Abstract
Kindlin-3 is an important integrin regulator that is mutated in the rare genetic disorder, leukocyte adhesion deficiency type III, a disorder characterized by defective neutrophil trafficking and platelet function, leading to recurrent bacterial infections and bleeding. Kindlin-3 is also known to regulate T cell adhesion in vitro and trafficking in vivo, but whether the integrin/kindlin interaction regulates T or B cell activation in vivo is unclear. In this study, we used TTT/AAA β2-integrin knock-in (KI) mice and TCR-transgenic (OT-II) KI mice, in which the integrin/kindlin connection is disrupted, to investigate the role of the integrin/kindlin interaction in T cell activation. We show that basal T cell activation status in these animals in vivo is normal, but they display reduced T cell activation by wild-type Ag-loaded dendritic cells in vitro. In addition, T cell activation in vivo is reduced. We also show that basal Ab levels are normal in TTT/AAA β2-integrin KI mice, but B cell numbers in lymph nodes and IgG and IgM production after immunization are reduced. In conclusion, we show that the integrin/kindlin interaction is required for trafficking of immune cells, as well as for T cell activation and B cell Ab responses in vivo. These results imply that the immunodeficiency found in leukocyte adhesion deficiency type III patients, in addition to being caused by defects in neutrophil function, may be due, in part, to defects in lymphocyte trafficking and activation.
Introduction
Integrins are heterodimeric cell surface adhesion molecules consisting of α and β subunits. Expression of the β2 (CD18) integrin subfamily is restricted to leukocytes, where they play important roles in cellular adhesion and migration in the immune system (1). The β2-integrin family member leukocyte function–associated Ag-1 (LFA-1; αLβ2, CD11a/CD18) is highly expressed in lymphocytes, namely B and T cells, and mediates binding to ICAMs on the surface of other cells. LFA-1 mediates firm adhesion to endothelial cells, which is necessary for extravasation of lymphocytes from the bloodstream into lymph nodes and sites of inflammation (2). Indeed, LFA-1–knockout mice display impaired lymphocyte homing to these sites (3–6).
In addition to its role in cellular migration, LFA-1 is a key component of the immunological synapse (IS) that forms between immune cells. For example, IS formation between APCs, such as dendritic cells (DCs), and CD4 T cells initiates T cell activation, and subsequent IS formation between CD4 T cells and B cells allows the provision of help for B cell Ab production. Specifically, LFA-1 forms part of the peripheral supramolecular activation cluster that surrounds the TCR or BCR cluster in the center, thus stabilizing the synapse and ensuring efficient lymphocyte activation (7). It is thought that LFA-1 downstream signaling may also contribute to the cellular activation signals, thus performing a costimulatory function, as was shown in T cells (8–10). However, the involvement of LFA-1 in T and B cell activation in vivo remains controversial because of the difficulty in segregating the roles of LFA-1 in migration versus activation.
Conformational changes in LFA-1 required for optimal ligand binding, as well as downstream integrin signaling, are regulated by the binding of cytoplasmic factors to the integrin subunit intracellular domains. Previously, we generated a mutant mouse line in which the threonine triplet in the β2-integrin tail was substituted with alanine residues (TTT/AAA β2-integrin knock-in (KI) mouse). This mutation abolishes binding of the important integrin regulator kindlin-3 to the integrin cytoplasmic domain (11), resulting in impaired integrin activation to its high-affinity state and, therefore, reduced integrin function. We showed that polyclonal activation of TTT/AAA β2-integrin KI T cells in vitro with soluble anti-CD3 results in a reduction in T cell activation and proliferation compared with wild-type (WT) T cells, whereas activation in response to plate-bound anti-CD3 is unaffected by the KI mutation (11). Ag-specific T and B cell activation in vivo in these mice remains largely unexplored.
Using integrin TTT/AAA β2-integrin KI and KI TCR-transgenic (OT-II) mouse models, we show that Ag-specific CD4 T cell activation in both an in vitro coculture system with DCs and in vivo in the spleen is dependent on the β2-integrin TTT site. We also reveal that optimal B cell numbers in lymph nodes and B cell Ab responses in vivo are dependent on fully functioning LFA-1 in leukocytes. These results indicate a vital role for LFA-1–mediated firm adhesion in lymphocyte activation, even in circumstances where LFA-1–mediated migration is unlikely to be involved.
