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 the breastmilk and peripheral blood of lactating individuals who received SARS-CoV-2 mRNA vaccination. Relative to blood, breastmilk contained higher frequencies of T effector and central memory populations that expressed mucosal-homing markers. T cell receptor (TCR) sequence overlap was limited between blood and breastmilk. Overabundan t breastmilk clones were observed in all individuals, were diverse, and contained CDR3 sequences with known epitope specificity including to SARS-CoV-2 Spike. Spike-specific TCRs were more frequent in breastmilk compared to blood and expanded in breastmilk following a third 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 infant passive protection.

One-sentence summary: The breastmilk T cell repertoire is distinct and enriched for SARS-CoV-2 Spike-specificity after maternal mRNA vaccination.

Fig. 1.. The T cell receptor (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). The frequencies of TCRβ clonotypes in the two compartments were compared using the immunoSEQ® Differential Abundance Tool. TCRβ repertoire overlap was analyzed using the Morisita index (M.I., value inset). As a control, TCRβ clonotypes from an individual’s PBMCs obtained 9 days and 17 days after 2^nd mRNA vaccine dose were compared (upper left plot).

Fig. 2.. Overabundant TCR clones in breastmilk are diverse.

(A) All TCRβ CDR3 amino acid sequences obtained from the BMC of each participant for which paired PBMC was available (n=11) were compared using tcrdist3; representative plots from two individuals are displayed. Black ticks denote TCRβ sequences significantly overabundant in BMC relative to PBMC. (B) Overabundant BMC TCRβ CDR3 amino acid sequences from 11 participants were compared to one another using tcrdist3. Overabundant TCRβ clones were 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. 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.

Fig. 3.. 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) is 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, green line indicates the position of the NYNYLYRLF epitope, and purple line indicates the position of the LTDEMIAQY epitope. (B) Spike-specific TCRβ templates are enriched in BMC relative to PBMC, incident rate ratio (IRR)=166, p=0.004.

Fig. 4.. SARS2 Spike-specific T cells expand in breastmilk following 3^rd mRNA vaccine dose.

BMC and PBMC from before and 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, n.s. (non-significant). Scatter plots of HLA-A*02_YLQ-positive (n=5) (A), HLA-A*01_LTD-positive (n=2) (B), and HLA-A*24_NYN-positive (n=1) (C) 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% to 2.8%, p=0.03; PBMC: 0.04% to 0.04%, p=0.9.