Itk controls the spatiotemporal organization of T cell activation

Abstract

During T cell activation by antigen-presenting cells (APCs), the diverse spatiotemporal organization of components of T cell signaling pathways modulates the efficiency of activation. Here, we found that loss of the tyrosine kinase interleukin-2 (IL-2)-inducible T cell kinase (Itk) in mice altered the spatiotemporal distributions of 14 of 16 sensors of T cell signaling molecules in the region of the interface between the T cell and the APC, which reduced the segregation of signaling intermediates into distinct spatiotemporal patterns. Activation of the Rho family guanosine triphosphatase Cdc42 at the center of the cell-cell interface was impaired, although the total cellular amount of active Cdc42 remained intact. The defect in Cdc42 localization resulted in impaired actin accumulation at the T cell-APC interface in Itk-deficient T cells. Reconstitution of cells with active Cdc42 that was specifically directed to the center of the interface restored actin accumulation in Itk-deficient T cells. Itk also controlled the central localization of the guanine nucleotide exchange factor SLAT [Switch-associated protein 70 (SWAP-70)-like adaptor of T cells], which may contribute to the activation of Cdc42 at the center of the interface. Together, these data illustrate how control of the spatiotemporal organization of T cell signaling controls critical aspects of T cell function.

Fig. 1

Itk accumulates at the center of the T cell–APC interface. (A) The numbers of cell couples formed between DO11.10 T cells expressing Itk PHTHSH3SH2-GFP and A20 APCs in the presence of Ova peptide (10 μM) that displayed accumulation of Itk PHTHSH3SH2-GFP with the indicated patterns [as previously defined (3)] are expressed as percentages of the total number of tight cell couples formed. Forty-one cell couples from multiple independent experiments were analyzed. Error bars represent the SEM. (B) Representative interaction of a transduced DO11.10 T cell expressing Itk PHTHSH3SH2-GFP with an APC in the presence of Ova peptide (10 μM) over the indicated time points (in minutes) relative to the time of formation of a tight cell couple. DIC images are shown in the top panels, whereas top-down, maximum projections of three-dimensional Itk PHTHSH3SH2-GFP fluorescence data are shown in the bottom panels in a rainbow-like, false color intensity scale (increasing in intensity from blue to red). A movie depicting the entire time frame is provided as movie S1 with the caption in table S1.

Fig. 2

Itk regulates multiple elements of the spatiotemporal organization of T cell signaling. (A and B) Cluster analysis of the patterning data of the indicated sensors during the activation of (A) wild-type (WT) and (B) Itk-deficient DO11.10 T cells. The percentages of cell couples that displayed the indicated patterns (C, central; Inv, invagination; D, diffuse; AC, asymmetric concentrated; P, peripheral; L, lamellal) relative to the total number of tight cell couples formed in shades of red from C-40 to L420 are shown in the top part of each panel. Time points range from 40 s before cell couple formation (−40 s) to 420 s after cell couple formation. Pattern changes normalized to 20-s intervals are shown in the bottom parts of each panel (C-40 to L300). Red indicates an increase and green a decrease in the percentage occurrence of a pattern relative to the previous time point. The cluster tree derived by cluster analysis based on Pearson’s correlation is given in pink. Principal groups of patterns as defined by the cluster analysis are denoted by boxes, with central or diffuse in red, peripheral in green, and lamellal in blue. (C and D) The numbers of cell couples that displayed accumulation of the sensor for active Cdc42 with the indicated patterns are expressed as percentages of the total numbers of (C) WT and (D) Itk-deficient DO11.10 T cells that formed tight cell couples. Sixty-six and 52 cell couples were analyzed for (C) and (D), respectively. (E and F) Representative interactions of (E) WT and (F) Itk-deficient DO11.10 T cells expressing the sensor for active Cdc42 with A20 APCs in the presence of Ova peptide (10 mM). Two T cells (T1 and T2) are shown in (E), and the time relative to tight cell couple formation refers to T1. Movies covering the entire time frames are provided as movies S2 (E) and S3 (F).

