T cell responses to SARS-CoV-2 infection and vaccination are elevated in B cell deficiency and reduce risk of severe COVID-19

Reza Zonozi¹, Lucy C Walters^{2

3

4}, Aaron Shulkin², Vivek Naranbhai^{5

6

7}, Pravarut Nithagon¹, Gabriel Sauvage¹, Clarety Kaeske², Katherine Cosgrove¹, Anusha Nathan^{2

8}, Rhoda Tano-Menka², Alton C Gayton², Matthew A Getz², Fernando Senjobe², Daniel Worrall², A John Iafrate⁹, Caroline Fromson¹⁰, Sydney B Montesi¹⁰, Deepak A Rao¹¹, Jeffrey A Sparks¹¹, Zachary S Wallace¹², Jocelyn R Farmer^{2

13}, Bruce D Walker^{2

6

14

15

16}, Richelle C Charles¹⁷, Karen Laliberte¹, John L Niles¹, Gaurav D Gaiha^{2

18}

Affiliations

¹ Vasculitis and Glomerulonephritis Center, Massachusetts General Hospital, Boston, MA 02114, USA.
² Ragon Institute of Mass General, MIT, and Harvard, Cambridge, MA 02139, USA.
³ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁴ Koch Institute for Integrative Cancer Research, Cambridge, MA 02139, USA.
⁵ Massachusetts General Hospital Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA.
⁶ Center for the AIDS Programme of Research in South Africa, Durban 4001, South Africa.
⁷ Monash University, Melbourne, VIC 3022, Australia.
⁸ Program in Health Sciences and Technology, Harvard Medical School and Massachusetts Institute of Technology, Boston, MA 02115, USA.
⁹ Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA.
¹⁰ Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.
¹¹ Division of Rheumatology, Inflammation, and Immunity, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
¹² Division of Rheumatology, Massachusetts General Hospital, Boston, MA 02114, USA.
¹³ Division of Allergy and Inflammation, Beth Israel Lahey Health, Boston, MA 02215, USA.
¹⁴ Broad Institute, Cambridge, MA 02142, USA.
¹⁵ Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
¹⁶ Institute for Medical Engineering and Science and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
¹⁷ Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA 02114, USA.
¹⁸ Division of Gastroenterology, Massachusetts General Hospital, Boston, MA 02114, USA.

PMID: 38019932
PMCID: PMC12949954
DOI: 10.1126/scitranslmed.adh4529

T cell responses to SARS-CoV-2 infection and vaccination are elevated in B cell deficiency and reduce risk of severe COVID-19

Reza Zonozi et al. Sci Transl Med. 2023.

. 2023 Nov 29;15(724):eadh4529.

doi: 10.1126/scitranslmed.adh4529. Epub 2023 Nov 29.

Authors

Affiliations

¹ Vasculitis and Glomerulonephritis Center, Massachusetts General Hospital, Boston, MA 02114, USA.
² Ragon Institute of Mass General, MIT, and Harvard, Cambridge, MA 02139, USA.
³ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁴ Koch Institute for Integrative Cancer Research, Cambridge, MA 02139, USA.
⁵ Massachusetts General Hospital Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA.
⁶ Center for the AIDS Programme of Research in South Africa, Durban 4001, South Africa.
⁷ Monash University, Melbourne, VIC 3022, Australia.
⁸ Program in Health Sciences and Technology, Harvard Medical School and Massachusetts Institute of Technology, Boston, MA 02115, USA.
⁹ Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA.
¹⁰ Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.
¹¹ Division of Rheumatology, Inflammation, and Immunity, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
¹² Division of Rheumatology, Massachusetts General Hospital, Boston, MA 02114, USA.
¹³ Division of Allergy and Inflammation, Beth Israel Lahey Health, Boston, MA 02215, USA.
¹⁴ Broad Institute, Cambridge, MA 02142, USA.
¹⁵ Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
¹⁶ Institute for Medical Engineering and Science and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
¹⁷ Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA 02114, USA.
¹⁸ Division of Gastroenterology, Massachusetts General Hospital, Boston, MA 02114, USA.

