Menstrual Blood as a Potential Source of Endometrial Derived CD3+ T Cells

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

The Predominant CD4+ Th1 Cytokine Elicited to Chlamydia trachomatis Infection in Women Is Tumor Necrosis Factor Alpha and Not Interferon Gamma

Chlamydia trachomatis infection is the most prevalent bacterial sexually transmitted infection and can cause significant reproductive morbidity in women. There is insufficient knowledge of C. trachomatis-specific immune responses in humans, which could be important in guiding vaccine development efforts. In contrast, murine models have clearly demonstrated the essential role of T helper type 1 (Th1) cells, especially interferon gamma (IFN-γ)-producing CD4+ T cells, in protective immunity to chlamydia. To determine the frequency and magnitude of Th1 cytokine responses elicited to C. trachomatis infection in humans, we stimulated peripheral blood mononuclear cells from 90 chlamydia-infected women with C. trachomatis elementary bodies, Pgp3, and major outer membrane protein and measured IFN-γ-, tumor necrosis factor alpha (TNF-α)-, and interleukin-2 (IL-2)-producing CD4+ and CD8+ T-cell responses using intracellular cytokine staining. The majority of chlamydia-infected women elicited CD4+ TNF-α responses, with frequency and magnitude varying significantly depending on the C. trachomatis antigen used. CD4+ IFN-γ and IL-2 responses occurred infrequently, as did production of any of the three cytokines by CD8+ T cells. About one-third of TNF-α-producing CD4+ T cells coproduced IFN-γ or IL-2. In summary, the predominant Th1 cytokine response elicited to C. trachomatis infection in women was a CD4+ TNF-α response, not CD4+ IFN-γ, and a subset of the CD4+ TNF-α-positive cells produced a second Th1 cytokine

CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription

Retroviruses pack multiple genes into relatively small genomes by encoding several genes in the same genomic region with overlapping reading frames. Both sense and antisense HIV-1 transcripts contain open reading frames for known functional proteins as well as numerous alternative reading frames (ARFs). At least some ARFs have the potential to encode proteins of unknown function, and their antigenic properties can be considered as cryptic epitopes (CEs). To examine the extent of active immune response to virally encoded CEs, we analyzed human leukocyte antigen class I–associated polymorphisms in HIV-1 gag, pol, and nef genes from a large cohort of South Africans with chronic infection. In all, 391 CEs and 168 conventional epitopes were predicted, with the majority (307; 79%) of CEs derived from antisense transcripts. In further evaluation of CD8 T cell responses to a subset of the predicted CEs in patients with primary or chronic infection, both sense- and antisense-encoded CEs were immunogenic at both stages of infection. In addition, CEs often mutated during the first year of infection, which was consistent with immune selection for escape variants. These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity

Memory marker expression.

<p>CCR7 and CD45RA surface staining was performed on both CD3+CD4+ and CD3+CD8+ T cells from PBMC and MBC. <b>A–D</b> 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.</p

A representative example of surface antigen expression.

<p>Cell surface expression of CD62L, HLA-DR and αEβ7 on <b>A.</b> CD3+CD4+ and <b>B.</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.</p

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

<p>Surface markers on CD3+CD4+ cells are shown for panels <b>A–C</b> and on CD3+CD8+ on panels <b>D–F</b>. 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.</p

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

<p>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.</p

Cell composition of menstrual blood cells (MBCs).

<p>The percentage of various cell types present in menstrual blood versus PBMC were resolved by staining with cell type-specific surface markers. <b>A.</b> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028894#pone-0028894-g001" target="_blank">Figure 1</a>. <b>B.</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. <b>C.</b> 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.</p