Diversity of cwp loci in clinical isolates of Clostridium difficile.

An increased incidence of Clostridium difficile infection (CDI) is associated with the emergence of epidemic strains characterized by high genetic diversity. Among the factors that may have a role in CDI is a family of 29 paralogues, the cell-wall proteins (CWPs), which compose the outer layer of the bacterial cell and are likely to be involved in colonization. Previous studies have shown that 12 of the 29 cwp genes are clustered in the same region, named after slpA (cwp1), the slpA locus, whereas the remaining 17 paralogues are distributed throughout the genome. The variability of 14 of these 17 cwp paralogues was determined in 40 C. difficile clinical isolates belonging to six of the currently prevailing PCR ribotypes. Based on sequence conservation, these cwp genes were divided into two groups, one comprising nine cwp loci having highly conserved sequences in all isolates, and the other five loci showing low genetic conservation among isolates of the same PCR ribotype, as well as between different PCR ribotypes. Three conserved CWPs, Cwp16, Cwp18 and Cwp25, and two variable ones, Cwp26 and Cwp27, were characterized further by Western blot analysis of total cell extracts or surface-layer preparations of the C. difficile clinical isolates. Expression of genetically invariable CWPs was well conserved in all isolates, whilst genetically variable CWPs were not always expressed at comparable levels, even in strains containing identical sequences but belonging to different PCR ribotypes. This is the first report on the distribution and variability of a number of genes encoding CWPs in C. difficile

Vaccine adjuvant MF59 promotes the intranodal differentiation of antigen-loaded and activated monocyte-derived dendritic cells

MF59 is an oil-in-water emulsion adjuvant approved for human influenza vaccination in European Union. The mode of action of MF59 is not fully elucidated yet, but results from several years of investigation indicate that MF59 establishes an immunocompetent environment at injection site which promotes recruitment of immune cells, including antigen presenting cells (APCs), that are facilitated to engulf antigen and transport it to draining lymph node (dLN) where the antigen is accumulated. In vitro studies showed that MF59 promotes the differentiation of monocytes to dendritic cells (Mo-DCs). Since after immunization with MF59, monocytes are rapidly recruited both at the injection site and in dLN and appear to have a morphological change toward a DC-like phenotype, we asked whether MF59 could play a role in inducing differentiation of Mo-DC in vivo. To address this question we immunized mice with the auto-fluorescent protein Phycoerythrin (PE) as model antigen, in presence or absence of MF59. We measured the APC phenotype and their antigen uptake within dLNs, the antigen distribution within the dLN compartments and the humoral response to PE. In addition, using Ovalbumin as model antigen, we measured the capacity of dLN APCs to induce antigen-specific CD4 T cell proliferation. Here, we show, for the first time, that MF59 promotes differentiation of Mo-DCs within dLNs from intranodal recruited monocytes and we suggest that this differentiation could take place in the medullary compartment of the LN. In addition we show that the Mo-DC subset represents the major source of antigen-loaded and activated APCs within the dLN when immunizing with MF59. Interestingly, this finding correlates with the enhanced triggering of antigen-specific CD4 T cell response induced by LN APCs. This study therefore demonstrates that MF59 is able to promote an immunocompetent environment also directly within the dLN, offering a novel insight on the mechanism of action of vaccine adjuvants based on emulsions

Immuno-Contexture and Immune Checkpoint Molecule Expression in Mismatch Repair Proficient Colorectal Carcinoma

Colorectal carcinoma (CRC) represents a lethal disease with heterogeneous outcomes. Only patients with mismatch repair (MMR) deficient CRC showing microsatellite instability and hyper-mutated tumors can obtain clinical benefits from current immune checkpoint blockades; on the other hand, immune- or target-based therapeutic strategies are very limited for subjects with mismatch repair proficient CRC (CRCpMMR). Here, we report a comprehensive typing of immune infiltrating cells in CRCpMMR. We also tested the expression and interferon-γ-modulation of PD-L1/CD274. Relevant findings were subsequently validated by immunohistochemistry on fixed materials. CRCpMMR contain a significantly increased fraction of CD163+ macrophages (TAMs) expressing TREM2 and CD66+ neutrophils (TANs) together with decrease in CD4−CD8−CD3+ double negative T lymphocytes (DNTs); no differences were revealed by the analysis of conventional and plasmacytoid dendritic cell populations. A fraction of tumor-infiltrating T-cells displays an exhausted phenotype, co-expressing PD-1 and TIM-3. Remarkably, expression of PD-L1 on fresh tumor cells and TAMs was undetectable even after in vitro stimulation with interferon-γ. These findings confirm the immune suppressive microenvironment of CRCpMMR characterized by dense infiltration of TAMs, occurrence of TANs, lack of DNTs, T-cell exhaustion, and interferon-γ unresponsiveness by host and tumor cells. Appropriate bypass strategies should consider these combinations of immune escape mechanisms in CRCpMMR

MF59 promotes differentiation and transient accumulation of Mo-DCs within the dLN.

