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. 2024 Jan 3:14:1258844.
doi: 10.3389/fimmu.2023.1258844. eCollection 2023.

Frequencies of activated T cell populations increase in breast milk of HCMV-seropositive mothers during local HCMV reactivation

Katrin Lazar  1 , Graham Pawelec  2   3 , Rangmar Goelz  4 , Klaus Hamprecht  1 , Kilian Wistuba-Hamprecht  2   5   6
Affiliations

Frequencies of activated T cell populations increase in breast milk of HCMV-seropositive mothers during local HCMV reactivation

Katrin Lazar et al. Front Immunol. .

Abstract

Background: Human cytomegalovirus (HCMV) can reactivate in the mammary gland during lactation and is shed into breast milk of nearly every HCMV-IgG-seropositive mother of a preterm infant. Dynamics of breast milk leukocytes during lactation, as well as blood leukocytes and the comparison between both in the context of HCMV reactivation is not well understood.

Methods: Here, we present the BlooMil study that aimed at comparing changes of immune cells in blood and breast milk from HCMV-seropositive- vs -seronegative mothers, collected at four time ranges up to two months post-partum. Viral load was monitored by qPCR and nested PCR. Multiparameter flow cytometry was used to identify leukocyte subsets.

Results: CD3+ T cell frequencies were found to increase rapidly in HCMV-seropositive mothers' milk, while they remained unchanged in matched blood samples, and in both blood and breast milk of HCMV-seronegatives. The activation marker HLA-DR was more strongly expressed on CD4+ and CD8+ T cells in all breast milk samples than matched blood samples, but HCMV-seropositive mothers displayed a significant increase of HLA-DR+ CD4+ and HLA-DR+ CD8+ T cells during lactation. The CD4+/CD8+ T cell ratio was lower in breast milk of HCMV-seropositive mothers than in the blood. HCMV-specific CD8+ T cell frequencies (recognizing pp65 or IE1) were elevated in breast milk relative to blood, which might be due to clonal expansion of these cells during local HCMV reactivation. Breast milk contained very low frequencies of naïve T cells with no significant differences depending on serostatus.

Conclusion: Taken together, we conclude that the distribution of breast milk leukocyte populations is different from blood leukocytes and may contribute to the decrease of breast milk viral load in the late phase of HCMV reactivation in the mammary gland.

Keywords: CD4+ T cells; CD8+ T cells; HCMV-specific T cells; breast feeding; effector memory T cells; monocytes; viral load; virus transmission.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
BlooMil study design and HCMV viral load in breast milk. (A) timeline of blood and breast milk sampling postpartum (p.p.). (B) Breast milk and blood sample processing for analysis. (C) viral load in milk whey of 18 HCMV-seropositive mothers measured by real time PCR. T1 – 10 to 15, T2 – 25 to 30, T3 – 40 to 45, and T4 – 55 to 60 days postpartum.
Figure 2
Figure 2
Gating strategy of (A) breast milk cells (BMC) and (B) peripheral blood mononuclear cells (PBMCs).
Figure 3
Figure 3
Longitudinal courses of CD14+ monocyte/macrophage frequencies in blood (red droplet) of HCMV-seronegative (A) and seropositive (B) mothers and in breast milk (white droplet) of HCMV-seronegative (C) and positive (D) mothers. The inserts in (A, B) show the biological control over all experimental days. Individual color-coding was applied as shown in Table 1. Mother 2 (red) was excluded from statistical analysis due to an HCMV primary infection during pregnancy. Empty squares as data points indicate probable mastitis at this time point. Time ranges were T1 – 10 to 15, T2 – 25 to 30, T3 – 40 to 45, and T4 – 55 to 60 days postpartum.
Figure 4
Figure 4
Longitudinal courses of CD3+ T cell frequencies in blood (red droplet) of HCMV-seronegative (A) and seropositive (B) mothers and in breast milk (white droplet) of HCMV-seronegative (C) and positive (D) mothers. The inserts in (A, B) show the biological control over all experimental days. Individual color-coding was applied as shown in Table 1. Mother 2 (red) was excluded from statistical analysis due to an HCMV primary infection during pregnancy. Empty squares as data points indicate probable mastitis at this time point. Time ranges were T1 – 10 to 15, T2 – 25 to 30, T3 – 40 to 45, and T4 – 55 to 60 days postpartum.
Figure 5
Figure 5
T cell subsets of HCMV seropositive and seronegative mothers. CD4+/CD8+ T cell ratio in blood (A) and breast milk (B) are shown. The activation marker HLA-DR was investigated on CD4+ (C) and CD8+ T cells (D). Longitudinal analysis of CD8+ HLA-DR+ T cells in blood (red droplet) of HCMV-seronegative (E) and seropositives (F), as well as in breast milk (white droplet) of HCMV seronegatives (G) and seroposiitves (H) are shown. The inserts in (E, F) show the biological control over all experimental days. Individual color-coding was applied as shown in Table 1. Mother 2 (red) was excluded from statistical analysis due to an HCMV primary infection during pregnancy. Empty squares as data points indicate probable mastitis at this time point. Statistical significances were indicated as followed: *p<0.05, **p<0.01, ***p<0.001.
Figure 6
Figure 6
T cell memory subsets in breast milk and blood. CD4+ TEMRA (A), naïve (B), effector memory (C) and central memory (D) T cells of blood and breast milk from HCMV seropositive and seronegative mothers. Additionally, CD8+ TEMRA (E), naïve (F), effector memory (G) and central memory (H) T cells of blood and breast milk from HCMV seropositive and seronegative mothers were shown. Memory subsets were determined using CCR7 and CD45RA. Time ranges were T1 – 10 to 15, T2 – 25 to 30, T3 – 40 to 45, and T4 – 55 to 60 days postpartum. Statistical significances were indicated as followed: *p<0.05, **p<0.01, ***p<0.001.
Figure 7
Figure 7
Heatmap of T cells in breast milk and HCMV-specific T cells. Clustering of breast milk cells (BMC) such as Monocytes, CD3+, CD4+ and CD8+ T cells etc. of HCMV-seropositive (purple) and seronegative (green) mothers at T1 and T4 (A) using the complex heatmap package (18, 19) is presented. It was calculated using maximum length apart for the row distance and the ward.D2-approach for clustering. Additionally, four mothers were analysed for their HCMV-specific T cell frequency using HLA-A*02 tetramer analysis (pp65 NLVPMVATV and IE1 VLEETSVML). Viral load in milk whey (B), as well as the pp65-specific T cell frequencies of those mothers at T1 – 10 to 15 (C) and T3 – 40 to 45 days postpartum (D) are shown. Furthermore, IE1-specific T cell frequencies at T1 – 10 to 15 (E) and T3 – 40 to 45 days postpartum (F) are presented.
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The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The study was financed by “Sonderlinie Medizin: Verbundprojekt Freiburg-Tuebingen-Ulm” by the Ministry of Science and Arts of Baden Wuerttemberg, grant number [KL 25280-0].
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