T cell responses to SARS-CoV-2 vaccination differ by disease-modifying therapy for multiple sclerosis

Abstract

Immune responses in people with multiple sclerosis (pwMS) receiving disease-modifying therapies (DMTs) have been of significant interest throughout the COVID-19 pandemic. Lymphocyte-targeting immunotherapies, including anti-CD20 treatments and sphingosine-1-phosphate receptor (S1PR) modulators, attenuate Ab responses after vaccination. Evaluation of cellular responses after vaccination, therefore, is of particular importance in these populations. In this study, we used flow cytometry to analyze CD4 and CD8 T cell functional responses to SARS-CoV-2 spike peptides in healthy control study participants and pwMS receiving 5 different DMTs. Although pwMS receiving rituximab and fingolimod therapies had low Ab responses after both 2 and 3 vaccine doses, T cell responses in pwMS taking rituximab were preserved after a third vaccination, even when an additional dose of rituximab was administered between vaccine doses 2 and 3. PwMS taking fingolimod had low detectable T cell responses in peripheral blood. CD4 and CD8 T cell responses to SARS-CoV-2 variants of concern Delta and Omicron were lower than to the ancestral Wuhan-Hu-1 variant. Our results indicate the importance of assessing both cellular and humoral responses after vaccination and suggest that, even in the absence of robust Ab responses, vaccination can generate immune responses in pwMS.

Keywords: COVID-19; Cellular immune response; Immunology; Multiple sclerosis; T cells.

Figure 1. Responses after 2 vaccine doses in pwMS on DMTs.

Individuals are grouped by DMT (healthy control participants [HC], and patients with MS who were treated with alemtuzumab [ALEM], cladribine [CLAD], natalizumab [NTZ], fingolimod [FIN], and rituximab [RTX]). (A) BAU values after 2 doses of vaccine. Responses below the lower limit of detection are shown as 0.5 BAU/mL; titers <5 BAU/mL are considered negative, 5–20 BAU/mL as very (V.) weak positives, 20–200 BAU/mL as weak positives, and >200 BAU/mL as positives. Individuals are shown as separate points; the box indicates median and IQR, whiskers indicate minimum and maximum values. Statistical analyses by Kruskal-Wallis test comparing DMT groups with HC with the Benjamini-Hochberg correction for multiple comparisons. (B) CD4 T cell (CD40L⁺ TNF-α⁺) and (C) CD8 T cell responses (IFN-γ⁺ and/or TNF-α⁺) to spike peptides before (V0) and after (V2) 2 doses of vaccine. Responses with zero events are plotted at 0.001% to indicate nonresponses. Samples from the same individual before and after vaccination are paired with a line. Statistical comparisons by Wilcoxon 2-tailed paired t tests. *P < 0.05, **P < 0.01, with the Holm-Šídák method for multiple comparisons. Patient numbers for each group are indicated along the x axis.

Figure 2. T cell and Ab responses in rituximab- and fingolimod-treated patients after third vaccine dose.

Ab responses (BAU/mL) and CD4 and CD8 T cell responses after third vaccine dose in rituximab-treated (A–C) and fingolimod-treated patients (D–F) (A, n = 43–61; B, n = 21–56; C, n = 21–54; D, n = 13–21; E, n = 6–13; F, n = 6–17). Dotted lines in (A) and (D) indicate classification of Ab responses as negative or positive, as described previously; box-and-whisker plots indicate the minimum and maximum, median, and IQR. (B, C, E, and F) Lines on scatter plots indicate the median. Statistical analyses by 2-tailed Wilcoxon signed-rank tests with Benjamini-Hochberg FDR correction for multiple comparisons. *P < 0.05, ****P < 0.0001. Flu, influenza; Unstim, without stimulation; V. very.

Figure 3. Readministration of rituximab (RTX) between vaccine doses affects Ab but not T cell responses.

Rituximab-treated individuals were grouped by whether they received a dose of RTX between V2 and V3. (A) Ab titers (BAU/mL) after the V2 and V3 vaccine doses for patients who did not receive RTX between vaccines (empty boxes) (n = 20–32) and patients who did receive RTX between vaccines (gray boxes) (n = 23–30). (B) CD4 T cell and (C) CD8 T cell responses without stimulation (unstim) or to SARS-CoV-2 spike or CMV peptides after third vaccine dose for patients without (n = 28) or with RTX administration (n = 28) between vaccine doses. Statistical analyses for paired responses by Wilcoxon signed-rank tests, unpaired responses by Mann-Whitney tests, and 2-tailed P values are shown, *P < 0.05. All t tests were corrected for multiple comparisons by Benjamini-Hochberg FDR method.

Figure 4. T cell responses to the Delta and Omicron SARS-CoV-2 variants after 3 vaccine doses.

(A) Schematic of mutated regions in the Alpha, Delta, and Omicron regions stimulated by peptides. The SARS-CoV-2 spike protein is 1273 aa long and consists of the signal peptide and S1 and S2 subunits; the RBD in S1 is indicated in red (59). Regions covered by the SARS-CoV-2 Prot_S (WT) peptide used for activation-induced marker assays are shown for reference. The control and mutant peptides for each variant cover the same loci but with the mutated (mutant) or Wuhan-Hu-1 variant (control). The aa mutations are listed in Methods. PBMCs from rituximab-treated patients after a third vaccine dose were stimulated with spike peptide pools from the mutated regions (blue circles) of the Alpha (n = 29), Delta (n = 41), and Omicron (n = 21) VOC and the same regions of the WT sequence (empty circles), and the CD4⁺ (B) and CD8⁺ (C) T cell responses were compared. Statistical differences were calculated by Wilcoxon signed-rank tests with the Holm-Šídák method for multiple comparisons. *P < 0.05, **P < 0.01.

Figure 5. Immune responses after 3 vaccine doses do not predict subsequent infection.

PwMS taking rituximab who were subsequently infected with SARS-CoV-2 up to February 2022 (gray boxes; n = 15) did not have significantly different lymphocyte counts (A), BAU levels (B), activated CD4 T cells (C), or activated CD8 T cells (D), compared with pwMS taking rituximab who were not infected in the same time period (white boxes; n = 45). Boxes represent the median and IQR, whiskers represent the minimum and maximum values; all individuals shown as separate points. (C and D) T cell responses show frequencies of spike-specific responses with unstimulated background subtracted. Statistical differences were calculated by Mann-Whitney unpaired t tests, All results were NS.