Fig. 3

Centrally targeted, active Cdc42 restores the accumulation of actin at the interface of Itk-deficient T cells. (A to C) The numbers of cell couples containing Itk-deficient DO11.10 T cells that displayed accumulation of retrovirally expressed (A) tat–Tec-PHTHSH3–GFP–Cdc42ca, (B) tat–ZAP-70 tandem SH2-GFP-Cdc42ca, and (C) tat-GFP-Cdc42ca with the indicated patterns are expressed as percentages of the numbers of tight cell couples formed. The numbers of cell couples analyzed for (A), (B), and (C) were 33, 32, and 59, respectively. (D to F) Representative interactions of DO11.10 T cells expressing (D) tat–Tec-PHTHSH3–GFP–Cdc42ca, (E) tat–ZAP-70 tandem SH2-GFP-Cdc42ca, and (F) tat-GFP-Cdc42ca with A20 APCs in the presence of Ova peptide (10 μM). Movies covering the entire time frames are provided as movie S4 (D), movie S5 (E), and movie S6 (F). (G to I) WT and Itk-deficient DO11.10 T cells, as indicated, were retrovirally transduced to express actin-GFP and were activated in the presence of the indicated concentrations of (G) tat–Tec-PHTHSH3–Cdc42ca, (H) tat–ZAP-70 tandem SH2-Cdc42ca, and (I) tat-Cdc42ca. The mean fluorescence intensity of actin-GFP at the T cell–APC interface relative to that of the background cell fluorescence in tight cell couples is shown. The same control data for WT and Itk-deficient T cells treated with buffer alone are included in each panel. On average, 41 (range, 29 to 74) cell couples were analyzed for each condition (570 total cell couples analyzed). Movies displaying representative T cell actin dynamics are provided as movies S17 and S18.

Fig. 4

Itk regulates the localization of Cdc42 GEFs. (A to L) The numbers of cell couples containing WT (A, E, and I) or Itk-deficient (C, G, and K) DO11.10 T cells that displayed the indicated patterns of accumulation of (A and C) GFP-SLAT, (E and G) Vav1-GFP, and (I and K) GFP–α-Pix are expressed as percentages of the numbers of tight cell couples formed. On average, 58 cell couples (range, 27 to 95) were analyzed for each condition (350 total). Representative interactions of WT (B, F, and J) and Itk-deficient (D, H, and L) DO11.10 T cells expressing (B and D) GFP-SLAT, (F and H) Vav1-GFP, or (J and L) GFP–α-Pix with A20 APCs in the presence of Ova peptide (10 μM) are shown. Two T cells (T1 and T2) are included in (B), and the time relative to tight cell couple formation refers to T1. Movies covering the entire time frames are provided as movie S7 (B), movie S8 (D), movie S9 (F), movie S10 (H), movie S11 (J), and movie S12 (L).

Fig. 5

SLAT knockdown results in a phenotype similar to that of Itk deficiency. (A to C) The numbers of cell couples that displayed accumulation of the sensor for active Cdc42 with the indicated patterns are expressed as percentages of the total number of tight cell couples formed involving WT or Itk-deficient DO11.10 T cells upon knockdown of (A and B) SLAT and in (C) cell couples containing WT T cells upon knockdown of Vav1. The numbers of cell couples analyzed for (A), (B), and (C) were 25, 16, and 33, respectively. (D) Mean fluorescence intensities of actin-GFP at the T cell–APC interface relative to that of the background cell fluorescence of tight cell couples containing WT or Itk-deficient DO11.10 T cells that were transfected to express either control or SLAT-specific shRNA, as indicated, with A20 APCs in the presence of Ova peptide (10 μM). The reference data for the WT and Itk-deficient T cells are from Fig. 3G. The numbers of cell couples analyzed in the context of SLAT knockdown were 22 and 33 for WT and Itk-deficient T cells, respectively. (E) The mean fluorescence intensities of actin-GFP at the T cell–APC interface relative to that of the background fluorescence in tight cell couples containing either WT or Itk-deficient DO11.10 T cells transfected to express either control or Vav1-specific shRNA, as indicated, and A20 APCs in the presence of Ova peptide (10 μM). The reference data for the WT and Itk-deficient T cells are from Fig. 3G. Thirty-five cell couples were analyzed in the context of Vav1 knockdown.

Fig. 6

The SH2 domain of Itk is critical for its regulation of TCR localization. (A to I) The numbers of cell couples that displayed accumulation of TCRζ-GFP with the indicated patterns in tight cell couples containing (A) WT DO11.10 T cells, (C) Itk-deficient DO11.10 T cells, (E) Itk-deficient T cells retrovirally transduced to express WT Itk, (F) Itk-deficient T cells retrovirally transduced to express the kinase-deficient mutant Itk K390R, (G) Itk-deficient T cells retrovirally transduced to express the SH2 mutant (SH2mt) Itk R265K, and (I) WT T cells retrovirally transduced to express the SH2 mutant Itk R265K are expressed as percentages of the total numbers of tight cell couples formed. An average of 59 cell couples (range, 32 to 98) were analyzed for each condition (352 total). Representative interactions of (B) WT or (D and H) Itk-deficient DO11.10 T cells expressing TCRζ-GFP alone (B and D) or together with the SH2 mutant Itk R265K (H) and A20 APCs in the presence of Ova peptide (10 μM). Movies covering the entire time frames are provided as movie S14 (B), movie S15 (D), and movie S16 (H).