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doi: 10.1371/journal.pone.0061034. Epub 2013 Apr 8.

The hedgehog receptor patched1 in T cells is dispensable for adaptive immunity in mice

Affiliations

The hedgehog receptor patched1 in T cells is dispensable for adaptive immunity in mice

Kai D Michel et al. PLoS One.2013.

Abstract

Hedgehog (Hh) signaling modulates T cell development and function but its exact role remains a matter of debate. To further address this issue we made use of conditional knock-out mice in which the Hh receptor Patched1 (Ptch) is inactivated in the T cell lineage. Thymocyte development was moderately compromised by the deletion of Ptch as characterized by reduced numbers of CD4 and CD8 single-positive cells. In contrast, peripheral T cells were not affected. Proliferation and IFNγ secretion by Ptch-deficient T cells were indistinguishable from controls irrespectively of whether we used strong or suboptimal conditions for stimulation. Analysis of CTL and Treg cell functions did not reveal any differences between both genotypes, and T cell apoptosis induced by glucocorticoids or γ-irradiation was also similar. Surprisingly, absence of Ptch did not lead to an activation of canonic Hh signaling in peripheral T cells as indicated by unaltered expression levels of Gli1 and Gli2. To test whether we could uncover any role of Ptch in T cells in vivo we subjected the mutant mice to three different disease models, namely allogeneic bone marrow transplantation mimicking graft-versus-host disease, allergic airway inflammation as a model of asthma and growth of adoptively transferred melanoma cells as a means to test tumor surveillance by the immune system. Nonetheless, we were neither able to demonstrate any difference in the disease courses nor in any pathogenic parameter in these three models of adaptive immunity. We therefore conclude that the Hh receptor Ptch is dispensable for T cell function in vitro as well as in vivo.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Flow cytometric analysis of thymocyte development.
Thymocytes were isolated fromPtchflox/flox control orPtchflox/flox CD4Cre+/− mutant mice, counted and stained for CD4, CD8, and CD44 to identify different developmental stages. (A) Mean number of total thymocytes ± SEM is shown for four mice of each genotype. (B) Percentages of CD4−CD8−CD44+ (DN stages 1 and 2) or CD4−CD8−CD44− (DN stages 3 and 4) thymocytes amongst total DN thymocytes are depicted. (C) Percentages of DN, DP and CD4 SP or CD8 SP cells amongst total thymocytes are shown. Panels B and C refer to the mean ± SEM of four mice of each genotype. Statistical analysis was performed by unpaired t-test (*: p<0.05). Differences were not statistically different unless otherwise indicated.
Figure 2
Figure 2. Gene expression and flow cytometric analysis of peripheral T cells.
Splenic T cells were purified fromPtchflox/flox control orPtchflox/flox CD4Cre+/− mutant mice and used to generate cDNA for qRT-PCR analysis. Alternatively, total splenocytes were isolated from mice of each genotype and stained for TCRβ, CD4, CD8, CD44 and CD69 to identify different peripheral T cell subpopulations. (A) Relative expression ofwt Ptch transcripts derived from thePtchflox allele,Ptchdel transcripts after Cre-mediated recombination of the Ptchflox allele,Gli1 andGli2 transcripts as well asPtch2 transcripts are depicted. As a positive control forPtch2 expression, cDNA from wt mouse embryos at stage E10.5 was used for PCR amplification. Gene expression was normalized toHPRT1 and the transcriptional level of each gene inPtchflox/flox control mice or E10.5 embryos, respectively, was set to 1.0. All panels show the mean ± SEM of fivePtchflox/flox and threePtchflox/flox CD4Cre+/− mice; n.d.: not detectable. Measurement of all samples was performed in duplicates. (B) Mean number of total splenocytes ± SEM is shown for tenPtchflox/flox and eightPtchflox/flox CD4Cre+/− mice. (C) The percentages of T cells amongst all splenocytes and of CD4+ or CD8+ T cells amongst total splenic T cells is depicted. (D) T cells showing a recently activated (CD69+) or effector memory (CD44hi) phenotype were identified amongst the CD4+ and CD8+ splenic T cells. Panels C and D show the mean ± SEM of fivePtchflox/flox and sevenPtchflox/flox CD4Cre+/− mice. Statistical analysis was performed by unpaired t-test (*: p<0.05).
