Nature MedicineArtic e https://doi.org/10.1038/s41591-024-03278-ytetanus vaccine. S2P IgA ASCs were also detected predominantly in PopAand PopB: a mean of 1.5% (1.46 ± 1.57) and 0.9% (0.90 ± 0.66), respec-tively, and were virtually absent in PopD: a mean of 0.03% (0.03 ± 0.06)(Extended Data Fig. 3b). On average, the fold changes of IgA ASC spe-cificities within PopD were 50.9 for Flu:S2P and 9.3 for Tet:S2P (Supple-mentary Table 3). For S2P specificity, the fold change of PopA:PopD was43.8 and of PopB:PopD was 27.0 (Supplementary Table 4). Thus, similarto IgG ASCs, other class-switched isotypes such as S2P IgA ASC are alsomostly excluded from PopD (albeit small sample size).Absence of SARS-CoV-2-specific IgG in LLPC culturesupernatantTo validate the antigen-specific ELISpot results, we measured secretedIgG from BM ASC subsets (Fig. 2a; see also Methods). Briefly, from eightindividuals who yielded sufficient sorted cells for all BM ASC subsets(PopA, PopB and PopD), we cultured ASCs in a specialized in vitro BMmimetic system overnight 16 and measured the cultured supernatantsfor secreted IgG specific for Flu, Tet and S2P by multiplex bead-bindingassays (MBBAs)34 (Extended Data Fig. 4). The results were similar to thePopAPopBPopDPopAPopBPopDPopAPopBPopDPopAPopBPopDPopAPopBPopDPopAPopBPopD0246810121416BM ASC in cultureBM Flu-, Tet-, S2P-IgGASC (% total)Purple: TetGreen: S2PBlue: FluFlu Tet S2PFlu Tet S2P0131323330.61 0.4429.07Non-LLPC:LLPC ratio, IgG ASC013132333Non-LLPC:LLPC ratio, sup IgGPurple: TetGreen: S2PBlue: Flu0246713192531BM ASC in cultureSup Flu-, Tet-, S2P-IgG (% total)0.66 0.4423.26PopD PopB PopASub 8Total S2PFlu TetLLPC Non-LLPCELISpotCellsMBBASupSorting forBM ASCBM donorsASC culture(overnight)BM MNC PopA, PopB andPopD (LLPC)ASC survivalmediumBM collectionand MNC isolationa bc de fBM ASCBM ASC supFig. 2 | Absence of SARS-CoV-2 BM IgG LLPC after SARS-CoV-2 mRNA vaccinesby detection of ASC and secreted IgG in the BM ASC culture supernatants.a, Summary of the techniques and the experimental designs for detection oftotal, Flu, Tet and S2P ASCs and secreted IgG by ELISpots and MBBA, respectively.MNC, mononuclear cells. b, Representative ELISpot scanned images. Thenumbers of input ASC that were incubated were ~52 K, ~12.1 K and ~10.1 K forPopA, PopB and PopD, respectively. Each symbol represents an individualvaccine subject for total IgG and antigen-specific ASC from PopA, PopB andPopD. c, ELISpots measuring BM IgG ASC specific for Flu, Tet and S2P. Data weregenerated from 8, 15 and 17 different SARS-CoV-2-vaccinated subjects for PopA,PopB and PopD, respectively. For individual ratios and statistic comparisonsbetween any two antigens for any subset or between any two subsets for anyantigen, see Supplementary Tables 1 and 2, respectively. d, Fold difference(ratios) when comparing different vaccine specificities between non-LLPCs(combined PopA and PopB) versus LLPCs (PopD). e, MBBA measuring IgGspecific for Flu, Tet and S2P (normalized to total IgG) from culture supernatant ofPopA, PopB and PopD. Supernatant preps were collected from 18–24-h culturesof BM ASCs after revival from the FACS sorters and were quantified for total IgGand vaccine-specific IgG in neat (undiluted). Data were generated from eightdifferent SARS-CoV-2-vaccinated subjects. For individual ratios and statisticcomparisons between any two antigens for any subset or between any twosubsets for any antigen, see Supplementary Tables 1 and 2, respectively. f, Thefold difference (ratios) when comparing normalized vaccine-specific IgG in thesupernatants from the culture of non-LLPCs (combined PopA and PopB) versusLLPCs (PopD). For ratio calculation, see Methods. For IgG standard versus MFIcurve, see Extended Data Fig. 4. Counts were provided by the sorters. LLPC, boxesin b, c, and e. Sub, subject; Sups, BM ASC culture supernatant preps. For details ofsubjects and samples, see Table 1.