PMID: 38019932
PMCID: PMC12949954
DOI: 10.1126/scitranslmed.adh4529

Abstract

Individuals with primary and pharmacologic B cell deficiencies have high rates of severe disease and mortality from coronavirus disease 2019 (COVID-19), but the immune responses and clinical outcomes after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination have yet to be fully defined. Here, we evaluate the cellular immune responses after both SARS-CoV-2 infection and vaccination in patients receiving the anti-CD20 therapy rituximab (RTX) and those with low B cell counts due to common variable immune deficiency (CVID) disease. Assessment of effector and memory CD4⁺ and CD8⁺ T cell responses to SARS-CoV-2 revealed elevated reactivity and proliferative capacity after both infection and vaccination in B cell-deficient individuals, particularly within the CD8⁺ T cell compartment, in comparison with healthy controls. Evaluation of clinical outcomes demonstrates that vaccination of RTX-treated individuals was associated with about 4.8-fold reduced odds of moderate or severe COVID-19 in the absence of vaccine-induced antibodies. Analysis of T cell differentiation demonstrates that RTX administration increases the relative frequency of naïve CD8⁺ T cells, potentially by depletion of CD8⁺CD20^dim T cells, which are primarily of an effector memory or terminal effector memory (TEMRA) phenotype. However, this also leads to a reduction in preexisting antiviral T cell immunity. Collectively, these data indicate that individuals with B cell deficiencies have enhanced T cell immunity after both SARS-CoV-2 infection and vaccination that potentially accounts for reduced hospitalization and severe disease from subsequent SARS-CoV-2 infection.

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Conflict of interest statement

Competing interests: S.B.M. reports research funding from Pliant Therapeutics, Merck, and Boehringer Ingelheim; consulting fees from DevPro Biopharma, Gilead Sciences, and Roche; advisory board fees from APIE Therapeutics and Pliant Therapeutics; and royalties form Wolters Kluwer. J.A.S. has received research support from Bristol Myers Squibb and performed consultancy for AbbVie, Amgen, Boehringer Ingelheim, Bristol Myers Squibb, Gilead, Inova Diagnostics, Janssen, Optum, and Pfizer unrelated to this work. J.R.F. is an ongoing consultant for Pharming and has received research funding from Pfizer, Bristol Myers Squibb, and Pharming with no direct relation to the work presented. G.D.G. reports research funding from Merck and Moderna and has filed patent application PCT/US2021/028245 titled “Highly Networked Coronavirus Immunogen Composition.” All other authors declare that they have no competing interests.

Figures

**Fig. 1.. Effector T cell reactivity and proliferative CD8⁺ T cell responses to SARS-CoV-2 antigens are elevated in RTX-treated individuals after SARS-CoV-2 infection.**
(A) Representative IFN-γ ELISpot wells for Non-RTX (black, n = 27) and RTX-treated (blue, n = 11) individuals after no stimulation (DMSO), anti-CD3 and anti-CD28 stimulation (positive control), or overlapping peptide pools of SARS-CoV-2 antigens (M, membrane; NCAP, nucleocapsid; spike protein; E, envelope; ORF3A; NSP7; NSP6; NSP8; NSP1; NSP5; NSP10; NSP12; NSP15; NSP16; ORF9B + ORF10). Values in the upper right corner of each well indicate detected spot-forming units. (B) Comparative IFN-γ ELISpot spot-forming units (SFUs) per 10⁶ peripheral blood mononuclear cells (PBMCs) in Non–RTX- and RTX-treated individuals to overlapping peptide pools for SARS-CoV-2 antigens. (C and D) Representative proliferative CD4⁺ T cell responses (C) and proliferative CD8⁺ T cell (D) responses for Non–RTX-treated (black, n = 24) and RTX-treated (blue, n = 10) individuals. (E) Comparative %CD4⁺ CFSE low cells in Non–RTX- and RTX-treated individuals to overlapping peptide pools for SARS-CoV-2 antigens. (F) Comparative %CD8⁺ CFSE low cells in Non–RTX- and RTX-treated individuals to overlapping peptide pools for SARS-CoV-2 antigens. Differences between groups were evaluated using the nonparametric Mann-Whitney U test. Boxes represent median values. Data are biological replicates. Calculated P values are presented as follows: ***P < 0.001, ****P < 0.0001.

