key: cord-0769924-snt58wsa
authors: Paniskaki, Krystallenia; Anft, Moritz; Thieme, Constantin J.; Skrzypczyk, Sarah; Konik, Margarethe Justine; Dolff, Sebastian; Westhoff, Timm H.; Nienen, Mikalai; Stittrich, Anna; Stervbo, Ulrik; Witzke, Oliver; Heine, Guido; Roch, Toralf; Babel, Nina
title: SARS-CoV-2 cross-reactive B and T cell responses in kidney-transplant patients
date: 2022-03-16
journal: Transplant Proc
DOI: 10.1016/j.transproceed.2022.02.016
sha: b2398dcdffbed431b7d138b3ab5f6c8afc0609e6
doc_id: 769924
cord_uid: snt58wsa

Background: Immune responses to seasonal endemic coronaviruses might have a pivotal role in protection against SARS-CoV-2. Those SARS-CoV-2-crossreactive T cells were recently described in immunocompetent individuals. Still, data on cross-reactive humoral and cellular immunity in kidney transplant recipients is currently lacking. Methods: The preexisting, crossreactive antibody, B, and T cell immune responses against SARS-CoV-2 in unexposed adults with kidney transplantation (Tx, n=14) and without (non-Tx, n=12) sampled before the pandemic were compared with 22 convalescent COVID-19 patients (Cp) applying ELISA and flow cytometry. Results: In both unexposed groups SARS-CoV-2 IgG antibodies were not detectable. Memory B cells binding spike (S) protein SARS-CoV-2 were detected in unexposed individuals (64% Tx, 50% non-Tx) and higher frequencies after infection (80% Cp). The numbers of SARS-CoV-2-reactive T cells were comparable between Tx and non-Tx. Of note, SARS-CoV-2-reactive follicular T helper (Tfh) cells were present in 61% of the unexposed cohort in both, Tx and non-Tx. Conclusions: Cross-reactive memory B and T cells against SARS-CoV-2 exist also in transplanted adults suggesting a primed adaptive immunity. The impact on disease course may depend on the concomitant immunosuppressive drugs.

Preexisting immunity to SARS-CoV-2, most likely resulting from cross-reactivity against seasonal corona viruses has been addressed early in the pandemic as a potential immunomodulatory factor affecting the immune response after SARS-CoV-2 infection or vaccination 1-5. Grifoni et al. and Nelde et al. found wild-type S-reactive CD4+ T cells in 40%-60% and 80% of unexposed individuals, respectively, suggesting a SARS-T cell immunity that is cross-reactive with HCoV 6-7. Despite the weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in prepandemic donors 8-9, independent study groups have detected cross-reactive preexisting SARS-CoV-2 B cell memory cells in healthy individuals unexposed to SARS-CoV-2 8-10. Preexisting cross-reactive T memory cells are recalled and expanded upon SARS-CoV-2 infection, reinforcing but not preventing a robust and persistent primary response to new epitopes of SARS-CoV-2 8, 11-12. However, still data on preexisting SARS-CoV-2 immunity among immunosuppressed patients, such as transplant recipients, are currently scarce. In this study we aimed to characterize SARS-CoV-2-reactive preexisting SARS-CoV-2-reactive T and B cells in a cohort of renal transplant patients under immunosuppressive therapy that were unexposed to SARS-CoV-2 in comparison to unexposed immunocompetent blood donors. Tables 1 and 2 .

Peripheral blood was collected in S-Monovette K3 EDTA blood collection tubes (Sarstedt).

Collected blood was prediluted in phosphate-buffered saline PBS/BSA (Gibco) at a 1:1 ratio and underlaid with 15 mL of Ficoll-Paque Plus (GE Healthcare). Tubes were centrifuged at 800 g for 20 min at room temperature. Isolated PBMCs were washed twice with PBS/BSA and stored at -80 °C. The cryopreserved PBMCs were thawed by incubating cryovials for 2-3 minutes at 37 °C in a bead bath, washed twice in 37 °C RPMI 1640 medium (Life Technologies) supplemented with 1% penicillin-streptomycin-glutamine (Sigma-Aldrich) and 10% fetal calf serum (FCS) (PAN-Biotech) and incubated overnight at 37 °C.

