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 detection of Gag-specific CD8+ cells in the GI tract of SIV infected Rhesus macaques

<p>Abstract</p> <p>Background</p> <p>SIV and HIV predominantly replicate in lymphoid tissue, but the study of virus specific CD8⁺T cells in intact lymphoid tissue is difficult, as traditional <it>in situ </it>tetramer staining requires fresh tissue.</p> <p>Results</p> <p>In this report, we demonstrate a novel technique using Qdot 655-conjugated peptide-MHC multimers to directly visualize SIV specific cells in cryopreserved tissue biopsies from chronically SIVmac239 infected Rhesus macaques. Qdot 655 multimers showed similar sensitivity and specificity to APC-conjugated tetramers by flow cytometry analysis, but yielded ten-fold higher signal intensity when imaged by fluorescence microscopy. Using this technique, we detected CD8⁺T cells which recognize an immunodominant epitope (Gag CM9) in the spleen, lymph nodes, ileum and colon. In all these tissues, the Gag CM9 positive cells were mainly located in the extra follicular T cell zone. In the ileum and colon, we found Gag CM9 positive cells concentrated in Peyer's patches and solitary lymphoid follicles, a pattern of localization not previously described.</p> <p>Conclusions</p> <p>The use of Qdot multimers provide an anatomic and quantitative evaluation of SIV specific CD8⁺T cell responses in SIV pathogenesis, and may prove useful to studies of SIV specific CD8⁺T cell responses elicited by vaccines and other immunotherapies in the non-human primate model.</p

Leukemia inhibitor factor (LIF) and gp130 in early defence against HIV-1 infection

Leukemia inhibitor factor (LIF) is a polyfunctional cytokine that belongs to the IL-6 family which mainly signals through the Jak/Stat pathway via the gp130/LIFR-á heterodimer. The focus of my research has been to investigate and understand if and how LIF exerts HIV1 suppressing activity. We therefore examined the expression of LIF, and its receptors (gp130 and LIFR-á) in lymphoid tissue biopsies from primary HIV-1 infected (PHI), chronic HIV-1 infected (cHI) individuals and from long term non progressors (LTNP). Furthermore, consecutively obtained HIV-plasma samples collected from PHI individuals were analysed for LIF and soluble gp130 levels. Our data showed that LIF is one of the mediators of the innate immune response during HIV-1 infection. High production of LIF and gp130 were detected both in lymphoid tissue and in plasma from individuals with primary HIV-1 infection. Assesment of LIF plasma levels at PHI did not predict low levels of HIV-1 viremia after discontinuation of anti-retroviral treatment. However, a positive correlation between levels of plasma HIV-1 viral load and the production of LIF in lymphoid tissue or in plasma was found. In addition, a positive correlation between plasma levels of HIV-1 RNA and IFNá, TNF-á, IL-1â, MIP-1á and MIP-1â were found in plasma from HIV-1 infected individuals that were in the primary phase of infection. After cessation of antiretroviral treatment the levels of cytokines, including LIF, and chemokines were reduced as compared to the levels seen during primary HIV-1 infection. HIV-1 infected individuals that controlled their infection after cessation of treatment showed higher plasma levels of IFN-ã and MIP-1â as compared to individuals that did not control their HIV-1 infection. This suggests that during primary HIV-1 infection there is not a lack of a certain immune mediator that leads to immune failure, it is more like "too much and too many". However, individuals that do control the infection appear to have a recall response to the virus, since they produce IFN-ã and MIP-10 which are suggested to be beneficial for the host to be abel to control the HIV-1 replication. We also found that even though more than 50% of the total CD4+ cells in lymphoid tissue expressed gp130, less than 5% of the total HIV-1 positive replicating cells (p24+) in lymphoid tissue were gp130+. Thus, LIF mediated a certain amount of control in CD4+gp130+ cells in lymphoid tissue. In addition, treatment of cMAGI cells with LIF prior to HIV-1 infection resulted in a dose dependent reduction in HIV-1 infected cells compared to untreated cells. Furthermore, both LIF and HIV-1 induced phosphorylation of Stat 3, and LIF pre-treatment resulted in a down modulation of the HIV-1 mediated Stat activation. Additionally, Jak/Stat inhibitors as well as siRNA against Stat 3 reduced HIV-1 replication. We suggest that the Jak/Stat pathway is important for HIV-1 replication and that LIF likely interferes with it. In conclusion, LIF like many other cytokine and chemokines, is bifunctional since it has both HIV1 suppressive action if present prior to HIV-1 infection, and HIV-1 enhancing activity if present after established HIV-1 infection

Infection of ectocervical tissue explants with HIV-1_BaL.