Figure 3
Figure 3. Proliferation and IFNγ production by activated T cells.
Total T cells were isolated from the spleens ofPtchflox/flox control orPtchflox/flox CD4Cre+/− mutant mice and 105 cells per well were stimulated with different concentrations of ConA or anti-CD3/CD28 antibodies in a total volume of 200 µl medium. For detection of IFNγ, 50 µl medium were removed at each time point and used for analysis by ELISA. For measurement of proliferation, an equal volume of fresh medium containing 37 kBq3H-thymidine was added to the same wells and incubated for another 16 hours. (A) Proliferation was assessed by scintillation counting. (B) Quantification of IFNγ levels was achieved by ELISA. Each panel shows the mean ± SEM of five individual experiments. Statistical analysis for each experimental condition was performed by unpaired t-test and no differences were found in any case.
Figure 4
Figure 4. Cytolytic capacity of CTLs and suppressive activity of Treg cells.
(A) Specific lysis of Ltk− target cells (H2k) by alloreactive CTLs derived fromPtchflox/flox control orPtchflox/flox CD4Cre+/− mutant mice (H2b), which had been immunized with splenocytes from C3H/HeN mice (H2k). Means of specific lysis of triplicates of the Ltk− cells were determined for eight individual mice of both genotypes. The diagram shows the mean of specific lysis ± SEM for both genotypes at several effector to target (E:T) ratios. Statistical analysis was achieved by 2-way ANOVA (p = 0.216). (B) Abundance and FoxP3 expression of splenic TCRβ+CD4+CD25+GITR+FoxP3+ Treg cells. Treg cells in the spleen were enumerated by FACS analysis and their percentage amongst the CD4+ T cells is depicted as the mean ± SEM from threePtchflox/flox and fivePtchflox/flox CD4Cre+/− mice (left panel). The levels of intracellular FoxP3 expression in Treg cells were quantified by FACS and the mean fluorescent intensity (MFI) is depicted as mean ± SEM for threePtchflox/flox and fourPtchflox/flox CD4Cre+/− mice (right). Both parameters were statistically similar based on analysis by unpaired t-test (C) Suppressive capacity of Treg cells fromPtch-deficient and control mice. CD4+CD25+ Treg cells were cocultured with CD4+CD25− Th cells at ratios of 1∶1, 1∶5 and 1∶10. Polyclonal activation was achieved using anti-CD3/CD28 antibodies and IL-2 levels were assessed in the cell culture supernatants after 48 hours. All values are normalized to cultures of activated CD4+CD25− Th cells which served as a positive control. Resting CD4+CD25− Th cells and activated CD4+CD25+ Treg cells served as negative controls. The figure shows the mean ± SEM of three (Ptchflox/flox) and four (Ptchflox/flox CD4Cre+/−) independent experiments, respectively. Based on the analysis by unpaired t-test, the suppressive capacity of Treg cells from both genotypes was similar.
Figure 5
Figure 5. Sensitivity of T cells to apoptosis induction.
(A) Apoptosis induction by γ-irradiation was assessed by culturing 2 x 105 T cells following exposure to a dose of 1 or 2 Gy or without any manipulation. Cell viability was assessed every 24 hours by FACS analysis using 7-AAD. Results represent the mean ± SEM of three independent experiments. (B) Glucocorticoid-induced apoptosis was investigated by culturing 2 x 105 splenic T cells fromPtchflox/flox control orPtchflox/flox CD4Cre+/− mutant mice in medium with or without 2 or 6 nM of water-soluble Dex for 4 days. Cell survival was determined as described above. Results represent the mean ± SEM of three independent experiments. (C) T cells were pre-activated with 1.5 µg/ml soluble anti-CD3 and anti-CD28 for 24 hours. Apoptosis was then induced by adding 1 µM of water-soluble Dex and cell viability was assessed up to four days after apoptosis induction similar as above. Results represent the mean ± SEM from threePtchflox/flox or fourPtchflox/flox CD4Cre+/− animals. Based on the analysis by unpaired t-test, apoptosis was not different between both genotypes.