Materials and Methods
Mice
Mice were bred and maintained at the University of Dundee or the University of Helsinki, in compliance with national and local rules. The TTT/AAA β2-integrin KI mouse line was described previously (11). The KI mice were crossed with OT-II mice (provided by Prof. Colin Watts, University of Dundee) to generate homozygote Itgb2 KI mice expressing the OT-II transgene. Expression of the v-α2 and v-β5 TCR subunits was confirmed by flow cytometry, and the KI mutation as confirmed by PCR. C57BL/6 mice were purchased from Charles River. In all experiments, age- and sex-matched mice were used.
Cell preparation
Bone marrow–derived dendritic cells (BMDCs) were generated by culturing mouse bone marrow for 10 d in 10 ng/ml GM-CSF (PeproTech) in nontissue culture–treated petri dishes. Cells were given fresh medium and growth factor on days 3, 6, and 8 and were harvested on day 10 using 4 mM EDTA solution. CD4 T cells were purified from mouse spleens and lymph nodes by positive selection using magnetic beads to CD4 (Miltenyi Biotec), according to the manufacturer’s instructions. Splenic B cells were purified by negative selection with anti-mouse CD43 magnetic beads (Miltenyi Biotec), according to the manufacturer’s instructions. The purity of B and T cells was determined by flow cytometry and was typically >95%. In some instances, purified CD4 T cells and B cells were labeled with 5 μM CFSE (Life Technologies) for 10–20 min at 37°C, according to the manufacturer’s guidelines. All cell cultures were performed in RPMI 1640 medium supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin-streptomycin, and 50 μM 2-ME. Both CD4 T cells and B cells expressed only the LFA-1 (αLβ2) β2-integrin family member (Supplemental Fig. 2).
BMDC–T cell coculture
BMDCs were either stimulated with 100 ng/ml LPS (Sigma-Aldrich) for 24 h or left unstimulated. Both stimulated and unstimulated BMDCs were loaded with peptide OVA Ag (OVA 323–339; AnaSpec) for 90 min. Ag-loaded BMDCs were cultured together with purified CD4 OT-II T cells at a ratio of 1:10, typically with 1 × 106 T cells in a total volume of 1 ml. Cells and supernatants were taken on days 1–5 of the coculture for analysis of T cell activation markers by flow cytometry and cytokine production by ELISA, respectively.
B cell proliferation
Purified, CFSE-labeled B cells were seeded on a 96-well plate (CELLSTAR) at a density of 1 × 106 cells/well. B cells were stimulated with LPS (Sigma-Aldrich, St. Louis, MO) or anti-mouse IgM (Jackson ImmunoResearch Europe, Suffolk, U.K.) or were left untreated. CFSE fluorescence was detected with flow cytometry 48 h after stimulation.
Adoptive transfer and immunizations
WT recipient mice received a total of 2 × 106 purified CFSE-labeled T cells i.v. in a volume of 200 μl. Twenty-four hours later, the recipient mice were immunized with 100 μg peptide OVA (AnaSpec) in 10 μg LPS (Sigma-Aldrich) in a total volume of 200 μl i.v. into the tail vein. The splenic T cell response was analyzed 5 d postimmunization.
To assess the B cell Ab response in WT and KI mice, mice were immunized s.c. in the hind legs with 100 μg protein OVA (Endofit; InvivoGen) in CFA (Sigma-Aldrich) in a volume of 50 μl/leg. Blood samples were taken prior to immunization and weekly thereafter, and serum was isolated and frozen for Ab analysis by ELISA.
Flow cytometry
Cells were stained with the following fluorescently labeled Abs: B220-FITC (RA3-6B2; BioLegend), CD4-PerCP (RM4-5), CD11a-PE (2D7), CD11b-allophycocyanin (M1/70), CD11c-PE-Cy7 (HL-3), CD18-FITC (C71/16), CD21/CD35-PE-Cy7 (eBio8D9; eBioscience), CD23–eFluor 660 (B3B4; eBioscience), CD25-allophycocyanin-Cy7 (PC61), CD40-PE (3/23), CD44-PE (IM7), CD62L-FITC (MEL-14), CD69-allophycocyanin (H1.2F3; BioLegend), CD80-allophycocyanin (16-10A1), CD86-FITC (GL1), IgM-allophycocyanin-eFluor 780 (II/41, eBioscience), IgD-PerCP-eFluor 710 (11-26c; eBioscience), and MHC class II–allophycocyanin-Cy7 (M5/114.15.2). Abs were from BD Biosciences unless otherwise mentioned. Fc block (4.4G2) was included in all stains. Data were acquired on a LSR II flow cytometer (Becton Dickinson) and analyzed using FlowJo software (TreeStar).
