Neuro-PASC is characterized by enhanced CD4+ and diminished CD8+ T cell responses to SARS-CoV-2 Nucleocapsid protein

Abstract

Introduction: Many people with long COVID symptoms suffer from debilitating neurologic post-acute sequelae of SARS-CoV-2 infection (Neuro-PASC). Although symptoms of Neuro-PASC are widely documented, it is still unclear whether PASC symptoms impact virus-specific immune responses. Therefore, we examined T cell and antibody responses to SARS-CoV-2 Nucleocapsid protein to identify activation signatures distinguishing Neuro-PASC patients from healthy COVID convalescents.

Results: We report that Neuro-PASC patients exhibit distinct immunological signatures composed of elevated CD4⁺ T cell responses and diminished CD8⁺ memory T cell activation toward the C-terminal region of SARS-CoV-2 Nucleocapsid protein when examined both functionally and using TCR sequencing. CD8⁺ T cell production of IL-6 correlated with increased plasma IL-6 levels as well as heightened severity of neurologic symptoms, including pain. Elevated plasma immunoregulatory and reduced pro-inflammatory and antiviral response signatures were evident in Neuro-PASC patients compared with COVID convalescent controls without lasting symptoms, correlating with worse neurocognitive dysfunction.

Discussion: We conclude that these data provide new insight into the impact of virus-specific cellular immunity on the pathogenesis of long COVID and pave the way for the rational design of predictive biomarkers and therapeutic interventions.

Keywords: COVID-19 immunity; IL-6; T cell memory; immunoregulation; long COVID; neuro-PASC; proteomics.

Figure 1

Study design and clinical data (A) Study design. (B) Demographic table for Neuro-PASC (NP), convalescent controls (CC), and healthy control (HC) study participants. (C) PROMIS-57 patient-reported outcome survey T scores for NP patients (n=36) and CC subjects (n=13). (D) NIH Toolbox cognitive T scores for NP patients (n = 55). Horizontal black line represents the U.S. national average T score of 50; p values relative to demographic-matched US national average by one sample Wilcoxon signed rank test. *p<0.05, ***p<0.005, ****p<0.0001 by two-tailed Student’s t test.

Figure 2

T cells from Neuro-PASC patients have elevated responses to select SARS-CoV-2 structural proteins compared to convalescent controls. (A) NP patients and CC subjects display similar IFN-γ responses to SARS-CoV-2 S peptides, but NP patients have enhanced N- and M peptide-specific responses. (B) Spike RBD antibody responses are similar in NP and CC subjects. (C) NP patients have elevated anti-N IgG titers compared to CC and HC controls. (D) NP patients have higher T follicular helper cell (Tfh) activation after N antigen stimulation compared to CC subjects. (E) Influenza A Haemagglutinin (HA) antibody responses are similar in all groups. +ctrl = plasma from patients who received the Influenza vaccine within 3 weeks before sample collection; -ctrl = plasma from patients collected pre-2019. Only unvaccinated subjects were examined for anti-Spike responses in A and B Horizontal black line in B,C,E = limit of detection. Data representative of 7 experiments with all conditions plated in duplicate. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001 by one-way ANOVA with Tukey’s posttest.

Figure 3

Neuro-PASC patients have elevated reactivity to the C-terminal region of N protein. (A) Diagram showing partition of SARS-CoV-2 N peptides into 3 pools comprising the N-terminal (N1), middle (N2), and C-terminal (N3) regions (top) and further splitting of N3 into 5 sub-pools A-E for ELISPOT experiments. (B) T cells from NP patients display enhanced reactivity to the C-terminal third of N protein. (C) T cell reactivity to N protein is mainly localized to aa 309-402. (D) Distribution of SARS-CoV-2-specific TCRs stratified by ORF specificity in unvaccinated NP and CC subjects. (E) Elevated proportion of N3-specific TCRs in NP patients. (F) Quantification of template copies from the top TCR clone specific for the N3 region in NP and CC subjects. (G) N3-specific IFN-γ production from a subset of NP and CC participants in E-F. (H) Percentage of N3-specific TCR templates from CD4 vs. CD8 T cells in NP vs. CC. (I) N antigen functionally stimulates more TNF-α production from CD4⁺ T cells in NP patients. (J) CDR3 sequence, TRBV, and TRBJ usage in top N3 clone from each subject. NP patients have higher TRBV07-09 usage, which is not observed in CC subjects. ELISPOT data combined from 6 independent experiments with the indicated n values. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001 using one-way ANOVA with Dunnett’s posttest (B, C, I) or two-tailed Student’s t Test with Welch’s correction (F, G). ns, not significant.

Figure 4

Elevated Nucleocapsid-specific CD4⁺ T cell and attenuated CD8⁺ memory T cell activation in Neuro-PASC patients. (A) CD4⁺ T cells from NP patients have enhanced N antigen-specific TNF-α production compared to CC. (B) CD8⁺ TEM from NP patients have enhanced IL-6 production after N antigen stimulation compared to CC subjects on a per-cell basis (mean fluorescence intensity; MFI). (C) Increased soluble IL-6 and IL6Rβ in NP patient plasma compared with CC subjects. (D) CD8⁺ TEM from NP patients show decreased activation after stimulation with N peptides. (E) Higher percentages of CD8⁺ TEMRA cells are found in NP patients compared to control groups. (F) CD8⁺ TEMRA cells from NP patients are less activated by N antigens compared with CC subjects. Data combined from 5 independent experiments with the indicated n values. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001 using two-tailed Student’s t test with Welch’s correction. ns, not significant.

Figure 5

Correlation of cognitive and psychiatric clinical measures with virus-specific immune responses in Neuro-PASC patients. (A) N3-specific IFN-γ production is negatively correlated with self-reported cognition scores (top) and positively correlated with anxiety scores (bottom) in NP patients. (B) NP patients with lower scores on Attention or Executive Function cognitive tests had higher N3-specific IFN-γ responses and RBD IgG titers. (C) High Pain Interference scores correlate with more IL-6 production from CD8⁺ T cells in response to S peptides. (D) High depression scores correlate with lower polyfunctionality in CD8⁺ TEM. Data representative of 5 independent experiments with n=39-51 for correlation data analysis (A, B) and n=8-9 NP subjects per quartile for SPICE analysis (C, D). Correlations calculated using simple linear regression (A), nonparametric Spearman rank correlation (B), or Permutation test (C, D). All pie graphs are background subtracted (unstimulated conditions). *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

Figure 6

Neuro-PASC patients have elevated immunoregulatory and decreased antiviral response-associated proteins in plasma correlating with enhanced symptom severity and cognitive dysfunction. (A) Gene set enrichment analysis (GSEA) demonstrating elevations in immunoregulatory pathway-related proteins (top left panel) in NP patients contrasting with elevated pro-inflammatory and antiviral pathway-related proteins (top right, bottom panels) in CC subjects. List of proteins analyzed in each pathway found in Tables S2 – S5 . (B) Quantification of individual immunoregulatory (top) and TASOR antiviral pathway-associated protein levels (bottom) between Neuro-PASC patients and healthy COVID convalescents. (C) Patient-reported outcomes of symptom severity and cognitive scores are significantly correlated with expression levels of immunoregulatory proteins (left) and TASOR pathway proteins (right). RFU: relative fluorescence units. FDR: false discovery rate. NES: normalized enrichment score. *p<0.05; **p<0.01; ***p<0.005; ****p<0.0001 by Student’s t test (B) or Pearson correlation (C).