[Show abstract][Hide abstract] ABSTRACT: Tumors with reduced expression of MHC class I (MHC-I) molecules may be unrecognized by tumor antigen-specific CD8(+) T cells and thus constitute a challenge for cancer immunotherapy. Here we monitored development of autochthonous melanomas in TiRP mice that develop tumors expressing a known tumor antigen as well as a red fluorescent protein (RFP) reporter knock in gene. The latter permits non-invasive monitoring of tumor growth by biofluorescence. One developing melanoma was deficient in cell surface expression of MHC-I, but MHC-I expression could be rescued by exposure of these cells to IFNγ. We show that CD8(+) T cells specific for tumor antigen/MHC-I were efficient at inducing regression of the MHC-I-deficient melanoma, provided that the T cells were endowed with properties permitting their migration into the tumor and their efficient production of IFNγ. This was the case for CD8(+) T cells transfected to express an active form of STAT5 (STAT5CA). The amount of IFNγ produced ex vivo from T cells present in tumors after adoptive transfer of the CD8(+) T cells was correlated with an increase in surface expression of MHC-I molecules by the tumor cells. We also show that these CD8(+) T cells expressed PD-1 and upregulated its ligand PDL-1 on melanoma cells within the tumor. Despite upregulation of this immunosuppressive pathway, efficient IFNγ production in the melanoma microenvironment was found associated with resistance of STAT5CA-expressing CD8(+) T cells to inhibition both by PD-1/PDL-1 engagement and by TGFβ1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients.
[Show abstract][Hide abstract] ABSTRACT: B. melitensis CβG induces cellular immunity in vivo. CD8+ Ly5.2 CFSE+ T cells were transferred intravenously (i.v.) into naive congenic C57Bl/6 Ly5.1 recipient mice. 24 h after OT-I adoptive transfer, recipient mice were immunized subcutaneously (s.c.) either with PBS (grey) or 30 µg OVA in PBS or 30 µg OVA mixed with 200 µg of CβG or 30 µg OVA mixed with 50 µg Poly I:C or 30 µg OVA mixed with 20 µg MPLA. At day 6 post-immunization, the OVA-specific OT-I T cell activation in the draining popliteal lymph nodes of immunized mice was investigated by analyzing the up-regulation of CD25 and the down-regulation of CD62L by flow cytometry. The median fluorescence for each marker is indicated under histograms. Endogenous CD8+ T cell population (in grey). Data are representative of one experiment among three different experiments.
[Show abstract][Hide abstract] ABSTRACT: MALDI-TOF mass spectrometry analysis of CβG preparations. The spectra show six main signals at m/z 2,795, 2,957, 3,119, 3,268, 3,444, and 3,607 consistent with those expected for CβG molecules composed of 17–22 glucose residues, as previously described. (A) Positive-ion MALDI-TOF MS. (B) Negative-ion MALDI-TOF MS. Mark the absence of the ion species at m/z 2073, 2145, 2173 that are characteristic of Brucella lipid A.
[Show abstract][Hide abstract] ABSTRACT: B. melitensis CβG is not immunogenic in mice. (A) Balb/C mice were immunized by either PBS or E. coli LPS, (10 µg/mouse) or B. melitensis LPS (Brucella LPS, 10 µg/mouse) or B. melitensis CβG (CβG, 10 µg/mouse). The primary antibody response (white bars) was measured 21 days after immunization. Mice were boosted 45 days with the same molecules (5 µg/mouse) to measure the secondary antibody response (black bars). 3 independent experiments were performed (n = 5), *p<0.05. (B) Levels of pro-inflammatory cytokines are strongly reduced in the sera of CβG-immunized mice. C57Bl/6 mice (n = 5) were immunized either with PBS, MPL, CβG, LPS or Poly I/C. After 6 h, 24 h and 72 h of immunization, mice were bled and the cytokine levels were measured in the sera by CBA. The levels of IL-6, IL-10, MCP-1, IFNγ, TNFα and IL-12p70 are presented. Data represent means ± standard errors from 5 samples. ***p<0.001, **p<0.01.
[Show abstract][Hide abstract] ABSTRACT: Bone marrow-derived murine dendritic cells infected with cgs- mutant display a low maturation profile. Mouse DC were infected for 24 h with Salmonella thyphimurium S12023 virulent strain, Brucella abortus 2308 virulent strain or isogenic cgs- B. abortus mutant. (A, B) IL-12 and TNFα levels in supernatant were measured by ELISA after infection, respectively. Data represent means ± standard errors of at least 3 independent experiments. *p<0.05 cgs- mutant compared to the wild type values, ns: not significant.
