Development of humoral and cellular immunological memory against SARS-CoV-2 despite B cell depleting treatment in multiple sclerosis

Abstract

B cell depleting therapies (BCDTs) are widely used as immunomodulating agents for autoimmune diseases such as multiple sclerosis. Their possible impact on development of immunity to severe acute respiratory syndrome virus-2 (SARS-CoV-2) has raised concerns with the coronavirus disease 2019 (COVID-19) pandemic. We here evaluated the frequency of COVID-19-like symptoms and determined immunological responses in participants of an observational trial comprising several multiple sclerosis disease modulatory drugs (COMBAT-MS; NCT03193866) and in eleven patients after vaccination, with a focus on BCDT. Almost all seropositive and 17.9% of seronegative patients on BCDT, enriched for a history of COVID-19-like symptoms, developed anti-SARS-CoV-2 T cell memory, and T cells displayed functional similarity to controls producing IFN-γ and TNF. Following vaccination, vaccine-specific humoral memory was impaired, while all patients developed a specific T cell response. These results indicate that BCDTs do not abrogate SARS-CoV-2 cellular memory and provide a possible explanation as to why the majority of patients on BCDTs recover from COVID-19.

Keywords: Immunology; Virology.

Graphical abstract

Figure 1

Study design CLD = cladribine; DMF = dimethyl fumarate; ECLIA = electro-chemiluminescence immunoassay; FGL = fingolimod; Glat. = glatirameracetate; HC = healthy controls; HSCT = hematopoietic stem cell transplantation; IFN = interferon-beta; No TX = no treatment; NTZ = natalizumab; OCR = ocrelizumab; OFA = ofatumumab; RTX = rituximab; TFN = teriflunomide.

Figure 2

Patients with MS treated with immunosuppressants develop SARS-CoV-2 humoral and cellular immunological memory (A) Correlation of SARS-CoV-2 antibody levels as measured by MFI of S1S2 or N with ΔIFN-γ SFUs after stimulation with S1 (n = 122) or N (n = 122) peptides, respectively. Ten samples that were only tested with ECLIA are also shown. (B) Number of ΔIFN-γ SFUs after stimulation with S1 (n = 132) or N peptides (n = 132) in seropositive or seronegative patients on anti-CD20 (n = 75) or other immunosuppressive treatment (n = 42) and in HC (n = 15). The percentage of persons with SARS-CoV-2-positive T cell immunity per serostatus and treatment group or HC. (C) Number of ΔIFN-γ SFUs after stimulation with S1 or N peptides in patients (n = 88) and HC (n = 11) with specific COVID-19-like symptoms. The percentage of SARS-CoV-2-positive T cell immunity per specific COVID-19-like symptom. (D) Anti-SARS-CoV-2 antibody MFI for S1S2 and N in persons with positive or negative T cell immunity and on anti-CD20 (n = 73) or other immunosuppressive treatment (n = 39) and in HC (n = 10). The percentage of positive and negative serology status in pwMS and HC with positive or negative SARS-CoV-2 T cell immunity is shown. (E and F) Correlation of MFI (n = 34) or ΔIFN-γ SFU (n = 42) measured with days between sampling and COVID-19-like symptoms. Spearman r and p values are shown. Dots represent individual data points while X represents samples that were only tested with ECLIA. Box plots represent median and 95% CI. See also Tables S1, S2 and Figure S1. ECLIA = electro-chemiluminescence immunoassay; HC = healthy controls; MFI = median fluorescent intensity; pwMS = persons with multiple sclerosis; SFU = spot forming unit.

Figure 3

SARS-CoV-2-specific T cells from patients with MS on anti-CD20 treatment are functional (A–C) Correlation of ΔIFN-γ SFUs measured by FluoroSpot with percentages of LFA-1⁺ IFN-γ⁺ T cells stimulated with anti-CD3 (n = 34) or four SARS-CoV-2 peptides (N, S, S1, and M, n = 34) or only N and S1 SARS-CoV-2 peptides in pwMS and HC (n = 30). (D) Representative dotplots of LFA-1 and IFN-γ expression of total CD3⁺ T cells with corresponding IFN-γ FluoroSpot image of one HC and one pwMS under anti-CD20 treatment. (E) Repartition of LFA-1⁺ IFN-γ⁺ specific T cells between CD4 and CD8 among pwMS with anti-CD20 treatment following stimulation as in (A–C) (n = 14 or 15) and with EBV peptides (n = 4). (F) Representative dotplots of CD4⁺ T cell expression of LFA-1 with CD40L, IFN-γ, TNF, and GM-CSF from one HC and one pwMS with anti-CD20 treatment following a culture with medium or in the presence of SARS-CoV-2 peptides N and S1. (G–J) Percentages of LFA-1⁺CD40L⁺, LFA-1⁺IFN-γ⁺, LFA-1⁺TNF⁺, and LFA-1⁺GM-CSF⁺ among CD4⁺ T cells in HC (n = 8) and pwMS with anti-CD20 treatment (n = 18) or with other therapies (n = 7). Spearman r and p values are shown. Dots represent individual data points. Box plots represent median and 95% CI. See also Figure S2. CLD = cladribine; DMF = dimethyl fumarate; DN = double-negative; DP = double-positive; EBV = Epstein-Barr virus; HC = healthy controls; MFI = median fluorescent intensity; NTZ = natalizumab; pwMS = persons with multiple sclerosis; RTX = rituximab; SFU = spot forming unit.

