Selection of T-cell receptors with a recurrent CDR3β peptide-contact motif within the repertoire of alloreactive CD8(+) T cells.

International audiencePeptide/MHC complexes recognized by alloreactive T lymphocytes (TLs) have been identified, but their contribution to in vivo allo-rejection is not known. We previously characterized the peptide pBM1, highly represented among endogenous H-2K(b) (K(b) )-associated peptides and critically required to induce full activation of H-2(k) monoclonal CD8(+) TLs expressing the cognate TCR-BM3.3. Here, we asked whether a pBM1/K(b) -specific TL subset could be detected within a polyclonal TL population rejecting allogeneic cells in vivo. We show that the proportion of pBM1/K(b) -binding CD8(+) TLs increased from <0.04% in naïve mice to 3% of activated CD44(+) CD8(+) TLs in H-2(k) mice rejecting K(b) -expressing cells. Among these, TCR-Vβ2 usage was greatly enriched, and 75% of them shared a TCR-Vβ2 CDR3β motif with the prototype TCR-BM3.3. Fewer than 5% of K(b) -reactive CD44(+) CD8(+) TLs not binding pBM1/K(b) displayed this CDR3β motif. We found that the recurrent CDR3β motif of pBM1/K(b) -binding TLs was assembled from distinct V/D/J recombination events, suggesting that it is recruited upon immunization for its optimal TCR-peptide/MHC fit. Thus, a CDR3β motif generated by a process akin to "convergent recombination" accounts for a sizable fraction of the alloreactive anti-K(b) TCR repertoire

Cleavage/alteration of interleukin-8 by matrix metalloproteinase-9 in the female lower genital tract.

OBJECTIVE:Interleukin-8 (IL-8, CXCL8) plays important roles in immune responses at mucosal sites including in the lower genital tract. Since several types of bacteria produce proteases that cleave IL-8 and many types of bacteria can be present in lower genital tract microbiota, we assessed genital fluids for IL-8 cleavage/alteration. STUDY DESIGN:Genital fluids collected by lavage from 200 women (23 HIV-seronegative and 177 HIV-seropositive) were tested for IL-8 cleavage/alteration by ELISA. RESULTS:IL-8 cleaving/altering activity was observed in fluids from both HIV-positive (28%) and HIV-negative women (35%). There was no clear relationship between the activity and the types of bacteria present in the lower genital tract as determined by high-throughput sequencing of the 16S rRNA gene. Protease inhibitors specific for matrix metalloproteinases (MMPs) reduced the activity and a multiplex assay that detects both inactive and active MMPs showed the presence of multiple MMPs, including MMP-1, -3, -7, -8, -9, -10 and -12 in genital secretions from many of the women. The IL-8-cleaving/altering activity significantly correlated with active MMP-9 as well as with cleavage of a substrate that is acted on by several active MMPs. CONCLUSIONS:These studies show that multiple MMPs are present in the genital tract of women and strongly suggest that MMP-9 in genital secretions can cleave IL-8 at this mucosal site. These studies suggest that MMP-mediated cleavage of IL-8 can modulate inflammatory responses in the lower genital tract

Total MMP levels in mucosal fluid samples.

<p>The concentration of MMPs 1, 3, 7, 8, 9, 10 and 12 were measured by Luminex immunoassay in mucosal fluid samples. 22 samples positive for IL-8 cleavage (square symbols) and 5 samples negative for IL-8 cleavage (x symbols) were evaluated.</p

Relationship of active MMPs and IL-8 cleavage.

<p>A. The amount of MMP activity in genital fluids 22 samples positive for IL-8 cleavage and 5 negative for IL-8 cleavage was measured using a fluorogenic substrate and plotted against the % cleavage of IL-8 for each sample. B. The amount of active MMP-9 in the 27 samples was plotted against the % cleavage of IL-8 for each sample.</p

The effect of protease inhibitors on the IL-8-cleaving activity in genital tract fluid.

<p>A. Two samples positive for IL-8 cleavage (samples 26 and 56) were incubated with recombinant IL-8 for 20 h at 37°C in the presence or absence (No I) of EDTA or a cocktail of protease inhibitors (general protease inhibitor, GPI). B. MMP inhibitors Marimastat and C471474 were tested. For both A and B, control is IL-8 incubated without genital fluid or inhibitors. Samples were analyzed at 20 h by IL-8 ELISA. Shown are the mean values ± SD. * p<0.05, **p<0.1, t test compared to no inhibitor.</p

Cleavage of IL-8 in genital mucosal fluids.

