Spike-specific T cells are enriched in breastmilk following SARS-CoV-2 mRNA vaccination

Abstract

Human breastmilk is rich in T cells; however, their specificity and function are largely unknown. We compared the phenotype, diversity, and antigen specificity of T cells in breastmilk and peripheral blood of lactating individuals who received SARS-CoV-2 messenger RNA (mRNA) vaccination. Relative to blood, breastmilk contained higher frequencies of T effector and central memory populations that expressed mucosal-homing markers. T cell receptor sequence overlap was limited between blood and breastmilk. Overabundant breastmilk clones were observed in all individuals, were diverse, and contained complementarity-determining regions in three sequences with known epitope specificity, including to SARS-CoV-2 spike. SARS-CoV-2 spike-specific T cell receptors were more frequent in breastmilk compared to blood and expanded in breastmilk following a 3^rd mRNA vaccine dose. Our observations indicate that the lactating breast contains a distinct T cell population that can be modulated by maternal vaccination with potential implications for passive infant protection.

Fig. 1

Antigen-experienced and mucosal-homing T cells are enriched in breastmilk. PBMC and BMC were analyzed by flow cytometry. Comparisons made with linear regression and clustering by individual, ***p < 0.001, **p < 0.01, *p < 0.05. (A) Scatter plots showing expression of CD45RO and CCR7 in CD4+ and CD8+ T cells from PBMC and BMC in one representative participant; (B) Frequencies of T_EM, T_CM, and T_N-like CD4+ and CD8+ T cells in PBMC and BMC (n = 17): CD4+ T_EM: 41% versus 84%, p < 0.001, CD4+ T_CM: 6% versus 11%, p = 0.006, CD4+ T_N-like: 11% versus 1%, p < 0.001; CD8+ T_EM: 38% versus73%, p < 0.001, CD8+ T_CM: 1% versus 1%, p = n.s., CD8+ T_N-like: 12% versus 0.2%, p < 0.001; (C) Scatter plots showing expression of CCR9 and CD103 in CD4+ and CD8+ T cells from PBMC and BMC in one representative participant; (D) Expression of CCR9 and CD103 within CD4+ and CD8+ T cell populations in PBMC and BMC: CD4+/CCR9+ (n = 11): 4% versus 38%, p < 0.001, CD4+/CD103+: 0.4% versus 7%, p < 0.001, CD4+/CCR9+/CD103+: 0.1 versus 5%, p < 0.001, CD8+/CCR9+: 3% versus 12%, p = 0.005; CD8+/CD103+: 3% versus 32%, p < 0.001, CD8+/CCR9+/CD103+: 0.4% versus 3%, p < 0.001. BMC = breastmilk cells; PBMC = peripheral blood mononuclear cells; n.s. = not significant; T_CM = central memory T cells; T_EM = T cell effector memory T cells; T_N-like = naïve-like T cells.

Fig. 2

The TCR repertoires in breastmilk and peripheral blood are distinct. Bulk TCRβ sequencing from BMC and PBMC individuals who had paired samples available and at least 1,000 TCRβ templates in the BMC sample (n = 11). Each clone is represented as a dot, with the relative frequency in BMC represented on the X axis and relative frequency in PBMC on the Y axis. TCRβ repertoire overlap was analyzed using the M.I. (value inset), with a value of 0 representing no repertoire overlap and a value of 1 representing complete overlap of the repertoires. The frequencies of each individual TCRβ clonotype in the two compartments were statistically compared using the immunoSEQ® Differential Abundance Tool. Gray dashed line indicates frequency equality in the two compartments. Blue dots represent clonotypes that are significantly more abundant in BMC relative to PBMC. Orange dots represent clonotypes that are significantly more abundant in PBMC relative to BMC. Gray dots represent clonotypes that were not significantly different in frequency between the two compartments. Pale gray dots represent clonotypes that fell below the frequency for valid statistical comparison. As a control, TCRβ clonotypes from an individual’s PBMC obtained nine days and 17 days after 2^nd mRNA vaccine dose were compared (upper left plot), demonstrating a high degree of repertoire overlap and only a few clones expanded at the 2^nd time point relative to the 1^st. BMC = breastmilk cells; M.I. = Morisita index; PBMC = peripheral blood mononuclear cells; TCR = T cell receptor; TCRβ = TCR beta chain.