In brief, as previously described 14, PBMCs were plated in 96-U-Well plates in RPMI 1640 medium (Life Technologies) and stimulated with SARS-CoV-2 S-peptide (Miltenyi Biotec) or left untreated as a control for 16 h. As a positive control, cells were stimulated with staphylococcal enterotoxin B (1 μg/mL, Sigma-Aldrich). After 2 h, brefeldin A was added. A detailed list of the antibody panel for general phenotyping and T cell activation ex vivo is shown in Table 3 . After stimulation overnight, the PBMCs were stained with optimal concentrations of antibodies for 10 min at room temperature in the dark. Stained cells were washed twice with PBS/BSA before preparation for intracellular staining using the Intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher Scientific) according to the manufacturer's instructions. Fixed and permeabilized cells were stained for 30 min at room temperature in the dark with an optimal dilution of antibodies against the intracellular antigen. All samples were immediately acquired on a CytoFLEX flow cytometer (Beckman Coulter). Quality control was performed daily using the recommended CytoFLEX daily QC fluorospheres (Beckman Coulter). No modification to the compensation matrices was required throughout the study. Antigen-reactive responses were considered positive after the nonreactive background was subtracted and greater than 0.01% was detectable.

Negative values were set to zero. In one exception to the abovementioned minimum limit of 0.01%, we evaluated all positive frequencies of CD4+CD154+CD137+CXCR5+ cells after the background was subtracted, as no large populations of Tfh cells were expected to be found in circulation.

As previously described 15, SARS-CoV-2 S1/S2-protein (henceforth referred to as Sprotein) (Sino Biological Inc.) was aliquoted into three samples. Sample 1 was left unlabeled for blocking, and samples 2 and 3 were coupled to fluorescein isothiocyanate (FITC) and Cy5 fluorochromes, respectively. PBMCs were divided into three samples (blocked, unblocked, and negative control samples). Blocking was performed by using a 10 times excess of unlabeled protein. After blocking, PBMCs were surface-stained with fluorochrome-labeled antibodies, as described in Table 4 . Finally, mixed FITC-and Cy5-labeled protein was added.

Cells were stained for 10 min at 4 °C. After washing with PBS, the samples were stored at 4 °C until measurement on a Cytoflex flow cytometer. Directly before analysis, the samples were stained with DAPI to differentiate live from dead cells. Antigen-reactive responses were considered positive after the blocked background was subtracted and greater than 0.001% was detectable. Negative values were set to zero.

Peripheral blood was collected in S-Monovette Z-Gel (Sarstedt). SARS-CoV-2 IgG titers were analyzed in purified serum using a SARS-CoV-2 IgG kit (Euroimmun, Lübeck, Germany). The test was performed according to the manufacturer's instructions. Briefly, serum samples were diluted 1:100 and added to plates coated with recombinant SARS-CoV-2 antigen.

Bound SARS-CoV-2 S1 protein-reactive IgG was detected by horseradish peroxidaseconjugated anti-human IgG. The absorbance was assessed on a microplate reader at 450 nm with a reference at 620 nm and evaluated as the ratio of the absorbance of the sample to the absorbance of the internal standard.

Flow cytometry data were analyzed using FlowJo version 10.6.2 (BD Biosciences); gating strategies are presented in figures 1 and 2. For the analysis of anti-SARS-CoV-2 T and B cells, a threshold of 0.01% and 0,001% was employed respectively, to define a detectable response. Single stains and fluorescence-minus-one controls were used for gating. Gates for each individual were adjusted according to the negative control. CD4+ T cells expressing CD154 and CD137 and CD8+ T cells expressing CD137 were defined as reactive T cells.