[Show abstract][Hide abstract] ABSTRACT: B. melitensis CβG induces mouse DC maturation in a dose-dependent manner. (A) Mouse DC were stimulated for 8 h (white) and 24 h (black) with medium, E. coli LPS (0.25 µM) or B. melitensis CβG (0.025 µM, 0.25 µM, 2.5 µM). MHC II, CD80, CD40 and CD86 surface levels were analyzed by flow cytometry. Graphs represent median of fluorescence ± standard error of four independent experiments. (B) Mouse DC were treated in triplicate for 8 and 24 h with either medium, E. coli LPS (0.25 µM) or B. melitensis CβG (0.025 µM, 0.25 µM, 2.5 µM). IL-12 and TNFα levels in the supernatants were measured by ELISA. Data represent means ± standard errors of at least 3 independent experiments. *p<0.05., ns: not significant.
[Show abstract][Hide abstract] ABSTRACT: CβG triggers a local skin inflammation. Mice were immunized either with PBS, MPL, CβG, LPS or Poly I/C. At 48 h post-treatment, both the adjuvant-treated and untreated ears were collected for histological analysis of cutaneous inflammation. Hematoxylin- and eosin-stained sections of adjuvant-immunized mice revealed marked increase in ear thickness accompanied by inflammatory cell infiltration.
[Show abstract][Hide abstract] ABSTRACT: Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella β 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella β 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8(+) T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4(+) and CD8(+) T cell responses including cross-presentation by different human DC subsets. Brucella β 1,2 cyclic glucans increased the memory CD4(+) T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.
[Show abstract][Hide abstract] ABSTRACT: Immunotherapy based on adoptive transfer of tumor antigen-specific CD8(+) T cell (TC) is generally limited by poor in vivo expansion and tumor infiltration. In this study, we report that activated STAT5 transcription factors (STAT5CA) confer high efficiency on CD8(+) effector T cells (eTC) for host colonization after adoptive transfer. Engineered expression of STAT5CA in antigen-experienced TCs with poor replicative potential was also sufficient to convert them into long-lived antigen-responsive eTCs. In transplanted mastocytoma- or melanoma-bearing hosts, STAT5CA greatly enhanced the ability of eTCs to accumulate in tumors, become activated by tumor antigens, and to express the cytolytic factor granzyme B. Taken together, these properties contributed to an increase in tumor regression by STAT5CA-transduced, as compared with untransduced, TCs including when the latter control cells were combined with infusion of interleukin (IL)-2/anti-IL-2 complexes. In tumors arising in the autochthonous TiRP transgenic model of melanoma associated with systemic chronic inflammation, endogenous CD8(+) TCs were nonfunctional. In this setting, adoptive transfer of STAT5CA-transduced TCs produced superior antitumor effects compared with nontransduced TCs. Our findings imply that STAT5CA expression can render TCs resistant to the immunosuppressive environment of melanoma tumors, enhancing their ability to home to tumors and to maintain high granzyme B expression, as well as their capacity to stimulate granzyme B expression in endogenous TCs.
[Show abstract][Hide abstract] ABSTRACT: iMCs retrieved from spleens of Amela-bearing mice migrate to the bone marrow (BM) in tumor-free mice and anti-Gr1 mAb treatment fails to deplete them from Amela-bearing hosts. (A) Spleen cells from Amela-bearing mice rich in iMCs were labeled with CFSE 10 µM and transferred in tumor-free or in induced Amela-bearing mice. 20 hrs later LN, spleen and BM were harvested and FACS analysis was performed on CFSE+ gated cells. % of CFSE+ cells expressing Gr1 and CD11b (Gr1CD11b) or CD3 (T cells) is indicated as the mean +/− s.d. for 3 mice per group. Enrichment in Gr1+CD11b+ cells in spleens and in LN was observed only in Amela-tumor bearing mice. (B) Control and induced Amela-bearing mice were treated with 300 µg anti-Gr1 mAb (RB6-8C5) injected i.p. every 2 days for 6 days, were sacrificed at day 8 and LN, spleen and BM cells were analyzed by FACS for expression of CD11b and Ly-6G mAb (1A8), which recognizes an epitope of the Ly-6G/Gr1 molecule that is distinct from that recognized by mAb RB6-8C5. High percentages of CD11b+Ly6−G+ (iMCs) were still present in LN and spleen of Amela-bearing mice. Representative of two mice per group.