**Fig. 2.. Effector T cell reactivity and proliferative CD4⁺ and CD8⁺ T cell responses to SARS-CoV-2 spike protein were elevated in RTX-treated individuals after SARS-CoV-2 vaccination.**
(A) Representative IFN-γ ELISpot wells for Non–RTX-treated (black) and RTX-treated (blue) individuals after no stimulation (DMSO), anti-CD3 and anti-CD28 stimulation (positive control), or an overlapping peptide pool for SARS-CoV-2 spike protein. Values in the upper right corner of each well indicate detected SFUs. (B and C) Comparative IFN-γ ELISpot SFUs per 10⁶ PBMCs in response to an overlapping peptide pool for SARS-CoV-2 spike protein in vaccinated, uninfected individuals (closed symbols; Non-RTX, n = 13; RTX, n = 15) (B) or in both vaccinated, uninfected individuals and individuals with hybrid immunity (open symbols; Non-RTX, n = 4; RTX, n = 7) (C). (D) Representative flow cytometry plots demonstrating proliferative CD4⁺ T cell responses for Non–RTX-treated (black) and RTX-treated (blue) individuals. (E and F) Comparative %CD4⁺ CFSE low cells in response to an overlapping peptide pool for SARS-CoV-2 spike protein in vaccinated, uninfected individuals (closed symbols; Non-RTX, n = 13; RTX, n = 15) (E) or in both vaccinated, uninfected individuals and individuals with hybrid immunity (open symbols; Non-RTX, n = 4; RTX, n = 7) (F). (G) Representative flow cytometry plots demonstrating proliferative CD8⁺ T cell responses for Non–RTX-treated (black) and RTX-treated (blue) individuals. (H and I) Comparative %CD8⁺ CFSE low cells in response to an overlapping peptide pool for SARS-CoV-2 spike protein in vaccinated, uninfected individuals (closed symbols; Non-RTX, n = 13; RTX, n = 15) (H) or in both vaccinated, uninfected individuals and individuals with hybrid immunity (open symbols; Non-RTX, n = 4; RTX, n = 7) (I). Differences between groups were evaluated using the nonparametric Mann-Whitney U test. Bars represent median values. Data are biological replicates. Calculated P values are presented as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant.

**Fig. 3.. Vaccination of RTX-treated individuals led to a reduction in hospitalization and severe disease after SARS-CoV-2 infection in the absence of anti-spike protein antibodies.**
(A) Comparison of anti-RBD IgG titers (binding antibody units per milliliter; BAU/ml) in Non–RTX-treated (black, n = 13) and RTX-treated (blue, n = 13) individuals. Differences between groups were evaluated using the nonparametric Mann-Whitney U test. Data are biological replicates. (B) Comparison of clinical outcomes after SARS-CoV-2 infection in B cell–depleted RTX-treated individuals who were unvaccinated, vaccinated with no detectable antibodies (Abs), vaccinated with minimal antibodies (<20 units), and vaccinated with moderate concentrations of antibodies (>20 units). Comparison of frequencies of mild and hospitalized/severe/critical/fatal disease was performed using Fisher’s exact test. Calculated P values are presented as follows: *P < 0.05; **P < 0.01; ****P < 0.0001.

**Fig. 4.. RTX treatment is associated with an increase in the naïve CD8⁺ T cell pool that correlates with the induction of spike protein–specific CD8⁺ T cell responses after vaccination.**
(A and B) Representative flow cytometry plots of CD4⁺ (A) and CD8⁺ (B) memory T cell phenotypes in Non–RTX-treated (black) and RTX-treated (blue) individuals. (C and D) Comparison of prevaccine CD4⁺ (C) and CD8⁺ (D) memory T cell phenotypes in Non–RTX-treated (black, n = 15) and RTX-treated (blue, n = 11) individuals. (E) Comparison of the frequency of naïve CD8⁺ T cells (%CD45RA⁺CD62L⁺) before and after vaccination in Non–RTX-treated (n = 3) and RTX-treated (n = 5) individuals. (F) Scatterplot of the magnitude of naïve CD8⁺ T cells (%CD45RA⁺CD62L⁺) before vaccine and the magnitude of proliferative memory spike protein–specific CD8⁺ T cells (%CD8⁺ CFSE low) after vaccine. (G) Representative staining of CD8⁺CD20^dim T cells in a Non-RTX control sample (red) and an RTX-treated sample (blue). FMO, fluorescence minus one control. (H) Comparison of the frequency of CD8⁺CD20^dim T cells in Non–RTX-treated (red, n = 9) and RTX-treated (blue, n = 9) individuals. (I) Representative T cell memory marker staining of CD8⁺CD20^dim T cells from a Non-RTX sample. (J) Comparison of the frequency of naïve (closed) and non-naïve (open) CD8⁺CD20^dim T cells in Non-RTX (n = 9) individuals. Differences between groups in (C), (D), (H), and (J) were evaluated using the nonparametric Mann-Whitney U test. Statistical analysis in (E) was performed using the nonparametric Wilcoxon matched-pairs signed-rank test. Correlations in (F) were calculated by Spearman’s rank correlation coefficient. Bars represent median values. Data are biological replicates. Calculated P values are presented as follows: *P < 0.05; **P < 0.01; ****P < 0.0001.

