deviations of dimethylsulfoxide (DMSO)-exposed wells. Staphylococcal Enterotoxin B (SEB) served as the positive control. The median time between second vaccination and sample collection was 17 days (IQR 14–21 days) in the vaccinated patient cohort and 18 days (IQR 14–80 days) in the healthy control cohort. The T-cell response rate in the vaccinated healthy cohort was 71% (5/7) and 58% in the patient cohort with previous or ongoing anti-CD20 treatment (29/50). In patients and healthy controls with an available sample prior to vaccination, we observed positive T-cell responses (6 out of 26 patients, 1 out of 9 healthy controls, data not shown). These positive T-cell responses without prior COVID-19 infection were interpreted as a crossreactive response of memory T cells after prior infection with common cold coronaviruses. (B) T-cell response rates according to the anti-S1 seroconversion after the second vaccination. The T-cell response rate of patients with a successful seroconversion was 70% (14/20). Patients without a seroconversion still showed a T-cell response in 50% (15/30). The difference in T-cell response rates between patients with and without a seroconversion was not statistically significant (Fisher’s exact test P = .2). (C) T-cell response rates after second vaccination according to the interval of last anti-CD20 treatment and first COVID-19 vaccination (<3 months: n = 15, >3 months and <12 months: post n = 12; >12 months: n = 23). The T-cell response rate was 57% (13/23), 67% (8/12), and 53% (8/15) when the last anti-CD20 treatment was administered >12 months, within >3 and <12 months, or <3 months prior to vaccination, respectively. No significant difference of the T-cell response between all 3 groups was observed (Fisher’s exact test: >12 months vs >3 months and <12 months, P = .7; >12 months vs <3 months, P = 1.0; >3 months and <12 months vs <3 months, P = .7). (D) T-cell responses in patients with a proven negative prevaccination T-cell status. For 26 patients, prevaccination samples (median time of sample collection: 218.5 days [IQR 4.5–309 days]) prior to vaccination were available and tested for T-cell response. Out of the 26 patients, 20 patients had a negative T-cell response prior to vaccination. Representative ELISpot images were shown for a patient with diffuse large B-cell lymphoma who was treated with rituximab-CHOP and received the last cycle 48 days prior to the first vaccination. This patient had no SARS-CoV-2-S1 antibody response after 2 vaccinations with BNT162b2 (BioNTech). The left ELISpot panel shows the negative T-cell response measured in the sample 1 day prior to vaccination. The right ELISpot panel shows a positive T-cell response after 2 COVID-19 vaccinations (sample collection: 14 days post-second vaccination). The right bar plot shows the T-cell response rate after V2 in patients with a confirmed negative T-cell response prior to vaccination (n = 20). A positive T-cell response after vaccination was observed in 50% of patients (10/20). V1, vaccination 1; V2, vaccination 2; ns, not significant.