Real-time quantitative PCR
Spleen tissue of WT and KI mice was homogenized with ULTRA-TURRAX T8 (IKA) in RA1 included in the NucleoSpin RNA II Total RNA isolation kit (MACHEREY-NAGEL; Bioline, London, U.K.). RNA was extracted according to the manufacturer’s instructions and used as a template for cDNA synthesis. cDNA was synthesized from 0.5 μg total RNA with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies). PCR primers and probes were obtained from Life Technologies. A TaqMan gene expression assay was performed with TaqMan Fast Advanced MasterMix, and quantitative real-time PCR was performed with a 7500 Fast Real-Time PCR System and SDS Software v.1.4.0 (Applied Biosystems). Gene expression was normalized with 18S rRNA, and target gene expression was calculated by the comparative CT method (Applied Biosystems).
ELISAs
Cytokine levels produced in T cell cultures, namely IL-2 and IFN-γ, were measured by ELISA using paired Ab kits (eBioscience), according to the manufacturer’s instructions.
Total and OVA-specific serum Ab levels were detected by ELISA. Briefly, 5 μg/ml protein OVA (InvivoGen) was coated onto Nunc MaxiSorp plates at 4°C overnight. Alternatively, for total Ab measurements, unlabeled Ig subclass–specific Abs (Southern Biotech) were coated onto plates. After washing, serum was added, and serial dilutions were performed and incubated at room temperature for 2 h. Ab subclass–specific AP-labeled Abs (Southern Biotech) were used for detection, according to standard protocols, and A405 was measured.
Western blot
T and B cells were activated with anti-CD3 or anti-IgM, respectively, for 0, 6, 30, or 60 min and lysed in 1% Tx-100, 150 mM NaCl, 50 mM Tris (pH 7.4), 10 mM EDTA in the presence of phosphatase and protease inhibitors (Pierce), and lysates were analyzed by Western blotting. Primary Abs against phospho-Syk/Zap70, total Syk/Zap, phospho-Erk1/2, phospho-Akt308, and total Akt were from Cell Signaling Technology.
Static adhesion assays
Static adhesion assays were performed as described (11). Briefly, the integrin ligands ICAM-1 (6 μg/ml; R&D Systems), fibronectin (10 μg/ml), and VCAM (6 μg/ml; R&D Systems) were coated onto 96-well MaxiSorp plates (Nunc) by overnight incubation at 4°C. Purified splenic B or T cells (1.5–2 × 106 cells/ml) were resuspended in adhesion medium (RPMI 1640 supplemented with 0.1% BSA, 40 mM HEPES, and 2 mM MgCl2) and added to the plate. Where appropriate, cells were stimulated with 200 nM PdBu (Sigma-Aldrich), 10 μg/ml anti-BCR (R&D Systems), 10 μg/ml anti-CD3 (R&D Systems), 0.2 μg/ml LPS (Sigma-Aldrich), or 5 mM MgCl2 + 1 mM EGTA immediately before being added to the plate. Cells were allowed to adhere for 30 min at 37°C before gentle washing and detection, as described (12).
Statistics
The Student t test and two-way ANOVA (GraphPad Prism) were used for statistical analysis. In all cases, p values are defined as *p < 0.05, **p < 0.01, and ***p < 0.001.
