Importance of integrin LFA-1 deactivation for the generation of immune responses

Abstract

The dynamic regulation of ligand binding is considered crucial for integrin function. However, the importance of activity regulation for integrin function in vivo is largely unknown. Here, we have applied gene targeting to delete the GFFKR sequence of the lymphocyte function-associated antigen-1 (LFA-1) alpha(L) subunit cytoplasmic domain in mouse germline. Lymphocytes from Lfa-1(d/d) mutant mice showed constitutive activation of LFA-1-mediated cell adhesion and impaired de-adhesion from intercellular adhesion molecule-1 that resulted in defective cell migration. In contrast, signaling through LFA-1 was not affected in Lfa-1(d/d) cells. T cell activation by superantigen-loaded and allogeneic APCs, cytotoxic T cell activity, T-dependent humoral immune responses, and neutrophil recruitment during aseptic peritonitis were impaired in Lfa-1(d/d) mice. Thus, deactivation of LFA-1 and disassembly of LFA-1-mediated cell contacts seem to be vital for the generation of normal immune responses.

Figure 1.

Generation of Lfa-1 ^d/d mutant mice. (a) Organization of the murine Lfa-1 genomic locus (top), targeting vector (second from top), targeted Lfa-1 ^d allele (second from bottom), and Lfa-1 ^d allele following intercross with the cre deleter strain (bottom). (b) Southern blot analysis of genomic DNA derived from a wild-type mouse (lane 1) and a heterozygous Lfa-1 ^d-neo/+ mouse (lane 2) before deletion of the neocassette using the 3′ flanking probe. (c) PCR analysis of genotypes from wild-type (+/+), heterozygous Lfa-1 ^d/+ (d/+), and homozygous Lfa-1 ^d/d (d/d) mice after deletion of the neocassette. To detect the mutant allele, a genomic fragment containing the newly introduced HpaI site in exon 31 was amplified by PCR and digested with HpaI.

Figure 2.

Integrin expression in Lfa-1 ^d/d mice. (a) Cell surface levels of LFA-1 and total cellular LFA-1 protein were measured by flow cytometry analyses of Lfa-1 ^d/d (red lines) and wild-type (blue lines) splenocytes. (b) Cell surface integrin expression was determined on Lfa-1 ^d/d (red lines) and wild-type (blue lines) splenic T cells (CD3⁺ cells), B cells (B220⁺ cells), and bone marrow neutrophils (Gr-1⁺ cells). Staining with isotype-matched controls is indicated as dotted lines (n = 3 independent experiments).

Figure 2.

Integrin expression in Lfa-1 ^d/d mice. (a) Cell surface levels of LFA-1 and total cellular LFA-1 protein were measured by flow cytometry analyses of Lfa-1 ^d/d (red lines) and wild-type (blue lines) splenocytes. (b) Cell surface integrin expression was determined on Lfa-1 ^d/d (red lines) and wild-type (blue lines) splenic T cells (CD3⁺ cells), B cells (B220⁺ cells), and bone marrow neutrophils (Gr-1⁺ cells). Staining with isotype-matched controls is indicated as dotted lines (n = 3 independent experiments).

Figure 3.

Lfa-1 ^d/d mutation enhances cell adhesion but impairs endothelial transmigration. Thymocytes were incubated with plates coated with purified ICAM-1(D1-2)-Fc (a) or VCAM-1-Fc (b) and the fraction of adherent cells was determined (n = 4–8 independent experiments). (c) Lymph node cells stimulated with PMA and ionomycin and rested in IL-2 were incubated with CD3 mAbs for the indicated periods (10 and 40 min) and adhesion to ICAM-1(D1-2)-Fc was determined. Results are presented as percentage increase of ICAM-1(D1-2)-Fc adhesion of CD3-stimulated as compared with unstimulated cells. (d) Splenocytes (SPL) or lymph node (LN) cells were plated on bEnd5 endothelioma cells that were unstimulated (medium) or treated with TNF for 16 h. The number of cells adherent per unit area were determined (n = 5 independent experiments). (e) Transmigration of unstimulated (medium) or TNF-treated bEnd5 endothelioma cells by splenocytes or lymph node cells (right panel) was measured (n = 6 independent experiments). *P < 0.05; #P < 0.01 (Student's t test).

Figure 4.

