PMC9537824_01
Post-acute sequelae of SARS-CoV-2 infection (PASC) patient exhibits persistent SARS-CoV-2 antigen positivity in nasopharynx. (A) Study visit timeline, including vaccination, infection, and intervention dates. (B) FlowFlex  SARS-CoV-2 antigen test results over time. Time p.i., weeks post-infection. (C) PASC symptoms vs. rheumatoid arthritis symptoms.
fmed-09-1003103-g001.jpg
PMC9537824_01_fmed-09-1003103-g001.jpg
F1
South Asian woman on bi-weekly 162 mg/ml tocilizumab injections for RA was enrolled in a Neuro-PASC research study at Northwestern University in Chicago (demographics in Figure 1A; study design in Figure 1B). Initially, all PASC symptoms resolved, and the patient tested antigen- 3 weeks after completion of nirmeltravir/ritonavir, but PASC symptoms and antigen positivity subsequently reappeared at 4 weeks (Figures 1B,C). She resumed tocilizumab and within 3 weeks, her fatigue and brain fog had resolved while the occipital headache decreased in severity (Figure 1C).
IFN-gamma T cell responses to SARS-CoV-2 vary over time. (A) IFN-gamma production from SARS-CoV-2 Spike- and Nucleocapsid-specific T cells at each visit as determined by ELISPOT. (B) Spike receptor-binding domain (RBD)-specific IgG titers (top) and Nucleocapsid-specific IgG (bottom) at each visit. LoD, limit of detection. All ELISPOT data in panel (A) from duplicate wells. Data representative of 2 individual experiments, *p < 0.05 by Student's t-test.
fmed-09-1003103-g002.jpg
PMC9537824_01_fmed-09-1003103-g002.jpg
F2
Resumption of tocilizumab subsequently decreased T cell responses to both viral antigens (Figure 2A). Antibody titers against Spike receptor-binding domain (RBD) followed similar kinetics (Figure 2B, top), while the patient never mounted an antibody response against Nucleocapsid (Figure 2B, bottom).
Virus-specific CD4+ and CD8+ T cell subset activation correlates with antiviral and tocilizumab treatment. (A) Flow cytometry showing elevated virus-specific CD4+ T helper cell (Tfh) cell activation after vaccine booster dose (V3; 2nd row) and stopping tocilizumab treatment (V5; 4th row). (B) Total CD4+ T cells (left), CD4+ T helper cells (Tfh, middle), and CD4+ memory T cells (TEM, right) have enhanced reactivity to SARS-CoV-2 structural (S, N) and non-structural (Orf1ab, Orf7) peptides after vaccine booster (V3) and stopping tocilizumab (V5), but low reactivity after nirmatrelvir/ritonavir treatment (V4) and resuming tocilizumab therapy (V6). (B) Total CD8+ T cells (right) and CD8+ memory T cell subsets (CD8+ TEMRA, TEM; middle, right) show increased activation after vaccine boost (V3) and stopping tocilizumab (V5), but low reactivity after nirmatrelvir/ritonavir treatment (V4) and resuming tocilizumab (V5). Data combined from 3 independent experiments.
fmed-09-1003103-g003.jpg
PMC9537824_01_fmed-09-1003103-g003.jpg
F3
Table 1) CD4+ T follicular helper cells (Tfh; involved in T cell help for antibody production) showed that antiviral T cell activation was highest at V3 (post-infection, post-boost) and V5 (post-infection, -boost, -nirmatrelvir/ritonavir, and halting tocilizumab treatment). Tfh activation determined by the activation-induced marker assay (AIM) was lowest 3 weeks after nirmatrelvir/ritonavir treatment and resumption of tocilizumab (Figure 3A). Similarly, total CD4+ and CD8+ T cells, CD4+ and CD8+ T effector memory (TEM) cells, and CD8+ TEM cells re-expressing CD45RA (CD8+ TEMRA; terminally differentiated and highly cytotoxic T cells) exhibited maximal SARS-CoV-2-specific activation to Spike, Nucleocapsid, Orf1ab, and Orf7 antigens at V3 and V5, with limited activation at V4 and V6 (Figure 3B).
Autoreactive T cell responses oscillate in coordination with SARS-CoV-2-specific responses after infection. (A) T cell production of IFN-gamma after stimulation with rheumatoid arthritis-associated cartilage autoantigen YKL-40 compared with Spike- and Orf1ab-specific activation. (B) Activation of T cell subsets over the course of the study after stimulation with the rheumatoid arthritis-associated antigen YKL-40 or SARS-CoV-2 Orf7 peptides. Stopping tocilizumab resulted in increased virus-specific and autoreactive T cell reactivity, while resuming tocilizumab suppressed autoreactivity in most T cell subsets. Data representative of 2 individual experiments, *p < 0.05, **p < 0.01 by one-way ANOVA with Tukey's post-test.
fmed-09-1003103-g004.jpg
PMC9537824_01_fmed-09-1003103-g004.jpg
F4
Comparison of IFN-gamma production from T cells in response to Spike and Orf1ab vs. the RA-associated cartilage antigen YKL-40 showed the highest activation at V3 and V5, and the lowest activation at V4 after nirmatrelvir/ritonavir and V6 after resuming tocilizumab treatment (Figure 4A). Flow cytometry revealed similar activation patterns in T cell memory and Tfh cell subsets (Figure 4B).