Retention of CXCR4 in the endoplasmic reticulum blocks dissemination of a T cell hybridoma

Abstract

The dissemination of T cell hybridomas to multiple nonhematopoietic tissues is blocked by pertussis toxin, suggesting the involvement of a chemokine. To study whether this chemokine is SDF-1, we employed a strategy proposed previously for gene therapy of AIDS, whereby the SDF-1 receptor CXCR4 (also a coreceptor for HIV) is retained in the endoplasmic reticulum (ER) and fails to reach the cell surface. We transfected SDF-1, carrying an ER retention sequence, into a T cell hybridoma. This altered chemokine is retained in the ER, where it binds CXCR4 and prevents the latter protein from reaching the surface. These cells failed to migrate toward SDF-1 or to invade fibroblast monolayers, although they could still migrate toward thymus and activation-regulated chemokine (TARC) and invade TARC-treated monolayers. Furthermore, the ability of the transfected cells to disseminate to multiple organs upon intravenous injection into mice was abolished. This dissemination reflects the in vivo migration patterns of activated and memory T cells into nonhematopoietic tissues, which is thus likely to depend on CXCR4. Attempts to block CXCR4 function as a therapy for AIDS may affect this migration with consequences for T cell function. Our results also suggest a decisive role for CXCR4 in the dissemination of hematopoietic malignancies expressing this receptor.

Figure 1

Removal of CXCR4 using an intrakine method. (a) Schematic representation of expression vectors. A cDNA encoding SDF-1 with a C-terminally attached KDEL-sequence was cloned into the retroviral vector pLZRS-IRES-Hyg-EGFP. The empty vector lacks the SDF-KDEL constructs but does contain the IRES sequence and the cDNA encoding the hygromycin resistance-EGFP fusion protein. (b) FACS analysis of EGFP expression of the SDF-1-KDEL clones. Filled histograms are EGFP levels, open histograms are from an untransfected control. Shown are two representative clones out of four. (c) Binding of ¹²⁵I-SDF to the SDF-1-KDEL clones compared with TAM2D2 cells and the empty vector-transfected control cells. Shown is one representative experiment out of three.

Figure 2

Migration toward SDF-1 and TARC of SDF-1-KDEL transfectants. Data are percentages of cells that have migrated in 2 hours through a filter to the lower chamber of a transwell containing 100 ng/ml of the chemokine. Data are averages ± SEM of three experiments performed.

Figure 3

Invasion of SDF-1-KDEL clones into REF monolayers, either pretreated or not pretreated with 100 ng/ml TARC. Data are the percentages of cells that have invaded within 1 hour and are averages ± SEM of five experiments performed.

Figure 4

Dissemination of SDF-1-KDEL clones. Shown are survival curves of nude mice injected intravenously with 5 × 10⁵ cells. Mice were killed when moribund or after 100 days. Five mice were injected with empty vector transfectants and ten and nine mice were injected with the SDF-K7 and SDF-K17 transfectants, respectively.

Figure 5

Reduction of CXCR4 surface levels by the nonsignaling mutant SDF-1-KDEL, SDF(K1R)-K. (a) Schematic representation of expression vectors. The first amino acid of the mature SDF-1 protein (K) was mutated to an arginine (R) to obtain a nonsignaling SDF-1 and linked to a KDEL sequence. The cDNA was expressed using the retroviral vector pLZRS-IRES-Hyg-EGFP. (b) FACS analysis of EGFP expression of SDF(K1R)-KDEL clones. Filled histograms are EGFP levels, open histograms are from an untransfected control. Shown are two representative clones out of five. (c) Binding of ¹²⁵I-SDF to mutated SDF(K1R)-KDEL clones as compared with TAM2D2 cells and the empty vector tranfectants. Shown is one representative experiment out of three.

Figure 6

Migration, invasion, and dissemination of the SDF(K1R)-KDEL clones. Shown are the results of two of five clones, SDF(K1R)-K10 and SDF(K1R)-K30. (a) Data are the percentage of cells that have migrated in 2 hours through a filter to the lower chamber of a transwell containing different concentrations of SDF-1 and are averages of five experiments performed. (b) Data are the percentages of cells that have invaded into the REF monolayer after 1 hour and are averages ± SEM of six experiments performed. (c) Survival of syngeneic AKR mice injected intravenously with 5 × 10⁵ cells is shown. Animals were killed when moribund or after 100 days.

Figure 7

Migration of the TARC-KDEL transfectant. Data are the percentages of cells that have migrated in 2 hours through a filter to the lower chamber of a transwell containing 100 ng/ml SDF-1 or TARC and are averages of eight experiments performed.

Figure 8

Expression of SDF-1 in different tissues of the mouse. SDF-1 is expressed in all tested tissues. Actin expression is shown as a control for the RT-PCR.