Detection of Intraepithelial and Stromal Langerin and CCR5 Positive Cells in the Human Endometrium: Potential Targets for HIV Infection

Both the upper (endocervix and uterus) and lower (ectocervix and vagina) female genital tract mucosa are considered to be target sites for sexual transmission of HIV. There are a few reports on the T cell and antigen-presenting cell distribution in human endometrial tissue however, there is little known about the expression of the HIV co-receptor CCR5 and HIV-binding C-type lectin receptors on endometrial cell subsets. We therefore assessed endometrial tissue sections from HIV seronegative women undergoing hysterectomy of a benign and non-inflammatory cause for phenotypic characterization of potential HIV target cells and receptors by immunohistochemistry. Langerin was expressed on intraepithelial CD1a+CD4+ and CD11c+CD4+ Langerhans cells. Furthermore, CCR5+CD4+CD3+ T cells, DC-SIGN+MR+CD11c+ myeloid dendritic cells and MR+CD68+ macrophages were found within or adjacent to the epithelium of the uterine lumen. In addition, occasional CD123+ BDCA-2+ plasmacytoid dendritic cells were detected deep in the endometrial stroma. Both T cells and several antigen-presenting cells were detected in lymphoid aggregate formations in close proximity to the epithelial lining. The finding of intraepithelial and stromal Langerin+ cells as well as CCR5+ CD4+ T cells is novel for human endometrium

Presence of CD8+ T Cells in the Ectocervical Mucosa Correlates with Genital Viral Shedding in HIV-Infected Women despite a Low Prevalence of HIV RNA–Expressing Cells in the Tissue

The female genital tract is a portal of entry for sexual HIV transmission and a possible viral reservoir. In this study, the ectocervical CD8+ T cell distribution was explored in situ and was related to expression of CD3 and HLA-DR and presence of HIV RNA. For this purpose, ectocervical tissue samples and genital secretions were collected from HIV-seropositive (HIV+) Kenyan female sex workers (FSWs) (n = 20), HIV-seronegative (HIV−) FSWs (n = 17), and HIV− lower-risk women (n = 21). Cell markers were assessed by in situ staining and by quantitative PCR. HIV RNA expression in tissue was analyzed by in situ hybridization, and viral shedding was assessed by quantitative PCR. The HIV+FSW group had a higher amount of total cells and CD8+, CD3+, and HLA-DR+ cells compared with the HIV−FSW group and HIV− lower-risk women. The majority of CD8+ cells were CD3+ T cells, and the numbers of CD8+ cells correlated significantly with plasma and cervical viral load. HIV RNA expression in situ was found in 4 of the 20 HIV+FSW women but did not correlate with cervical or plasma viral load. Thus, the HIV+ women displayed high numbers of CD8+, CD3+, and HLA-DR+ cells, as well as a limited number of HIV RNA+ cells, in their ectocervical mucosa; hence, this localization cannot be neglected as a potential viral reservoir. The elevated levels of CD8+ T cells may play a role in the immunopathogenesis of HIV in the female genital tract

Presence of CD8+ T Cells in the Ectocervical Mucosa Correlates with Genital Viral Shedding in HIV-Infected Women despite a Low Prevalence of HIV RNA–Expressing Cells in the Tissue