<p>A. Recombinant IL-8 was added to either saline (negative control) or genital fluids (diluted 1:4) from 10 HIV-seropositive subjects and incubated at either 4°C or 37°C for 20 h. Levels of IL-8 were then measured by ELISA. The mean ± SD are shown. B. Recombinant IL-8 was added to genital fluid from subject 26 diluted 1:4 or 1:40 and incubated for 4 or 24 h before detection by ELISA.</p

Comparison of Lower Genital Tract Microbiota in HIV-Infected and Uninfected Women from Rwanda and the US

<div><p>Introduction</p><p>Previous studies have shown that alterations of the bacterial microbiota in the lower female genital tract influence susceptibility to HIV infection and shedding. We assessed geographic differences in types of genital microbiota between HIV-infected and uninfected women from Rwanda and the United States.</p><p>Methods</p><p>Genera of lower genital tract bacterial microbiota were identified by high-throughput pyrosequencing of the 16S rRNA gene from 46 US women (36 HIV-infected, 10 HIV-uninfected) and 40 Rwandan women (18 HIV-infected, 22 HIV-uninfected) with similar proportions of low (0–3) Nugent scores. Species of <i>Lactobacillus</i> were identified by assembling sequences along with reference sequences into phylogenetic trees. Prevalence of genera and <i>Lactobacillus</i> species were compared using Fisher's exact tests.</p><p>Results</p><p>Overall the seven most prevalent genera were <i>Lactobacillus</i> (74%), <i>Prevotella</i> (56%), <i>Gardnerella</i> (55%), <i>Atopobium</i> (42%), <i>Sneathia</i> (37%), Megasphaera (30%), and <i>Parvimonas</i> (26%), observed at similar prevalences comparing Rwandan to US women, except for <i>Megasphaera</i> (20% vs. 39%, p = 0.06). Additionally, Rwandan women had higher frequencies of <i>Mycoplasma</i> (23% vs. 7%, p = 0.06) and <i>Eggerthella</i> (13% vs. 0%, p = 0.02), and lower frequencies of <i>Lachnobacterium</i> (8% vs. 35%, p<0.01) and <i>Allisonella</i> (5% vs. 30%, p<0.01), compared with US women. The prevalence of <i>Mycoplasma</i> was highest (p<0.05) in HIV-infected Rwandan women (39%), compared to HIV-infected US women (6%), HIV-uninfected Rwandan (9%) and US (10%) women. The most prevalent lactobacillus species in both Rwandan and US women was <i>L. iners</i> (58% vs. 76%, p = 0.11), followed by <i>L. crispatus</i> (28% vs. 30%, p = 0.82), <i>L. jensenii</i> (20% vs. 24%, p = 0.80), <i>L. gasseri</i> (20% vs. 11%, p = 0.37) and <i>L. vaginalis</i> (20% vs. 7%, p = 0.10).</p><p>Discussion</p><p>We found similar prevalence of most major bacterial genera and <i>Lactobacillus</i> species in Rwandan and US women. Further work will be needed to establish whether observed differences differentially impact lower genital tract health or susceptibility to genital infections.</p></div

Comparison of the most prevalent genera among women with genital microbiota having 0–49% of sequences corresponding to <i>Lactobacillus</i>.

†<p>Percentage of women with genital microbiota having ≥1% of sequences corresponding to each genus listed. All genera with prevalence ≥19% in at least one group were included.</p><p>*Reported <i>P</i> values were obtained from Fisher's exact tests (only <i>P</i> values <0.10 are shown).</p

Study selection and analytic design.

<p>Study selection and analytic design.</p

Comparison of demographic and behavioral characteristics.

<p>Note: %, column percent; Med, median; IQR, interquartile range; Contractual sex, exchanged sex for money, drugs, or shelter; STD, sexually transmitted disease; PID, pelvic inflammatory disease.</p>†<p>Low: 0–49% of sequences correspond to <i>Lactobacillus</i>; high: ≥50% of sequences correspond to <i>Lactobacillus</i>.</p>‡<p>Reported by participant at baseline visit.</p><p>*Reported <i>P</i> values were obtained from Fisher's exact tests for categorical variables and from Mann-Whitney <i>U</i> tests for co.</p