<p>(<b>a</b>) Left panel: flow cytometry dot plots of dLN cell suspensions to identify APCs are reported for each time point and each treatment (as indicated). Macrophages (MΦ) are depicted in blue, whereas the rest of the APCs are in black. The transient appearance of a CD8α_F4/80 double positive APC subset (red gates) between 8 hrs and 18 hrs after immunization (red quadrant) is shown. Representative results of one experiment out of three are shown. Right panel: bar graph histogram that reports the number x10³ (per million of live/singlet cells acquired by flow cytometry) of CD8α_F4/80 double positive APCs at 8 hrs and 18 hrs after immunization with PE (white bars) and PE + MF59 (grey bars). Data from three independent experiments (single numbered bars) and the arithmetic mean of these values (black horizontal line) are shown. Statistical analysis: parametric one-tailed T-test was used to calculate the P-value. **<i>P</i><0.01. (<b>b</b>) Flow cytometry dot plots of dLN cell suspensions obtained 18 hrs after immunization with PE or PE + MF59 are shown to analyze the expression of the surface protein CD64 on the different APC subsets. CD8α_F4/80 double positive APCs are depicted red, LN DCs in green and MΦs in blue. Representative results of one experiment out of the three independent experiments reported in panel (a) are shown. (<b>c</b>) Flow cytometry dot plots of dLN cell suspensions obtained 8 hrs and 18 hrs after immunization with MF59 reveal that the CD8α_F4/80 double positive APC subset (red cells) expresses the CD11c DC marker 18 hrs but not 8 hrs after the immunization. Representative results of one experiment out of the three reported in panel (a) are shown. (<b>d</b>) Flow cytometry dot plots of the dLN cell suspensions to identify APCs derived from mice immunized with MF59, whose LNs were explanted from both legs 8 hrs after the immunization, but: the LNs from the left legs (left dot plots) were immediately processed (8 hrs), whereas the LNs from the right legs (right dot plots) were processed after 10 hrs of in vitro culture (8 hrs + 10 hrs (in culture)). Representative results of one experiment out of three are shown.</p

Mo-DCs are the major source of antigen-loaded and activated APCs within the dLN when immunizing with MF59.

<p>Popliteal dLN cell suspensions from mice immunized with PE or PE + MF59 analyzed by flow cytometry 18 hrs after immunization. (<b>a</b>) Upper panels: fow cytometry dot plots that show the uptake of PE and MF59 by MΦs (blue cells), DCs (green cells) and monocytes or Mo-DCs (red cells) from one representative experiment in mice immunized with PE or PE + fluorescently labelled-MF59 are reported as example. Middle and lower panels: bar graph histograms report the percentage (middle panels) and the number (per million of live/singlet cells acquired by flow cytometry) (lower panels) of PE positive cells in MΦs (blue bars), DCs (green bars) and monocytes or Mo-DCs (red bars). Data from three independent experiments (single numbered bars) and the arithmetic mean of these values (black horizontal line) are shown. Statistical analysis: parametric one–way ANOVA test (Dunnett’s multiple comparison using Mo-DCs as control column) has been applied to calculate the P-value.<i>*P</i>˂0.05; <i>***P</i>˂0.001; <i>****P</i>˂ 0.0001. (<b>b</b>) Bar graph histogram reports the average (+ standard deviation) number (per million of live/singlet cells acquired by flow cytometry) of PE positive MΦs (blue bars), DCs (green bars) and Mo-DCs vs. monocytes (red bars) in mice immunized with PE (dotted bars) or PE + MF59 (filled bars) in the three independent experiments reported in panel (a). Statistical analysis: parametric one-tailed T-test was used to calculate the P-value. <i>*P</i>˂0.05; **<i>P</i><0.01. (<b>c</b>) Upper panels: flow cytometry histograms of CD80 expression by MΦs (blue), DCs (green) and Mo-DCs vs. monocytes (red) in mice immunized with PE (thin lines) or PE + MF59 (thick lines) from one experiment out of the three shown in panel (a). Lower panel: bar graph histogram shows the difference in CD80 Mean Fluorescence Intensity (MFI) of MΦs (blue bars), DCs (green bars) and Mo-DCs vs. monocytes (red bars) from mice PE immunized with and without MF59 (ΔMFI). Data of the same three independent experiments reported in panel (a) (single numbered bars) and the arithmetic mean of these values (black horizontal line), are shown. Statistical analysis: parametric one-tailed T-test was used to calculate the P-value. <i>**P</i>˂0.01.</p

Co-localization of antigen, MF59 and DCs within the medullary compartment of the dLN.

<p>(<b>a</b>) Confocal microscopy images of dLNs collected from mice 18 hrs after treatment with PBS, PE or PE + MF59 and labeled to detect DCs and the medullary compartment of the LN. Signals which detect the LN medulla (blue, F4/80), PE (red) and DCs (yellow, CD11c) are shown separately and merged (as indicated). The magnification shows the co-localization of antigen and DCs within the LN medulla. The image of one section is shown, in each panel, as example of consecutive sections of a whole LN, which is representative of the organs of all immunized mice. Original magnification: 5X. Scale bar: 1 mm. Results of one representative experiment out of three are reported. (<b>b</b>) Confocal microscopy images of dLNs collected from mice 18 hrs after treatment with PE + fluorescently labelled-MF59 and labeled to detect DCs and the medullary compartment of the LN. Signals which detect the LN medulla (blue), MF59 (green), PE (red) and DCs (yellow) are shown separately and merged (as indicated). The magnification shows the co-localization of antigen, MF59 and DCs within the LN medulla. The image of one section is shown, in each panel, as example of consecutive sections of a whole LN, which is representative of the organs of all immunized mice. Original magnification: 5X. Scale bar: 1 mm. Representative results of one representative experiment out of two are reported.</p