The female genital tract is a portal of entry for sexual HIV transmission and a possible viral reservoir. In this study, the ectocervical CD8+ T cell distribution was explored in situ and was related to expression of CD3 and HLA-DR and presence of HIV RNA. For this purpose, ectocervical tissue samples and genital secretions were collected from HIV-seropositive (HIV+) Kenyan female sex workers (FSWs) (n = 20), HIV-seronegative (HIV−) FSWs (n = 17), and HIV− lower-risk women (n = 21). Cell markers were assessed by in situ staining and by quantitative PCR. HIV RNA expression in tissue was analyzed by in situ hybridization, and viral shedding was assessed by quantitative PCR. The HIV+FSW group had a higher amount of total cells and CD8+, CD3+, and HLA-DR+ cells compared with the HIV−FSW group and HIV− lower-risk women. The majority of CD8+ cells were CD3+ T cells, and the numbers of CD8+ cells correlated significantly with plasma and cervical viral load. HIV RNA expression in situ was found in 4 of the 20 HIV+FSW women but did not correlate with cervical or plasma viral load. Thus, the HIV+ women displayed high numbers of CD8+, CD3+, and HLA-DR+ cells, as well as a limited number of HIV RNA+ cells, in their ectocervical mucosa; hence, this localization cannot be neglected as a potential viral reservoir. The elevated levels of CD8+ T cells may play a role in the immunopathogenesis of HIV in the female genital tract

In Situ Distribution of HIV-Binding CCR5 and C-Type Lectin Receptors in the Human Endocervical Mucosa

The endocervical mucosa is believed to be a primary site of HIV transmission. However, to date there is little known about the distribution of the HIV co-receptor CCR5 and the HIV-binding C-type lectin receptors, including Langerin, dendritic cell (DC)-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN) and mannose receptor (MR) at this site. We therefore characterized the expression of these molecules in the endocervix of HIV seronegative women by computerized image analysis. Endocervical tissue biopsies were collected from women (n = 6) undergoing hysterectomy. All study individuals were diagnosed with benign and non-inflammatory diseases. CCR5+ CD4+ CD3+ T cells were found within or adjacent to the endocervical epithelium. The C-type lectin Langerin was expressed by intraepithelial CD1a+ CD4+ and CD11c+ CD4+ Langerhans cells, whereas DC-SIGN+ MR+ CD11c myeloid dendritic cells and MR+ CD68+ macrophages were localized in the submucosa of the endocervix. The previously defined immune effector cells including CD8+, CD56+, CD19+ and IgD+ cells were also found in the submucosa as well as occasional CD123+ BDCA-2+ plasmacytoid dendritic cells. Understanding the spatial distribution of potential HIV target cells and immune effector cells in relation to the endocervical canal forms a basis for deciphering the routes of HIV transmission events in humans as well as designing HIV-inhibiting compounds

HIV-Neutralizing Activity of Cationic Polypeptides in Cervicovaginal Secretions of Women in HIV-Serodiscordant Relationships

HIV exposed seronegative (HESN) women represent the population most in need of a prophylactic antiviral strategy. Mucosal cationic polypeptides can potentially be regulated for this purpose and we here aimed to determine their endogenous expression and HIV neutralizing activity in genital secretions of women at risk of HIV infection.Cervicovaginal secretions (CVS) of Kenyan women in HIV-serodiscordant relationships (HESN, n = 164; HIV seropositive, n = 60) and low-risk controls (n = 72) were assessed for the cationic polypeptides HNP1–3, LL-37 and SLPI by ELISA and for HIV neutralizing activity by a PBMC-based assay using an HIV primary isolate. Median levels of HNP1–3 and LL-37 in CVS were similar across study groups. Neither HSV-2 serostatus, nor presence of bacterial vaginosis, correlated with levels of HNP1–3 or LL-37 in the HESN women. However, an association with their partner's viral load was observed. High viral load (>10,000 HIV RNA copies/ml plasma) correlated with higher levels of HNP1–3 and LL-37 (p = 0.04 and 0.03, respectively). SLPI was most abundant in the low-risk group and did not correlate with male partner's viral load in the HESN women. HIV neutralizing activity was found in CVS of all study groups. In experimental studies, selective depletion of cationic polypeptides from CVS rendered the remaining CVS fraction non-neutralizing, whereas the cationic polypeptide fraction retained the activity. Furthermore, recombinant HNP1–3 and LL-37 could induce neutralizing activity when added to CVS lacking intrinsic activity.These findings show that CVS from HESN, low-risk, and HIV seropositive women contain HIV neutralizing activity. Although several innate immune proteins, including HNP1–3 and LL-37, contribute to this activity these molecules can also have inflammatory properties. This balance is influenced by hormonal and environmental factors and in the present HIV serodiscordant couple cohort study we show that a partner's viral load is associated with levels of such molecules

Non-cationic proteins are associated with HIV neutralizing activity in genital secretions of female sex workers.