**Fig. 5.. RTX treatment is associated with a reduction in preexisting viral immunity.**
(A) Representative IFN-γ ELISpot wells for samples from individuals with prior infection treated with RTX or not, from a vaccinated Non-RTX control individual, and from an RTX-treated, uninfected individual before and after vaccination. ELISpots were read after no stimulation (DMSO); anti-CD3 and anti-CD28 stimulation (positive control); or stimulation with an overlapping peptide pool for CMV, EBV, and influenza (CEF). Values in the upper right corner of each well indicate detected SFUs. (B) Comparison of IFN-γ ELISpot SFUs per 10⁶ PBMCs to the CEF peptide pool in prior SARS-CoV-2–infected, unvaccinated Non–RTX-treated (black, n = 27 of 28) and RTX-treated (blue, n = 11 of 14) individuals. Open symbols represent individuals who were unvaccinated and uninfected (Non-RTX, n = 1 of 28; RTX, n = 3 of 14). (C) Comparison of IFN-γ ELISpot SFUs per 10⁶ PBMCs to the CEF peptide pool in vaccinated Non–RTX-treated (black, n = 17) and RTX-treated (blue, n = 22) individuals. Open symbols represent individuals with hybrid immunity (Non-RTX, n = 4 of 17; RTX, n = 7 of 22). (D) Representative flow cytometry plots show proliferative CD8⁺ T cell responses (%CD8⁺ CFSE low) to the CEF peptide pool in prior infected, unvaccinated Non–RTX-treated (black) and RTX-treated (blue) individuals. (E) Representative flow cytometry plots show proliferative CD8⁺ T cell responses (%CD8⁺ CFSE low) to the CEF peptide pool in Non–RTX-treated (black) and RTX-treated (blue) individuals before (pre-vax) or after (post-vax) vaccination. Samples are from the same donor. (F) Comparison of proliferative CD8⁺ T cell responses (%CD8⁺ CFSE low) to the CEF peptide pool in prior SARS-CoV-2–infected, unvaccinated Non–RTX-treated (black, n = 27 of 28) and RTX-treated (blue, n = 11 of 14) individuals. Open symbols represent individuals who were uninfected and unvaccinated (Non-RTX, n = 1 of 28; RTX, n = 3 of 14). (G) Comparison of proliferative CD8⁺ T cell responses (%CD8⁺ CFSE low) to the CEF peptide pool in vaccinated Non–RTX-treated (black, n = 17) and RTX-treated (blue, n = 22) individuals. Open symbols represent individuals with hybrid immunity (Non-RTX, n = 4 of 17; RTX, n = 7 of 22). (H) Comparison of proliferative CD8⁺ T cell responses (% CD8⁺ CFSE low) to the CEF peptide pool in paired samples collected before and after mRNA vaccination (n = 5). Differences between groups were evaluated using the nonparametric Mann-Whitney U test. Statistical analysis in (H) was performed using the nonparametric Wilcoxon matched-pairs signed-rank test. Bars represent median values. Data are biological replicates. Calculated P value is presented as follows: *P < 0.05.

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T cell responses to SARS-CoV-2 infection and vaccination are elevated in B cell deficiency and reduce risk of severe COVID-19

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T cell responses to SARS-CoV-2 infection and vaccination are elevated in B cell deficiency and reduce risk of severe COVID-19

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