Figure 4

Circulating follicular T helper cells correlate with SARS-CoV-2 antibody levels. PwMS and HC that were SARS-CoV-2 positive in either serological and/or T cell assays are included (A) Correlation matrix representing Spearman r (left panel) and the corresponding p values (right panel) between the COVID-19-specific antibody levels (n = 40), ΔIFN-γ levels measured by FluoroSpot (n = 50), and percentages of LFA-1⁺IFN-γ⁺ T cells (n = 14), with the percentage of immune cells determined by FACS in COVID-19-positive pwMS and HC. (B) Correlation between percentages of memory CD27⁺ B cells (left images) and Tfh cells (right images) with COVID-19-specific antibody levels (MFI) for spike S1S2 (upper images) and nucleocapsid C (lower images). (C) Correlation matrix representing Spearman r and their corresponding p value between the COVID-19-specific antibody levels immune response (n = 26), IFN-γ levels measured by FluoroSpot (n = 28) and percentages of LFA-1⁺IFN-γ⁺ T-cells (n = 13), with the percentage of immune cells determined by FACS in Covid⁺ (n = 13) pwMS with anti-CD20 treatment. (D) Correlation between percentages of Tfh cells with COVID-19-specific antibody levels for nucleocapsid C in COVID-19-positive pwMS with anti-CD20 treatment (n = 11). DMF = dimethyl fumarate; HC = healthy controls; ICS = intracellular staining; memB = memory B; MFI = median fluorescence intensity; NTZ = natalizumab; OCR = ocrelizumab; pwMS = persons with multiple sclerosis; RTX = rituximab; TCM cells = central memory T cells; TEM cells = effector memory T cells; Tfh = T follicular helper cells; Treg = T regulatory; TTD cells = terminally differentiated T cells.

Figure 5

Immunological memory to SARS-CoV-2 is evident in RTX-treated pwMS irrespective of B cell depletion status. Patients that were SARS-CoV-2 positive in either serological and/or T cell assays are included (A) Correlation of SARS-CoV-2 antibody levels for S1S2 or N with ΔIFN-γ SFUs for S1 (n = 22) or N (n = 22) peptides, respectively. (B and C) Anti-SARS-CoV-2 antibody levels for S1S2 and N and number of ΔIFN-γ SFUs for S1 or N peptides in patients with B cell repletion, partial repletion, and depletion. No significant differences were seen. (D) Percentages of LFA-1⁺IFN-γ⁺ in patients with B cell repletion (n = 1), partial repletion (n = 4), and depletion (n = 5). (E) Correlation of antibody levels for S1S2 and N with number of RTX infusions. (F) Correlation of ΔIFN-γ SFUs for S1 and N peptides with number of RTX infusions. (G–J) Comparison of seropositive (n = 14) and seronegative (n = 8) patients regarding number of ΔIFN-γ SFUs for S1 (p = 0.803) or N (p = 0.815) or days from symptom onset to sampling (p = 0.140) or days from last RTX dose to symptom onset (p = 0.009) or number of RTX infusions (p = 0.048). (A–C, E–J) B cell depletion status; n = 3 repletion, n = 6 partial repletion, n = 13 depletion. Dots represent individual data points. Box plots represent median and 95% CI. Mann-Whitney test was used for statistical analysis and p values ≤ 0.05 were considered significant. See also Table S3 and Figure S3. MFI = median fluorescent intensity; ns = non-significant; pwMS = persons with multiple sclerosis; RTX = rituximab; SFU = spot forming unit.

Figure 6

Development of T cellular immune response to the spike protein of SARS-CoV-2 in anti-CD20-treated pwMS after vaccination (A) Images of the IFN-γ FluoroSpot after stimulation with the positive control (anti-CD3), negative control (medium only), and SARS-CoV-2 specific peptides (N and S1) in one pwMS on anti-CD20 before and 4 weeks after SARS-CoV-2 vaccination. (B) Serology status and number of ΔIFN-γ SFUs after stimulation with N or S1 peptides four (n = 10) and twelve (n = 4) weeks after SARS-CoV-2 vaccination in pwMS on anti-CD20. (C) Serology status and number of ΔIFN-γ SFUs after stimulation with S1 peptides 4 weeks after vaccination (n = 10) and days from last anti-CD20 infusion to first vaccine dose. (D) Percentages of B cells in seropositive (n = 7) and seronegative (n = 3) pwMS 4 weeks after vaccination. Samples with <0.2% B cells of live lymphocytes are marked with a black circle. pwMS = persons with multiple sclerosis; SFU = spot forming unit.