T cell adhesion and migration on immobilized ICAM-1-Fc. (a) Adhesion of spleen T cells onto immobilized ICAM-1 in the presence of 6 mM Mg²⁺ was determined after 30 min (mean + SD). (b) T cells migrating on 50 μg/ml immobilized ICAM-1-Fc with calculation of speed over 10 min. The speed of Lfa-1 ^d/d T cells is expressed as percentage of wt T cell speed + SEM (***P < 0.01). (c) Phase images of migrating T cells. The red circle indicates the position of the T cell at 0 s; the yellow line details the T cell trajectory over 180 s. White bars, 10 μm. (d) Migration of T cells was tracked over 600 s; each line represents one T cell (wt, n =20; Lfa-1 ^d/d, n = 21). Experiments are representative of n =2 (a) and n = 6 (b–d).

Figure 5.

Lfa-1 ^d/d mutation impairs T cell proliferation stimulated by APCs. Splenocytes from wild-type or Lfa-1 ^d/d mice were stimulated with the indicated concentrations of SEB (a) or irradiated BALB/c splenocytes or autologous cells (b). ³[H]-thymidine incorporation was measured 72 h later (n = 6 independent experiments). (c) Splenocytes were incubated with SEB (10 μg/ml) for 16 h. The percentage of CD4 T cells from wild-type and Lfa-1 ^d/d mice expressing CD25 and CD69 was determined by flow cytometry (n = 4–5 independent experiments). (d) Enriched splenic T cells were stimulated with plate-bound CD3 antibody, and ³[H]-thymidine incorporation was measured after 72 h (n = 4 independent experiments). *P < 0.05; #P < 0.01 (Student's t test).

Figure 6.

Lfa-1^d/d mutation does not alter T cell apoptosis and LFA-1–mediated signaling. (a) Splenocytes from wild-type or Lfa-1 ^d/d mice were stimulated with SEB (10 μg/ml) and the percentage of Vβ8⁺ T cells that stained positive for annexin V was determined by flow cytometry (n = 4 independent experiments). (b) The fraction of Vβ8⁺ CD4 and CD8 T cells was determined in popliteal lymph nodes of mice left untreated (Ctrl) and 24 h after s.c. injection of 10 μg SEB into each hind footpad. n = 4 mice per group; #P < 0.01, *P < 0.05 (ctrl vs. SEB). (c) Lymph node cells from Lfa-1 ^d/d (d/d) and wild-type mice (+/+) were unstimulated or incubated with 10 μg/ml purified soluble ICAM-1(D1-2)-Fc for 30 min in the presence of 1 mM Mn²⁺. Phosphorylation and total protein levels of Jnk and Erk-1/2 were determined by Western blot analyses of cell lysates. (d) Nuclear extracts were prepared from thymocytes of Lfa-1 ^d/d (d/d) and wild-type mice (+/+), and protein levels of p27^Kip1 and laminA/C were determined by Western blot analysis.

Figure 7.

Distinct effects of the Lfa-1^d/d mutation on the lytic activity and generation of cytotoxic T cells. (a) Lfa-1^d/d and wild-type mice were immunized with OVA using CpG-DNA as adjuvant. Draining lymph node cells were harvested 4 d later and cultured with rIL-2 for an additional 4 d. CTL activity was assayed using untreated (dotted lines) or SIINFEKL-pulsed (solid lines) EL4 cells (n = 3–4 independent experiments). (b) Lfa-1^d/d (d/d) and wild-type mice (wt) were immunized with OVA and CpG-DNA. Draining lymph nodes were harvested 9 d later and CD8 T cells were analyzed for SIINFEKL/H-2K^b tetramer and CD62L antibody staining (n = 6 independent mice per group). *P < 0.05; #P < 0.01 (Student's t test).

Figure 8.

Impaired T cell–dependent antibody responses in Lfa-1 ^d/d mice. T cell-dependent production of TNP-specific antibody isotypes was measured in serum of wild-type (diamonds) and Lfa-1 ^d/d mice (circles) after immunization of mice with TNP-CGG adsorbed to alum (n = 7–9 independent mice per group). *P < 0.05; #P < 0.01 (Student's t test).

Figure 9.

Delayed neutrophil recruitment during peritonitis in Lfa-1 ^d/d mice. Lfa-1 ^d/d and wild-type mice were injected i.p. with thioglycollate and infiltrating neutrophils were identified by their Gr-1^hiMac-1^hi-staining profile in flow cytometry analysis n = 4–6 independent mice per group. *P < 0.05 (Student's t test).