Menstrual blood as a potential source of endometrial derived CD3+ T cells

Abstract

Studies of T cell-mediated immunity in the human female genital tract have been problematic due to difficulties associated with the collection of mucosal samples. Consequently, most studies rely on biopsies from the lower female genital tract or remnant tissue from hysterectomies. Availability of samples from healthy women is limited, as most studies are carried out in women with underlying pathologies. Menstruation is the cyclical sloughing off of endometrial tissue, and thus it should be a source of endometrial cells without the need for a biopsy. We isolated and phenotyped T cells from menstrual and peripheral blood and from endometrial biopsy-derived tissue from healthy women to determine the types of T cells present in this compartment. Our data demonstrated that T cells isolated from menstrual blood are a heterogeneous population of cells with markers reminiscent of blood and mucosal cells as well as unique phenotypes not represented in either compartment. T cells isolated from menstrual blood expressed increased levels of HLA-DR, αEβ7 and CXCR4 and reduced levels of CD62L relative to peripheral blood. Menstrual blood CD4+ T cells were enriched for cells expressing both CCR7 and CD45RA, markers identifying naïve T cells and were functional as determined by antigen-specific intracellular cytokine production assays. These data may open new avenues of investigation for cell mediated immune studies involving the female reproductive tract without the need for biopsies.

Figure 1. Gating strategy for menstrual and peripheral blood cells.

A. PBMC and B. MBC. Initial gating is performed from the SSC-A vs. FSC-A to determine the total lymphocyte gate (small gate) and the larger gate is used to further gate for monocytes (CD14). From the lymphocytes, live cells are gated, followed by gating B cells (CD19), T cells (CD3), CD4+CD3+ and CD8+CD3+ cells. From the CD3- gate, NK cell phenotypes are selected. CD16-CD56+(immunoregulatory cells) and CD16+CD56+(Cytotoxic cells). Percentages of positive cells for a given marker are indicated above/near gate.

Figure 2. Cell composition of menstrual blood cells (MBCs).

The percentage of various cell types present in menstrual blood versus PBMC were resolved by staining with cell type-specific surface markers. A. The total lymphocytes, were determined by the percentage of cells within a lymphocyte gate (small cells from FSC vs SSC plots) and monocytes (N = 8), are CD14+ cell from a leuokocyte gate (all cells within the larger FSC vs SSC plot), refer to Figure 1. B. Lymphocyte subsets present in menstrual versus peripheral blood. From the live cell gate, B cells were gated on CD19, T cells were gated on CD3 and T cell subsets were further divided into CD4+CD3+ and CD8+CD3+ T cells. C. Natural killer cell subsets (N = 8) gated from CD3- cells. CD16+CD56+ (cytotoxic NK cells) and CD16-CD56+ (immunoregulatory NK cells). The median is shown for data from 12 healthy women, except for monocytes and NK cells, where 8 samples were analyzed. Statistically significant differences (p<0.05) were obtained using Wilcoxon Signed Rank test.

Figure 3. A representative example of surface antigen expression.

Cell surface expression of CD62L, HLA-DR and αEβ7 on A. CD3+CD4+ and B. CD3+CD8+ T cells from PBMC, MBC and endometrial tissue (ENDO) is shown. Percent of positive cells for a given marker are indicated above the gate.

Figure 4. Surface marker expression on PBMC, MBC, and cells from endometrial tissue.

Surface markers on CD3+CD4+ cells are shown for panels A–C and on CD3+CD8+ on panels D–F. The median data from peripheral and menstrual blood is shown for 12 healthy women. Endometrial tissue from 7 women was available for several analyses; however, for some samples, data from only four samples was obtained. Comparisons were made using Wilcoxon Signed Rank test and Mann Whitney U test for paired and unpaired samples, respectively.

Figure 5. Memory marker expression.

CCR7 and CD45RA surface staining was performed on both CD3+CD4+ and CD3+CD8+ T cells from PBMC and MBC. A–D represent samples gated on different populations of cells CCR7 and CD45RA expression. The median and interquartile range data is shown for 12 healthy women. Tcm = central memory, Tem = effector memory, and Temra = effector memory RA+. Comparisons were made using Wilcoxon Signed Rank test.

Figure 6. CMV-specific T cells derived from the menstrual blood.

A. PBMC B. MBC from the same healthy woman (#1). Cells were stimulated with overlapping peptides from CMV pp65 and PMA/I as a positive control. Both IFN-γ and TNF-α secretion are shown. Percent of cytokine positive cells (IFN-γ or TNF-α) are shown above the gate.

Figure 7. Reduced frequency of CMV-specific T cells derived from the menstrual blood.

PBMC and MBC from the same women were stimulated with overlapping peptides from CMV pp65 or CMV lysate and stained for IFN-γ. Individual symbols represent paired samples from the same individual. Open symbols represent CD8 T cell responses, closed symbols represent CD4 T cell responses and gray symbols are responses from the healthy volunteer. Statistical comparisons were made using Wilcoxon Signed Rank test.