Statistical analysis was performed using GraphPad Prism v7. Categorical variables are summarized as numbers and frequencies; quantitative variables are reported as medians and interquartile ranges. Normality tests were performed with D'Agostino & Pearson, Shapiro-Wilk and Anderson-Darling tests. All applied statistical tests were two-sided. The frequencies of SARS-CoV-2-reactive B and T cells in recovered COVID-19 patients and immunocompetent donors were compared using an exact two-tailed Mann-Whitney test, and for grouped data, the Mann-Whitney test was used. Unexposed and exposed patient age was compared using an unpaired two-tailed t-test, and sex was compared using a twotailed Fisher's exact test. p values below 0.050 were considered significant; only significant p values are reported in the figures. p values were not corrected for multiple testing, as this study was of an exploratory nature.

We analyzed 26 unexposed individuals unexposed to SARS-CoV-2, of which 14 were Tx and 12 non-Tx, and 22 Cp (Table 1 ). The median age of the unexposed individuals at the time of study inclusion was 69 years, with participant ages ranging from 37 to 91 years, with 58% males and 42% females. Tx patients (12 kidney transplant, 1 liver transplant, 1 combined kidney/liver transplant recipient) were significantly younger, with a median age of 55 years (range of 37-75 years, p=0.0069), compared to the non-Tx patients with a median age of 73 years (range of 49-91 years). We compared the Tx patients to immunocompetent Cp patients at a median time of 110 days after diagnosis or onset of symptoms (range of days). All included Cp patients were confirmed to be SARS-CoV-2-positive by PCR. The median age of the Cp group was 54.5 years (range of 28-89 years) and not significantly different from that of the unexposed group (median age 69 years, range of 37-91 years) (p=0.5182, two-tailed unpaired t-test). There were no significant differences regarding sex between the unexposed and COVID-19 patients (p=0.5626, two-tailed Fisher's exact test).

Demographic characteristics are provided in Tables 1 and 2.

As applied in previous studies 14, 16, antigen-reactive T cell responses were considered positive after the background was subtracted and greater than 0.01% was detectable. The exception to this rule was the detection of circulating follicular CD4+ T helper cells, for which no minimum numerical limit was set, due to the extremely low number of this cell population normally in circulation. We found detectable SARS-CoV-2 S-protein-reactive CD4+ and CD8+ T cells in 94% and 22% of unexposed individuals, respectively. The frequencies of SARS-CoV-2-reactive CD4+ T cells in the exposed cohort were higher, without statistical significance regarding SARS-CoV-2-reactive CD4+ T cells (p=0.19, Mann-Whitney test) (Fig.   3A ). However, the frequencies of SARS-CoV-2-reactive CD8+ cells were significantly higher in the Cp cohort (p=0.0002, Mann-Whitney test) (Fig. 3B ). Inferferon γ (IFNγ)-producing Sprotein-reactive CD4+ T cells showed significantly higher frequencies in Cp patients than in unexposed individuals (p=0.006). For all other cytokines determined in our study, no significant differences were observed regarding the frequencies of cytokine-producing CD4+ T cells between the Cp and unexposed study participants (Fig. 3C ).

Tfh cells directly interact with B cells, indicate maturation of the humoral immune response and are crucial for the establishment of antigen-reactive B memory cells, which provide long-term immunity 17. We characterized circulating SARS-CoV-2 S-protein-reactive Tfh cells in the unexposed cohort by the expression of CXCR5 (Fig. 3D) . In one exception to the abovementioned minimum limit of 0,01%, we evaluated all positive frequencies of CD4+CD154+CD137+CXCR5+ cells after the background was subtracted, as no large populations of Tfh cells were expected to be found in circulation. Among the total population of unexposed individuals, 61% (n=11) showed positive frequencies for CD4 + CXCR5 + T cells.

To explore whether B cells reactive against SARS-CoV-2 S-protein were detectable in unexposed individuals, we analyzed the frequencies of SARS-CoV-2 S-protein-reactive B cells by flow cytometry using FITC-and Cy5-labeled S-protein as previously described 15.