<p>Infection of ectocervical explants with HIV-1_BaL was independently performed after seminal plasma (SP) 25% treatment (post-SP,red), or in the presence of SP25% (SP-mix, blue) (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006402#ppat.1006402.s001" target="_blank">S1B Fig</a>). In selected experiments, explants infected in the presence of SP were treated with lamivudine (3TC) 10μM throughout culture time. <b>A)</b> Kinetics of HIV-1_BaL replication in explants treated with SP (colored line) or culture medium (CM) (black line). Virus replication was evaluated as p24_gag concentration in explant culture medium over 18 days. Represented are mean values with s.e.m. (n = 9 for post-SP; n = 8 for SP-mix; n = 3 for SP-mix+3TC). <b>B)</b> Cumulative p24_gag production over culture time. Lines connect measurements obtained from donor-matched explants. <b>C)</b> N-fold change in cumulative p24_gag production in SP-treated explants compared to donor-matched untreated explants. Bars indicate median values. p<0.05 denotes a significant difference with CM (Wilcoxon signed rank test). <b>D)</b> HIV-1 DNA quantification in explants infected with a mix of HIV and SP or CM, cultured in the presence or the absence of 3TC, and harvested at the end of culture (day18). HIV-1 DNA copy numbers were normalized to the amount of the single-copy gene <i>HBB</i>. Lines connect measurements obtained from donor-matched explants. <b>E)</b> N-fold change in HIV-1 DNA copy numbers in SP-treated explants compared to donor-matched untreated explants. The bar indicates median value. p<0.05 denotes a significant difference with CM (Wilcoxon signed rank test).</p

Transwell migration of peripheral blood leukocytes (PBL).

<p>Mononuclear cells and granulocytes were isolated from blood, pooled (i.e. PBL) and incubated in a transwell system for 2 h with explant conditioned medium (ECM) from donor-matched ectocervical explants incubated with culture medium (CM-ECM) or seminal plasma (SP-ECM). Transmigrated cells were immunophenotyped and enumerated by flow cytometry (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006402#ppat.1006402.s005" target="_blank">S5 Fig</a>). <b>A)</b> Fraction of transmigrated cells out of total number of PBL loaded into a transwell insert for each analyzed cell population (input). PBL were incubated with CM-ECM (white), SP-ECM (red), and with medium only (grey) and medium supplemented with FBS 10% (yellow) as negative and positive controls respectively. Bars represent mean with s.e.m (n = 6). <b>B)</b> N-fold change in the fraction of transmigrated PBL incubated with SP-ECM compared to that of donor-matched CM-ECM. Bars indicate median values. p<0.05 denotes a significant difference with CM (Wilcoxon signed rank test). <b>C)</b> Expression levels of the chemokine receptor CCR5 on transmigrated monocytes. Top, peaks represent PBL untreated cultured (ctrl, gray), cultured with CM-ECM (CM, white), cultured with SP-ECM (SP, red), and unstained control (black) from one representative experiment. Middle, CCR5 mean fluorescence intensity (MFI). Bars indicate median values. Bottom, n-fold change in CCR5 MFI on PBL cultured with ECM compared to untreated cultured PBL (ctrl). Lines connect measurements obtained from donor-matched ECM. p<0.05 denotes a significant difference with ctrl (Wilcoxon signed rank test).</p

Infection of ectocervical tissue explants with transmitted/founder (T/F) HIV-1.

<p>Infection of ectocervical explants with pCH077.t/2627 (pCH077), pRHPA.c/2635 (pRHPA), and pTHRO.c/2626 (pTHRO) was performed after an initial treatment with seminal plasma (SP) 25% (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006402#ppat.1006402.s001" target="_blank">S1B Fig</a>). <b>A-C)</b> Kinetics of virus replication in explants treated with SP (colored line) or culture medium (CM) (black line). Virus replication was evaluated as p24_gag concentration in explant culture medium over 18 days. Represented are mean values with s.e.m. (n = 3 for pCH077; n = 3 for pRHPA; n = 4 for pTHRO). <b>D-E)</b> HIV-1 DNA copy numbers in infected explants harvested at the end of culture (day18). Values were normalized to the amount of the single-copy gene <i>HBB</i>. <b>E)</b> Donor-matched explants were independently infected with HIV-1_BaL and at least one T/F virus (n = 3). Donor-matched explants are indicated with the same symbol. <b>F)</b> Exogenously activated PBMCs were infected with T/F HIV-1 produced in 293T cells. HIV-1 replication was evaluated as p24_gag concentration in culture supernatant (red) and HIV-1 DNA copy numbers (black) in cells harvested at the end of culture (day 8–10 post-infection) (n = 1).</p

Cytokine concentration in ectocervical tissue explant conditioned medium (ECM).

<p><b>A)</b> Cytokine concentration (pg/ml) was measured in ECM of ectocervical explants incubated with culture medium (CM), seminal plasma (SP) 25% or SP50% in the presence or absence of indomethacin (indo) 10μM for 4 h, followed by an additional 12 h-incubation with medium only. Bars represent median with interquartile range (IQR). + excluded from the analysis (TGF-β1). <b>B)</b> N-fold change in ECM cytokine concentration of explants treated with SP and/or indomethacin for 4 or 12 h, compared to donor-matched explants incubated with CM. Bars indicate median values. Asterisks denote a statistically significant difference with CM (Wilcoxon signed rank test, p<0.05). CM (white, n = 11), CM+indomethacin (gray, n = 7), SP25% (orange, n = 7), SP50% (red, n = 11), and SP50%+indomethacin (brown, n = 7).</p