CAPRISA, 2015.Abstract available in pdf

HIV-Specific Antibodies Capable of ADCC Are Common in Breastmilk and Are Associated with Reduced Risk of Transmission in Women with High Viral Loads

There are limited data describing the functional characteristics of HIV-1 specific antibodies in breast milk (BM) and their role in breastfeeding transmission. The ability of BM antibodies to bind HIV-1 envelope, neutralize heterologous and autologous viruses and direct antibody-dependent cell cytotoxicity (ADCC) were analyzed in BM and plasma obtained soon after delivery from 10 non-transmitting and 9 transmitting women with high systemic viral loads and plasma neutralizing antibodies (NAbs). Because subtype A is the dominant subtype in this cohort, a subtype A envelope variant that was sensitive to plasma NAbs was used to assess the different antibody activities. We found that NAbs against the subtype A heterologous virus and/or the woman's autologous viruses were rare in IgG and IgA purified from breast milk supernatant (BMS) – only 4 of 19 women had any detectable NAb activity against either virus. Detected NAbs were of low potency (median IC50 value of 10 versus 647 for the corresponding plasma) and were not associated with infant infection (p = 0.58). The low NAb activity in BMS versus plasma was reflected in binding antibody levels: HIV-1 envelope specific IgG titers were 2.2 log10 lower (compared to 0.59 log10 lower for IgA) in BMS versus plasma. In contrast, antibodies capable of ADCC were common and could be detected in the BMS from all 19 women. BMS envelope-specific IgG titers were associated with both detection of IgG NAbs (p = 0.0001)and BMS ADCC activity (p = 0.014). Importantly, BMS ADCC capacity was inversely associated with infant infection risk (p = 0.039). Our findings indicate that BMS has low levels of envelope specific IgG and IgA with limited neutralizing activity. However, this small study of women with high plasma viral loads suggests that breastmilk ADCC activity is a correlate of transmission that may impact infant infection risk

