Figure 1.
ITK(K390R) is expressed in Itk-KD T-cells.
Western Blot (WB) following immune precipitation (IP) of lysates from purified T & B cells (combined lymph node & spleens) of Wild Type (WT) and Itk-KD mice. Monoclonal antibody anti-ITK (clone 2F12) was used for the IP and polyclonal rabbit anti-ITK for WB.
Figure 2.
ITK(K390R) retains scaffold function.
CD4+ cells were isolated from the spleens of naïve and Itk-KD mice and incubated with anti-CD3 coated beads. Capping of actin was revealed by staining with Alexa-488-phalloidin (a). The images are single confocal sections of 0.4 µm thickness and the scale bar = 10 µm. (b) Image analysis of actin capping. Polymerised actin surrounding the anti-CD3 coated beads was quantified as described in Materials and Methods. Data represents the means +/- S.E.M. from four determinations.
Figure 3.
Splenocytes from naïve Itk-KD mice fail to secrete cytokines.
Splenocytes from WT or Itk-KD mice were activated in vitro with increasing concentrations of soluble anti-CD3 for 72 h. Cell culture supernatants were collected and secreted cytokines quantified by MSD assays. Results are shown as mean +/− S.E.M. (n = 6 mice).
Figure 4.
Increased memory markers on CD4+ cells.
There is an increase in CD4 positive cells that express memory markers CD62LloCD44hi in the spleen of naïve Itk-KD mice compared to WT controls. Results are shown as the mean +/− S.E.M. n = 6 mice).
Figure 5.
Cytokine secretion by splenocytes following PMA/ionomycin stimulation.
Splenocytes from WT or Itk-KD mice were activated in vitro with PMA (50 ng/ml)/ionomycin (1 µg/ml) for 5 h and intracellular IFNγ and IL-4 determined by flow cytometry. There is an increase in the proportion of CD4+ cells from the spleens of Itk-KD mice which stain positively for IFNγ and IL-4 than WT. Results represent the mean +/− S.E.M. n = 6 mice.
Figure 6.
Antibody levels in the plasma of naïve mice.
Itk-KD mice exhibit increased IgE, IgG1, IgG2a and IgG2b. Results are expressed as mean +/− S.E.M. n = 5 mice, * P<0.05, ** P<0.01 (Fishers LSD, Hochberg correction).
Figure 7.
Investigation of γδ cells from the spleens of naïve mice.
(a) Purified γδ cells from the spleens of naïve Itk-KD mice have an increased percentage of CD4+ cells. Spleens from 6 mice per genotype were pooled prior to purification of γδ cells. Following purification, 96% of the live CD3 cells were γδ positive, and of these cells 5.2% were CD4+ from Itk-KD mice compared to 1.9% for the WT (Box A). (b) Purified γδ cells from Itk-KD mice show reduced cytokine release compared to WT controls following in vitro activation with anti-CD3. Results are expressed as mean +/− S.E.M. of triplicate cultures of γδ cells from the spleens of 6 mice per genotype.
Figure 8.
Upregulated expression of ICOS.
ICOS expression is upregulated on the surface of CD3+ αβ cells in Itk-KD splenocytes compared to WT mice. This difference is further enhanced when gating on CD3+ CD4+ αβ cells. αβ cells were identified as CD3+ cells negative for γδ. Results are expressed as mean +/− S.E.M. (n = 6 mice). ** P<0.01 (Fishers LSD, Hochberg correction).
Figure 9.
Airway hyper-reactivity (AHR) in OVA model.
Female WT and Itk-KD mice were sensitised to OVA emulsified in aluminium hydroxide by intraperitoneal injection on day 0 and 14. On day 24, 25 and 26, mice were challenged by the intranasal route with either OVA (WT-OVA and Itk-OVA) or saline (WT-saline or Itk-saline). AHR to a 5HT challenge was assessed on day 27 using whole body plethysmography (PenH). Itk-KD mice develop significantly reduced airway hyper-reactivity following sensitisation and challenge to OVA compared to WT (p<0.01, Tukey). Results are expressed as mean+/− S.E.M. (n = 10 for Itk-KD and n = 12 for WT).
Figure 10.
BAL cellular infiltration in OVA model.
Cell populations in the BAL of OVA-sensitised Itk-KD and WT mice, 48 h after intranasal challenge with OVA or saline. Significant reductions of eosinophils and B cells and non-significant reductions in Th1, Th2 and Treg cells were seen in the BAL of Itk-KD mice. Results are expressed as mean +/− S.E.M. (n = 6), * p<0.05, **P<0.01 (Anova with post hoc planned comparison to wild type (un-paired students T test).
Figure 11.
Cytokine levels in the BAL of OVA-sensitised Itk-KD and WT mice, 5 h after intranasal challenge with OVA or saline. Itk-KD mice show reduced levels of IFNγ, IL-2, IL-5 and IL-13 but not IL-4 or IL-10 in the BAL of mice 5 hours after intranasal challenge with OVA. Results are expressed as mean +/− S.E.M. (n = 6), **P<0.01, ***P<0.001 (Tukey).
Figure 12.
Histopathology of lungs from OVA model.
Reduced inflammation was observed in the lung of Itk-KD compared to WT mice following sensitisation and challenge with OVA. All animals were sensitised to OVA, and culled 4 hours or 48 hours after intranasal challenge with OVA or saline. Upper panels Itk-KD, lower panels WT. Scale bar = 200 µm.
Figure 13.
Reactivation of mediastinal lymph node cells from OVA model.
Following sensitisation and challenge of mice with OVA, mediastinal lymph node cells from WT and Itk-KD mice were reactivated in vitro with OVA for 72 hours. In WT mice this induces a concentration-dependent increase in cytokine release, measured in culture supernatants. However, under the same conditions mediastinal lymph node cells from Itk-KD mice fail to secrete cytokines. Results are shown as mean +/− S.E.M. (n = 12 mice).
Figure 14.
Antibody levels in the serum from OVA model.
Serum immunoglobulin levels were determined in WT and Itk-KD mice following sensitisation with OVA and challenge with either saline (Saline) or OVA (OVA). Results are expressed as mean +/− S.E.M.. (n = 5 mice for saline control, n = 6 mice for OVA challenge) * P<0.05, **P<0.01 compared to the WT control (significance between the OVA groups is shown in blue and WT in black; Fishers LSD, Hochberg correction).