Fig. 3

Overabundant TCR clones in breastmilk are diverse. (A) For each individual participant with paired PBMC available (n = 11), all TCRβ CDR3 amino acid sequences obtained from the BMC were compared to one another using tcrdist3; representative plots from two individuals are displayed. Black ticks denote TCRβ sequences significantly overabundant in BMC relative to PBMC, demonstrating that overabundant clones were distributed across the full breastmilk TCR repertoire for each individual (i.e. did not cluster by sequence); (B) Overabundant BMC TCRβ CDR3 amino acid sequences from 11 participants were compared to one another across participants using tcrdist3. Overabundant clones did not cluster by individual, and most clones were private, emphasizing the diversity of overabundant clones across individuals. Overabundant TCRβ clones were further compared by CDR3 amino acid sequence and V gene usage against available public databases of known TCR epitope specificity. Clones matching pathogen-specific epitopes are marked with colored ticks. Epitope matches were considered a direct match if the CDR3 amino acid and TRβV gene were identical and were considered a predicted match if the CDR3 amino acid sequences were identical, but V gene usage was mismatched. Only the minority of clone specificity could be assigned, and characterized clones were tightly clustered across all individuals. For participant 10, only CDR3 amino acid sequences enriched by a factor ≥ 50 relative to PBMC or with epitope specificity were included to reduce data skewing from this participant. BMC = breastmilk cells; CDR = complementarity-determining regions; EBV = Epstein-Barr virus; Orf = open reading frame; Mtb = Mycobacterium tuberculosis; PBMC = peripheral blood mononuclear cells; TCR = T cell receptor; TCRβ = TCR beta chain.

Fig. 4

Candidate SARS2 spike-specific T cells are enriched in the breastmilk relative to the peripheral blood of vaccinated individuals. The frequency of candidate spike-specific clones in all sequenced PBMC and BMC (n = 14 paired, n = 2 BMC only) was expressed relative to all clones predicted to bind to SARS2 antigens. Spike template frequency was compared using a negative binomial model, **p < 0.01. (A) TCRβ sequences predicted to bind to SARS2 were identified in BMC and PBMC using the ImmunoSEQ® T-MAP COVID Search Tool and are mapped by their epitope binding location on the spike protein. Gold line indicates the amino acid position of the spike peptide pool that includes the YLQPRTFLL epitope (HLA-A*02 restricted), green line indicates the position of the NYNYLYRLF epitope (HLA-A*24 restricted), and purple line indicates the position of the LTDEMIAQY epitope (HLA-A*01 restricted); (B) Spike-specific TCRβ templates are enriched in BMC relative to PBMC, incident rate ratio = 1.66, p = 0.004. BMC = breastmilk cells; CDR = complementarity-determining regions; HLA = human leukocyte antigen; PBMC = peripheral blood mononuclear cells; TCRβ = T cell receptor beta chain.

Fig. 5

SARS2 spike-specific T cells expand in breastmilk following 3^rd mRNA vaccine dose. BMC and PBMC from before and ∼ 1 week after receipt of the 3^rd dose of SARS2 mRNA vaccine were stained with SARS2 spike epitope-loaded class I tetramers and analyzed by flow cytometry to quantify spike-specific CD8+ T cells. Comparisons made with paired t tests, * p <0.05. Scatter plots of (A) HLA-A*02_YLQ-positive (n = 5); (B) HLA-A*01_LTD-positive (n = 2); and (C) HLA-A*24_NYN-positive (n = 1) CD8+ T cells in breastmilk obtained before (top) and after (bottom) 3^rd mRNA vaccine dose are shown from HLA concordant individuals. Frequencies of tetramer+ cells of CD8+ T cells inset; (D) Frequencies of tetramer+ cells of CD8+ T cells in BMC (n = 8, blue) and PBMC (n = 7, red). Triangle = HLA-A*02_YLQ, circle = HLA-A*01_LTD, square = HLA-A*24_NYN. BMC: 0.8-2.8%, p = 0.03; PBMC: 0.04-0.04%, p = 0.9. BMC = breastmilk cells; CDR = complementarity-determining regions; HLA = human leukocyte antigen; mRNA = messenger RNA; n.s. = non-significant; PBMC = peripheral blood mononuclear cells; TCRβ = T cell receptor beta chain.