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. 2013 Jan;24(1):27-37.
doi: 10.1089/hum.2012.130. Epub 2012 Nov 6.

Enhanced function of redirected human T cells expressing linker for activation of T cells that is resistant to ubiquitylation

Naoki Kunii  1 , Yangbing Zhao, Shuguang Jiang, Xiaojun Liu, John Scholler, Lakshmi Balagopalan, Lawrence E Samelson, Michael C Milone, Carl H June
Affiliations

Enhanced function of redirected human T cells expressing linker for activation of T cells that is resistant to ubiquitylation

Naoki Kunii et al. Hum Gene Ther. 2013 Jan.

Abstract

It is likely that the enhancement of signaling after antigenic stimulation, particularly in the tumor microenvironment, would improve the function of adoptively transferred T cells. Linker for activation of T cells (LAT) plays a central role in T cell activation. We hypothesized that the ubiquitylation-resistant form of LAT in cells would enhance T cell signaling and thus augment antitumor activity. To test this, human CD4(+) or CD8(+) T cells were electroporated with small interfering RNA (siRNA) to repress endogenous LAT and ubiquitylation-resistant LAT 2KR or wild-type LAT mRNA was introduced for reexpression. Significantly enhanced phosphorylation of LAT and phospholipase C-γ (PLCγ) was observed, and augmented calcium signaling after T cell receptor (TCR) triggering was observed in LAT 2KR-expressing T cells. TCR-induced calcium signaling was abrogated in LAT knockdown cells, but the baseline was higher than that of control siRNA-electroporated cells, suggesting a fundamental requirement of LAT to maintain calcium homeostasis. Redirected LAT 2KR T cells expressing a chimeric antigen receptor or an MHC class I-restricted TCR showed augmented function as assessed by enhanced cytokine secretion and cytotoxicity. These results indicate that interruption of LAT ubiquitylation is a promising strategy to augment effector T cell function for adoptive cell therapy.

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Figures

FIG. 1.
FIG. 1.
Substitution of endogenous linker for activation of T cells (LAT) by ubiquitylation-resistant LAT in primary T cells. (A) Expression of LAT small interfering RNA (siRNA) and yellow fluorescent protein (YFP)-fused LAT 2KR, 4YF, or wild-type (WT) in vitro-transcribed (IVT) RNA-electroporated, freshly isolated CD4+ T cells was analyzed by Western blot 24 or 48 hr after electroporation. Control siRNA (1 nmol) was used for negative control cells (first lane). (B and C) YFP expression of cells in (A) was confirmed by flow cytometry. The histogram and mean fluorescence intensity (MFI) are shown. (D and E) CD8+ T cells were stimulated with anti-CD3/CD28 beads for 10 days. After 10 days of culture, the in vitro-expanded cells were harvested and cryopreserved, and later these cells were thawed and incubated for 24 hr before electroporation. LAT expression of LAT siRNA and YFP-fused LAT 2KR, WT, and 4YF IVT RNA-electroporated CD8+ cells was analyzed by Western blot 24 hr after electroporation. (E) On the basis of the Western blot analysis of LAT siRNA and LAT–YFP RNA-electroporated CD8+ T cells 24 hr after electroporation, relative signal intensities of LAT were calculated. Averages and standard deviations (SD) from three different donors are shown. Statistical differences are presented with asterisks: *p<0.01 (Cochran–Cox); **p<0.01 (Student t test).
FIG. 2.
FIG. 2.
Enhanced T cell signaling of LAT-2KR-substituted CD8+ T cells after T cell receptor (TCR) and CD28 stimulation. (A) Phosphorylation of downstream molecules in LAT-substituted in vitro-expanded CD8+ T cells was analyzed by Western blot at three time points (0, 10, and 30 sec) after anti-CD3/CD28 bead stimulation, and the values of phospho-LAT191/LAT–YFP and phospho-PLCγ783/LAT–YFP were calculated (B). (C and D) LAT 2KR- and LAT WT-substituted CD8+ T cells were stained with Indo-1, and intracellular free ionized calcium concentrations were measured by flow cytometry after stimulation with anti-CD3 antibody (triangle). YFP expression of these cells is shown in (C).
FIG. 3.
FIG. 3.
Increased frequency of helper T cell type 1 (Th1) cytokine secretion by CD4+ T cells expressing LAT ubiquitylation mutants. (A) Single-cell ELISPOT assay with LAT-substituted freshly isolated CD4+ T cells from donor 1 was performed after 16 hr of stimulation with anti-CD3/CD28 beads (solid columns) or no stimulation (shaded columns). The average and SD of three wells are shown. Statistically significant differences are indicated by asterisks: *p<0.001; **p<0.01 (Student t test). (B) Intracellular cytokine staining of LAT-substituted CD4+ T cells from three different donors was measured by flow cytometry after 4 hr of stimulation with immobilized anti-CD3 antibodies. The frequency of cells expressing IFN-γ (solid columns) or IL-4 (shaded columns) is plotted.
FIG. 4.
FIG. 4.
Cellular proliferation and Th1 differentiation of LAT-substituted CD4+ T cells. (A and B) LAT-substituted CD4+ T cells were stained with PKH26 at 24 hr after electroporation and cultured on an anti-CD3 antibody-coated plate for 72 hr. (A) Histogram of PKH26 expression from one donor; (B) mean proliferative index [T(X) plus SD] from three different donors. NS, not significant. (C) LAT-substituted CD4+ T cells were cultured on anti-CD3 antibody-coated plates for 72 hr, and then the cytokine producing profiles were evaluated by intracellular staining after a 4-hr stimulation in the presence of phorbol myristate acetate (PMA) and ionomycin.
FIG. 5.
FIG. 5.
Enhanced cytotoxicity of TCR and chimeric antigen receptor (CAR)-redirected CD8+ T cells expressing LAT ubiquitylation mutants. (A, C, and D) Flow-based cytotoxicity assays against NY-ESO-1-transduced Nalm-6 target cells were performed after a 4-hr incubation with effector cells. Effector cells were anti-NY-ESO-1 TCR expressed in CD8+ T cells with endogenous LAT knockdown (A); anti-NY-ESO-1 TCR expressed in CD8+ T cells with the indicated LAT mutants without endogenous LAT knockdown (C); and anti-CD19-ζ CAR expressed in CD8+ T cells with endogenous LAT knockdown (D). The average±SD of triplet samples was plotted. Statistically significant differences are indicated by asterisks: *p<0.01 (Student t test); **p<0.05 (Cochran–Cox). (B) CD107a mobilization assay against 624.38 (NY-ESO-1+HLA-A2+) or 526 (NY-ESO-1−HLA-A2+) melanoma target cells was performed with anti-NY-ESO-1 TCR-expressing LAT-substituted T cells. CD8+ gated cells are shown.
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