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

CD49a Expression Defines Tissue-Resident CD8+ T Cells Poised for Cytotoxic Function in Human Skin

Tissue-resident memory T (Trm) cells form a heterogeneous population that provides localized protection against pathogens. Here, we identify CD49a as a marker that differentiates CD8(+) Trm cells on a compartmental and functional basis. In human skin epithelia, CD8(+)CD49a(+) Trm cells produced interferon-γ, whereas CD8(+)CD49a(−) Trm cells produced interleukin-17 (IL-17). In addition, CD8(+)CD49a(+) Trm cells from healthy skin rapidly induced the expression of the effector molecules perforin and granzyme B when stimulated with IL-15, thereby promoting a strong cytotoxic response. In skin from patients with vitiligo, where melanocytes are eradicated locally, CD8(+)CD49a(+) Trm cells that constitutively expressed perforin and granzyme B accumulated both in the epidermis and dermis. Conversely, CD8(+)CD49a(–) Trm cells from psoriasis lesions predominantly generated IL-17 responses that promote local inflammation in this skin disease. Overall, CD49a expression delineates CD8(+) Trm cell specialization in human epithelial barriers and correlates with the effector cell balance found in distinct inflammatory skin diseases

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

Monocyte-derived dendritic cells express and secrete matrix-degrading metalloproteinases and their inhibitors and are imbalanced in multiple sclerosis

Dendritic cells (DC) are antigen-presenting cells (APC) that most efficiently initiate and control immune responses. Migration processes of blood DC are crucial to exert their professional antigen-presenting functions. Matrix-degrading metalloproteinases (MMP) are proteolytic enzymes, which are considered to be key enzymes in extracellular matrix (ECM) turnover and mediators of cell migration. Tissue inhibitors of metalloproteinases (TIMP) are important regulators of MMP activity. Here we investigate whether blood monocyte-derived immature DC (iDC) and mature DC (mDC) express, produce and secrete functionally active MMP-1, -2, -3 and -9 and their inhibitors TIMP-1 and -2, and examine their involvement in multiple sclerosis (MS). On mRNA level, we observed high numbers of MMP-2 and TIMP-2 mRNA expressing iDC in MS. On protein level, high percentages of MMP-1, -2 and -9 expressing iDC by flow cytometry, and high MMP-1 secretion by Western blot together with high MMP-2 and -9 activities in iDC supernatants as studied with zymography were observed. Similarly, MS is associated with high percentages of MMP-2 and -3 and of TIMP-1 expressing mDC by flow cytometry together with high MMP-3 secretion and high MMP-9 activity in culture supernatants. Spontaneous migratory capacity of both iDC and mDC over ECM-coated filters was higher in MS compared to healthy controls (HC). In conclusion, blood monocyte-derived iDC and mDC express, produce and secrete several MMP and TIMP. Alterations in these molecules as observed in MS may be functionally important for DC functioning

Correction: Seminal plasma induces inflammation and enhances HIV-1 replication in human cervical tissue explants.

[This corrects the article DOI: 10.1371/journal.ppat.1006402.]

Seminal plasma induces inflammation and enhances HIV-1 replication in human cervical tissue explants

<div><p>The most immediate and evident effect of mucosal exposure to semen <i>in vivo</i> is a local release of proinflammatory mediators accompanied by an influx of leukocytes into the female genital mucosa (FGM). The implication of such response in HIV-1 transmission has never been addressed due to limitations of currently available experimental models. Using human tissue explants from the uterine cervix, we developed a system of mucosal exposure to seminal plasma (SP) that supports HIV-1 replication. Treatment of ectocervical explants with SP resulted in the upregulation of inflammatory and growth factors, including IL-6, TNF, CCL5, CCL20, CXCL1, and CXCL8, and <i>IL1A</i>, <i>CSF2</i>, <i>IL7</i>, <i>PTGS2</i>, as evaluated by measuring protein levels in explant conditioned medium (ECM) and gene expression in tissue. SP treatment was also associated with increased recruitment of monocytes and neutrophils, as observed upon incubation of peripheral blood leukocytes with ECM in a transwell system. To evaluate the impact of the SP-mediated response on local susceptibility to HIV-1, we infected ectocervical explants with the CCR5-tropic variant HIV-1_BaL either in the presence of SP, or after explant pre-incubation with SP. In both experimental settings SP enhanced virus replication as evaluated by HIV-1 p24_gag released in explant culture medium over time, as well as by HIV-1 DNA quantification in explants infected in the presence of SP. These results suggest that a sustained inflammatory response elicited by SP soon after coitus may promote HIV-1 transmission to the FGM. Nevertheless, ectocervical tissue explants did not support the replication of transmitted/founder HIV-1 molecular clones, regardless of SP treatment. Our system offers experimental and analytical advantages over traditional models of HIV-1 transmission for the study of SP immunoregulatory effect on the FGM, and may provide a useful platform to ultimately identify new determinants of HIV-1 infection at this site.</p></div

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