1. CD28 provides T-cell costimulation and enhances PI3K activity at the immune synapse independently of its capacity to interact with the p85/p110 heterodimer
by Fabien Garçon, Daniel T. Patton, Juliet L. Emery, Emilio Hirsch, Robert Rottapel, Takehiko Sasaki, and Klaus Okkenhaug
Blood
Volume 111(3):
February 1, 2008
©2008 by American Society of Hematology

2. p110δ is essential for PIP3 accumulation to the plasma membrane.
p110δ is essential for PIP3 accumulation to the plasma membrane. (A) Images showing fluorescence and transmitted light from a movie of AktPH-GFP+ WT, p110δD910A/D910A, p110γ−/−, CD28−/−, and CD28Y170F CD4+ T-cell blasts interacting with LPS-activated APCs presenting OVA peptide. In some experiments, WT cells were pretreated with PI3K inhibitor (LY294002) for 15 minutes before stimulation. (B) Quantification of AktPH-GFP redistribution in WT (n = 11), LY pretreated WT (n = 8), p110δD910A/D910A (n = 9), p110γ−/− (n = 12), CD28−/− (n = 9), and CD28Y170F (n = 7) CD4+ T cells. A few LY treated T cells showed apparently normal AktPH-GFP accumulation (Figure S3). It is possible that a subset of T cells express sufficient levels of multidrug resistance protein transporter and could potentially thus evade inhibition.81,82 Therefore, to establish a realistic baseline for minimal PI3K activity, these outliers were excluded from the analysis, but they are shown in Figure S3. The recruitment of AktPH-GFP at the plasma membrane is expressed as a ratio between the fluorescence measured at the contact area and the average fluorescence within the cell. Fluorescence was measured on at least 2 independent days. (C) Average ratio of PIP3 at membrane during the plateau phase of the response (approximately seconds). The mean fluorescence values were determined as described in “Methods” (**P < .01, ***P < .001). (D) Acute inhibition of PI3K activity with the p110δ-selective inhibitor IC Two T cell–B cell conjugates are shown after the addition of IC87114 as indicated by arrows. PIP3 depletion from the membrane occurred rapidly in both cases.
Fabien Garçon et al. Blood 2008;111:
©2008 by American Society of Hematology

3. CXCL12-induced (500 ng/mL) accumulation of PIP3 at the plasma membrane of CD4+ T-cell blasts is dependent on p110γ but not p110δ.
CXCL12-induced (500 ng/mL) accumulation of PIP3 at the plasma membrane of CD4+ T-cell blasts is dependent on p110γ but not p110δ. (A) Representative images showing the translocation of the AktPH-GFP probe to the plasma membrane. (B) Graphical representation of the percentage of total fluorescence at the plasma membrane as function of time. Data are representative of 2 independent experiments (WT, n = 6; p110δD910A/D910A, n = 4; p110γ−/−, n = 5).
Fabien Garçon et al. Blood 2008;111:
©2008 by American Society of Hematology

4. CD28, but not p110δ, regulates PKCθ localization at the synapse.
CD28, but not p110δ, regulates PKCθ localization at the synapse. (A) Conjugates between naive WT, WT + LY294002, p110δD910A/D910A, p110γ−/−, CD28−/−, or CD28Y170F CD4+ T cells and OVA –pulsed APCs were stained with antibodies against PKCθ and the TcRβ. Representative images of the distribution of AktPH-GFP, TcR, and PKCθ during conjugate formation are shown. (B) Quantification of AktPH-GFP recruitment at the plasma membrane (WT, n = 72; WT + LY294002, n = 31; p110δD910A/D910A, n = 53; p110γ−/−, n = 23; CD28−/−, n = 51; CD28Y170F, n = 62), determined as described in “Methods” (**P < .01, ***P < .001). (C) Recruitment of PKCθ to the contact area (WT, n = 76; WT + LY294002, n = 24; p110δD910A/D910A, n = 28; p110γ−/−, n = 28; CD28−/−, n = 33; CD28Y170F, n = 67), expressed as the ratio between the fluorescence at the contact area and the fluorescence within the cell. Only the means of the values for the CD28−/− and CD28Y170F values were significantly different from the WT values (***P < .001). (D) A 3-dimensional reconstruction of the contact area was done and representative pictures of the interface projection and the mean area (± SEM, *P < .05, **P < .001, Student 2-tailed t test; WT, n = 38; p110δD910A/D910A, n = 18; CD28−/−, n = 23; CD28Y170F, n = 21) occupied by PKCθ at the immune synapse are shown. Data are representative of 2 independent experiments. (E) The YMNM motif of CD28 is not required for NF-κB nuclear translocation. Conjugates between WT, CD28−/−, or CD28Y170F CD4+ T cells and OVA –pulsed APCs were stained for p65 NF-κ, and nuclei were stained with 7-AAD. The conjugates were scored for the frequency of nuclear localization of NF-κB. The p110δ inhibitor IC87114 blocked NF-κB nuclear translocation in CD28−/− and CD28Y170F T cells. Data represent mean (± SEM) with more than 30 cells analyzed in each of 3 experiments.
Fabien Garçon et al. Blood 2008;111:
©2008 by American Society of Hematology

5. Proliferation of CD28 and p110δ double mutant T cells.
Proliferation of CD28 and p110δ double mutant T cells. (A) Anti-CD3– and anti-CD28–dependent proliferation of WT, p110δD910A/D910A, CD28−/−, CD28Y170F, p110δD910A/D910ACD28−/−, and p110δD910A/D910ACD28Y170F lymph node T cells stimulated with 0.1 μg/mL anti-CD3 and with anti-CD hybridoma supernatant (1/100). (B) Proliferation of T cells from the mutants OT-II+ transgenic WT, p110δD910A/D910A, CD28−/−, CD28Y170F, p110δD910A/D910ACD28−/−, and p110δD910A/D910ACD28Y170F CD4+ T cells in response to 0.01μM, 0.1μM, or 1.0μM OVA peptide.
Fabien Garçon et al. Blood 2008;111:
©2008 by American Society of Hematology

6. Impaired humoral immune responses in p110δD910A/D910A CD28−/− mice.
Impaired humoral immune responses in p110δD910A/D910A CD28−/− mice. (A) WT, p110δD910A/D910A, CD28−/−, CD28Y170F, p110δD910A/D910A CD28−/−, and p110δD910A/D910ACD28Y170F mice were immunized with DNP-coupled KLH adsorbed onto alum. The mice were rechallenged with the same dose of antigen 78 days later. Anti-DNP–specific antibodies were detected by enzyme-linked immunoabsorbent assay. Each dot represents one mouse. (B) CD28 and p110δ are indispensable for efficient killing of allogeneically mismatched lymphocytes in vivo. Recipient mice on the B6 background (H-2b) were injected with a mixture of 5 × 106 CFSEhigh B6 lymphocytes and CFSElow CB6 F1 lymphocytes (H-2b/k). The ratio of CFSElow to CFSEhigh was normalized to 1 on day 1, and the average from 3 recipients from each genotype is shown (± SD).
Fabien Garçon et al. Blood 2008;111:
©2008 by American Society of Hematology