Figure 6
Figure 6. Morbidity and mortality after GvHD induction.
GvHD was induced in irradiated Balb/c mice (8.5 Gy) by transferring 107 T cell-depleted syngeneic bone marrow cells plus 2×106 T cells from eitherPtchflox/flox control orPtchflox/flox CD4Cre+/− mutant C57BL/6 donor mice (n = 29 for each genotype). Control animals received T cell depleted bone marrow only (n = 13). Mice were monitored every other day for clinical symptoms (A) and survival (B). The figures show the combined data of five independent experiments. Statistical analysis of the disease courses was performed by Mann Whitney test; in the case of the survival curves the log-rank test was employed. In both cases, no statistical significant difference between both genotypes was found.
Figure 7
Figure 7. Lung infiltration, antibody production and T cell function after induction of allergic airway inflammation.
Ptchflox/flox control andPtchflox/flox CD4Cre+/− mutant C57BL/6 mice were sensitized against Ova by repeated intraperitoneal injection of antigen plus adjuvant. Control mice (Ptchflox/flox) received adjuvant without Ova. Antigen challenge was achieved by intranasal delivery of dissolved Ova for three consecutive days and analysis was performed after a two day resting phase. (A) Lungs were washedin situ and total cell counts in the broncheoalveolar lavage fluid (BALF) were determined using light microscopy. (B) Identification of different populations of lung-infiltrating cells within the BALF by using FACS. (C) Lung histology of mice 48 hours after the last challenge. A control lung obtained from a non-sensitized mouse challenged with Ova is shown along with lungs ofPtchflox/flox andPtchflox/flox CD4Cre+/− mice which were both sensitized and challenged with antigen. No pathohistological signs of inflammation could be detected in control mice whereas a clear and massive cell influx was seen in sensitized and challenged mice. (D) Serum concentrations of Ova-specific IgG1, IgG2a and IgE immunoglobulins were quantified using ELISA and are depicted in the form of optical densities (OD). (E) Splenocytes were isolated and restimulatedex vivo with 10 µg/ml Ova for 72 hours and proliferation was assessed by3H-thymidine incorporation assay. Results represent the mean ± SEM from nine non-sensitized control mice as well as thirteenPtchflox/flox and fourteenPtchflox/flox CD4Cre+/− animals, respectively. Statistical analysis of all parameters was performed by unpaired t-test and no differences were found in any case.
Figure 8
Figure 8. Tumor incidence and size after inoculation of B16 melanoma cells.
C57BL/6 mice were inoculated with 104 B16-F10 melanoma cells into the right flank and tumor growth was monitored over a period of three weeks. The incidence of palpable tumors (left panel) and the mean tumor size ± SEM (right panel) are depicted for both experimental setups. (A) Results of tumor incidence and size are shown for elevenPtchflox/flox control and ninePtchflox/flox CD4Cre+/− mutant C57BL/6 mice in which tumorigenesis had been induced without additional manipulation. (B) To generate protective immunity, C57BL/6 mice were vaccinated with 106 irradiated B16-F10 cells together with adjuvant subcutaneously into the left flank. Ten days later the vaccinated mice were challenged with 104 viable B16-F10 cells subcutaneously into the right flank. Tumor incidence rate and mean tumor size ± SEM are shown for fourteenPtchflox/flox andPtchflox/flox CD4Cre+/− mice each. Statistical analysis of tumor incidence and size was performed by Mann Whitney test and not found to be different between both genotypes.
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This work was supported by grants from the Deutsche Forschungsgemeinschaft (RE1631/8-1 and HA2197/6-1) and Deutsche Krebshilfe (108713). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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