Specificity was controlled by blocking with excess unlabeled SARS-CoV-2 S-protein (Fig. 2) 18-19. Double-positive S-protein-FITC-and S-protein-Cy5-reactive B cells were considered to specifically bind to the S-protein when the frequency was above 0.001% after the frequency of the blocked sample was subtracted.

We observed detectable S-reactive B cells in 80% of the Cp control group and in 58% of the unexposed individuals. The control group of Cp patients showed significantly higher frequencies of SARS-CoV-2-reactive B cells compared to unexposed individuals (p=0.0047, exact two-tailed Mann-Whitney Test) (Fig. 3E) . Out of the 18 unexposed individuals with characterized T and B cell responses, 11 individuals presented preexisting SARS-CoV-2specific Tfh cells, 7 of whom had a detectable SARS-CoV-2-specific B cell response.

The frequencies of SARS-CoV-2-reactive and cytokine-producing CD4 + T cells were similar among unexposed Tx patients and non-Tx individuals (Fig. 4A) . Similarly, CD4 + CXCR5 + cells among Tx patients and non-Tx participants showed no significant difference (Fig. 4C) . The frequencies of SARS-CoV-2-reactive B cells in the Tx group were not significantly different compared to the immunocompetent participants (Fig. 4D, p=0.1588 ). Of note, SARS-CoV-2reactive B cells were found more frequently among Tx patients, as 64% (n=9) Tx patients showed SARS-CoV-2-reactive B cells compared to 50% (n=6) of non-Tx participants demonstrating SARS-CoV-2-reactive B cell immunity.

Here, we report cross-reactive and preexisting B and T cell immunity to SARS-CoV-2 in a cohort of unexposed individuals, including immunocompetent individuals and renal transplant recipients. Our study suggests that renal transplant patients are able to generate a preexisting SARS-CoV-2 response that is comparable to immunocompetent adults. A limitation of our study was the small number of patients, which makes robust assumptions challenging. Subsequent studies should enroll larger patient cohorts with a greater demographic variability to include individuals of all ages from different social levels and environments. Also multi-center design should be performed to exclude a local bias (ethnicity, environmental/seasonal corona viruses, treatment). The significant age gap between the Tx and non-Tx cohorts should also be taken into consideration. Overall, our study demonstrates preexisting SARS-CoV-2 immunity among the transplant cohort, which is comparable to the immunocompetent study group. Independent working groups demonstrate the poor immune response and waning of antibodies after SARS-CoV-2 infection or vaccination among transplant recipients 28-29. Taking also into consideration the emerging SARS-CoV-2 variants of concern understanding the influence of preexisting cross-reactive immunity to SARS-CoV-2 on the adaptive immune response is of critical importance.

This work was supported by grants of Mercator Foundation, the BMBF e:KID (01ZX1612A), and BMBF NoChro (FKZ 13GW0338B).

The study was approved by the Ethics Committee of the Ruhr University Bochum and University Hospital Essen (20-9214-BO). Written informed consent was obtained from all participants. The authors have no relevant financial or non-financial interests to disclose. As reactive SARS-CoV-2 T cells are defined the CD4+CD154+CD137+ and CD8+CD137+ cells.

Negative controls were subtracted from reactive stimulated samples to exclude unreactive activation. Statistical comparison was done with Mann-Whitney-test. P<0.05 was considered significant, only significant p values are documented in the figures.

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Characterization of SARS-CoV-2 reactive T and B cells in Tx and non-Tx donors. (A) CD4+CD154+CD137+ showed no significant statistical difference among Tx and non-Tx. (B) Analysis of the monofunctional CD4+CD154+CD137+ cells. (C) Correlation of CD4+CD154+CD137+CXCR5+ among Tx and non-Tx (D)Analysis of fluorochrome labelled SARS-CoV-2 S-protein binding B cells among the unexposed

Statistical comparison was done with Mann-Whitney-test. P<0.05 was considered significant, only significant p values are documented in the figures