[Show abstract][Hide abstract] ABSTRACT: Modifications of the stromal network in Amela-TDLN. LN sections from control mice and TDLN from Mela- and Amela-bearing mice were stained with (A) anti-B220-FITC (green), anti-Desmin/donkey anti-rabbit-Alexa555 (red) or (B) with anti-ER-TR7/chicken anti-rat-Alexa647 (green) and anti-Desmin/donkey anti-rabbit-Alexa555 (red). In (B) mice had received 106 CFSE-labeled B10.D2 TL (withe), as in Fig.2, 20hrs before their sacrifice. In the magnification (left), the TL (withe) can be seen to interact with the Desmin+ER-TR7+ FRC in the Mela-TDLN.
[Show abstract][Hide abstract] ABSTRACT: Heatmap output for the 80 most differentially expressed transcripts between Amela and Mela tumors. Each row represents a gene and each column represents a sample. Each experimental sample is represented by 2 or 3 values associated to 2 or 3 different hybridizations. Expression values are represented as colors, where the range of colors (red, pink, light blue, dark blue) shows the range of expression values (high, moderate, low, lowest). These genes were provided in the GSEA plots shown in Fig. 2.
[Show abstract][Hide abstract] ABSTRACT: Mice. On the B10.D2 (B10.D2/nOlaHsd, H-2d) background, the TiRP-10B Ink4a/Arfflox/flox B10.D2 mice housed in the CIML facility develop, after 4OH-tamoxifen treatment (injection s.c. twice two weeks apart of 4 mg 4OH-tamoxifen dissolved in ethanol and brought to 20 mg/ml with autoclaved sunflower oil as described (Huijbers et al. 2006)), single melanomas, either strongly pigmented (Mela) or unpigmented (Amela) with similar latency (on average 160 days) (Soudja et al. 2010). The incidence of Amela development was about 4 fold higher than that of Mela tumors. Mela- and Amela-tumor bearing mice analyzed in the present study each carried a single tumor, the size of the latter being on average twice that of the former. For examples, see Fig.S8.
[Show abstract][Hide abstract] ABSTRACT: Comparaison of representation of DC subsets in LN from control, Mela- and Amela-bearing mice. Analysis of DC subpopulations in TDLN (left) or non-draining LN (NDLN) (right) from Amela- (A) or Mela- (B) bearing or LN from control (C) mice. After gating on CD11c+NK1.1−CD25−CD45R− cells, CD11c versus MHCII staining identifies MigDC (CD11c+MHCIIhigh) and ResDC (CD11chighMHCIIintermediate) and shows a relative depletion of the MigDC population selectively in TDLN of Amela-bearing mice. (D) Immunohistology of sections from control LN, Mela-TDLN and Amela-TDLN showing anti-MHCII (white), anti-B220 (green; B cells) and anti-CD3 (blue; T cells).
[Show abstract][Hide abstract] ABSTRACT: Stromal cells present in the splenic red pulp of Amela-bearing mice express the VEGFR2. Spleen sections from control and from Amela-bearing mice were stained with anti-collagen IV antibody (red) and with goat anti-mouseVEGFR2 (Flk-1) antibody from R&D Systems (green). Single stainings and merge images are shown. A magnification of the merged image is shown (far rigth).
[Show abstract][Hide abstract] ABSTRACT: conserved numbers of TL in spleens of mice developing Amela-melanomas. (a) Spleen cells harvested from control, Mela- or Amela-bearing mice were counted and stained for CD45, CD4, CD8 and B220 expression by FACS as described (Soudja et al. 2010). N = number of mice analyzed. (b) % CD45+ cells among live spleen cells are given +/− SD. Corresponding cell numbers are given in bold. (c) % CD8 TL among CD45+ spleen cells +/− SD. Corresponding cell numbers are given in bold. (d) % CD4 TL among CD45+ spleen cells +/− SD. Corresponding cell numbers are given in bold. (e) % B220+ cells among CD45+ spleen cells +/− SD. Corresponding cell numbers are given in bold.
[Show abstract][Hide abstract] ABSTRACT: Gr1+CD11b+ iMCs are recruited to SLOs and tumor in Amela-bearing mice. Analysis of spleen (left), LN (middle) and skin or tumor (right) sections from control mice (upper) and Amela-bearing mice (lower). The sections were stained for the proliferation marker Ki-67 (green), Gr1 (red) and the nuclei marker Topro3 (blue) as described (Soudja et al. 2010).