Mucosal immune responses in HIV-1 exposed uninfected individuals

The development of HIV vaccines and immunotherapeutics remains hampered by our lack of understanding correlates of immune protection to infection and disease. The major (perhaps only) piece of evidence indicating that long-term, solid resistance to HIV infection is indeed possible comes from HIV exposed yet uninfected individuals (EUI). In these cohorts of commercial sexworkers and discordant couples, frequent exposure to HIV infection occurs without productive infection taking place. These groups have attracted substantial attention since they may provide clues to the immune correlates of HIV protection. Mucosal and systemic HIV-specific. immune responses have been described in some, EUI groups; these include HIV-specific CD4+ and CD8+ T cells and HIV neutralizing IgA antibodies. However, the role of HIV specific IgA antibodies in mucosal protection must be further evaluated, especially in relation to innate immune factors. The potential value of protective mediators, including interferon alpha (IFNα), regulated upon activation, normal T-cell expressed and secreted (RANTES), secretory leukocyte protease inhibitor (SLPI), leukemia inhibitory factor (LIF) and defensins, still remains to be validated. The present thesis aims to evaluate mucosal immune responses in individuals who seem to resist HIV-1-infection despite repeated exposure to the virus. Mucosal and plasma samples were collected from EUI female sex workers (FSWs) from the slum areas Pumwani and Kibera of Nairobi, Kenya and ectocervical biopsies were collected from Nigerian EUI FSWs. HIV-1 positive and healthy low-risk HIV-1 negative individuals were included as controls. The mucosal and plasma samples were quantified for IgA1/IgA2 and innate immune factors. Also, the neutralizing capacity of fractions representing IgA and innate factors was evaluated in classical PBMC neutralization assays and dendritic cell (DC)/PBMC co-cultures. Cervical biopsies were stained for cellular markers, cytokines/chemokines and innate molecules at the single cell level by immunohistochemistry and quantified by computerized in situ imaging. We detected a clear association between HIV neutralizing IgA in the genital tract and subsequent protection against sexual HIV acquisition in a prospective, nested case-control study. Also, we detected an HIV-1 neutralizing activity in mucosal samples of EUI FSWs that was neither due to IgA1 nor correlated with high levels of SLPI. Furthermore, we have shown that mucosal and plasma samples from HIV-1 infected individuals inhibit the transfer of HIV-1 R5 primary isolates from DC to T cells, in contrast, EUI seem to lack this ability. Finally, we have described the expression of a number of innate immune molecules with proposed anti-HIV-1 activity at the single cell level in the cervical tissue of FSWs at risk of HIV-1 infection. We detected a higher expression of IFNα and RANTES in these women as compared to low-risk HIV-1 uninfected controls. The conclusions we draw from our studies are; i) HIV neutralizing IgA in the genital tract may indeed protect individuals from subsequent sexual HIV acquisition, ii) preexisting IFNa and RANTES in cervical mucosa may contribute to protection of sexual HIV transmission in subjects with higher risk-behaviors, iii) due to previous encounter with HIV, mucosal tissues of EUI may contain pre-existing and-carbohydrate antibodies and/or a triggered innate immunity, and iv) EUI may block HIV infection at an earlier phase of viral transmission through the genital mucosa, before DC-T cell interaction. Our studies on the lower female genital tract of groups with different HIV-1 encountering risk behaviors will hopefully elevate our knowledge about potential microbicide targeting sites and have clear implications for HIV vaccine development

HIV-binding CLRs and CCR5 are expressed in the human endocervix.

<p>a: Langerin+, Scale bar: 100 µm. b: CD11c+ (red), Langerin+ (green), CD11c+ Langerin+ (yellow), Scale bar: 25 µm. c: CD4+ (red), Langerin+ (green), CD4+ Langerin+ (yellow), Scale bar: 25 µm. d: MR+, Scale bar: 100 µm. e: DC-SIGN+, Scale bar: 100 µm. f: MR+ (red),CD68+ (green), MR+CD68+ (yellow), Scale bar: 25 µm. g: DC-SIGN+ (red), CD68+ (green), DC-SIGN+CD68+ (yellow), Scale bar: 25 µm. h: MR+ (red), CD11c+ (green), MR+CD11c+ (yellow), Scale bar: 25 µm. i: DC-SIGN+ (red), CD11c+ (green), DC-SIGN+CD11c+ (yellow), Scale bar: 25 µm. j: CD1a+ (green), CCR5+ (red), Scale bar: 25 µm. k: CD4+ (green), CCR5+ (red), CD4+CCR5+ (yellow), Scale bar: 25 µm. l: CD11c+ (green), CCR5+ (red), CD11c+CCR5+ (yellow), Scale bar: 100 µm. Arrows indicate single positive cells; arrowheads indicate double positive cells.</p

CD4 expression is present on T cells and antigen-presenting cell subsets.

<p>a: CD4+ (red),CD3+ (green), CD4+CD3+ (yellow), I: 3 µm, Scale bar: 25 µm. b: CD4+ (red), CD1a+ (green), CD4+CD1a+ (yellow), I: 24 µm, Scale bar: 25 µm. c: CD4+ (red), CD11c+ (green), CD4+CD11c+ (yellow), Scale bar: 25 µm. d: CD11c+ (red), CD1a (green), CD11c+CD1a+ (yellow), Scale bar: 25 µm. e: CD123+ (red), CD4+ (green,) CD123+CD4+ (yellow), Scale bar: 25 µm. f: CD123+ (red), BDCA-2+ (green,) CD123+BDCA-2+ (yellow), Scale bar: 25 µm. Arrows indicate single positive cells; arrowheads indicate double positive cells.</p