Results
Impaired activation of KI T cells in vitro
To investigate the role of the β2-integrin TTT site in immune cell activation and function, we made use of a novel TTT/AAA β2-integrin KI mouse that we described recently (11). These mice have a mutated TTT site in the β2-integrin subunit cytoplasmic domain, meaning that the binding of the important integrin regulator, kindlin-3, is lost, and downstream integrin signaling is ablated (11). We reported previously that activation of KI T cells with plate-bound anti-CD3 or with phorbol ester in vitro appears normal, but there is a reduction in T cell activation (expression of CD69 and CD25) and a delay in T cell proliferation when soluble anti-CD3, in conjunction with IL-2, is used as a stimulus (11). We show in this article that increasing the amount of soluble anti-CD3 (Fig. 1A, 1B) or IL-2 (Fig. 1C) did not rescue the defect in T cell activation with soluble anti-CD3 seen in the KI cells. Higher doses of anti-CD3 also did not have any effect on T cell activation (data not shown). Early TCR signaling (p-Zap-70, p-Erk) appeared normal in the KI T cells (Fig. 1D). However, there was a reduction in KI T cell aggregation in response to anti-CD3 (Fig. 1E), indicating that cell–cell contact mediated, presumably, by LFA-1 and ICAM-1 on the surface of the T cells was reduced, which may influence T cell activation in this system. Indeed, addition of LFA-1 blocking Ab to the WT T cell cultures reduced T cell activation, showing that this process was LFA-1 dependent (Fig. 1F). Therefore, under conditions in which LFA-1 is engaged with ICAM-1 (using soluble anti-CD3 as a stimulus), polyclonal activation of KI T cells is impaired, and this impairment cannot be overcome by using increasing amounts of anti-CD3 or IL-2 in the cultures.
Impaired activation of TTT/AAA β2-integrin KI T cells in vitro. Polyclonal T cells from WT and KI mice were cultured with various concentrations of soluble anti-CD3 for 24 h; the expression of the activation markers CD69 and CD25 was assessed by flow cytometry (A), and IL-2 production was quantified by ELISA (B) (n = 4 mice). (C) Polyclonal WT and KI T cells were cultured with anti-CD3 (0.167 μg/ml) plus various concentrations of IL-2 for up to 2 d, and expression of CD69 and CD25 was assessed (n = 3 mice). The differences between WT and KI values are statistically significant (p < 0.05) for all data points with the exception of day 0. (D) WT and KI T cell signaling in response to anti-CD3 stimulation was assessed by Western blotting. Data are representative of n = 3 mice. (E) Representative images of T cell cultures showing cell clustering after 24 h of culture with various amounts of anti-CD3 and IL-2. Scale bars, 400 μm. (F) Polyclonal T cells from WT and KI mice were cultured for 24 h in the presence of soluble anti-CD3 (2.5 μg/ml), with or without blocking LFA-1 Ab (15 μg/ml), or were left untreated. The expression of CD69 and CD25 was assessed by flow cytometry (n = 4 mice). All error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, Student t test.
Impaired activation of TTT/AAA β2-integrin KI T cells in vitro. Polyclonal T cells from WT and KI mice were cultured with various concentrations of soluble anti-CD3 for 24 h; the expression of the activation markers CD69 and CD25 was assessed by flow cytometry (A), and IL-2 production was quantified by ELISA (B) (n = 4 mice). (C) Polyclonal WT and KI T cells were cultured with anti-CD3 (0.167 μg/ml) plus various concentrations of IL-2 for up to 2 d, and expression of CD69 and CD25 was assessed (n = 3 mice). The differences between WT and KI values are statistically significant (p < 0.05) for all data points with the exception of day 0. (D) WT and KI T cell signaling in response to anti-CD3 stimulation was assessed by Western blotting. Data are representative of n = 3 mice. (E) Representative images of T cell cultures showing cell clustering after 24 h of culture with various amounts of anti-CD3 and IL-2. Scale bars, 400 μm. (F) Polyclonal T cells from WT and KI mice were cultured for 24 h in the presence of soluble anti-CD3 (2.5 μg/ml), with or without blocking LFA-1 Ab (15 μg/ml), or were left untreated. The expression of CD69 and CD25 was assessed by flow cytometry (n = 4 mice). All error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, Student t test.
Baseline in vivo T cell activation is normal in TTT/AAA β2-integrin KI mice
Because in vitro KI T cell activation with soluble anti-CD3 and IL-2 was reduced, we next investigated T cell activation in vivo. Other investigators identified a role for LFA-1 in providing a costimulatory signal to T cells and contributing to their activation in vivo (8–10). Conversely, in the absence of β2-integrins, an increase in baseline T cell activation was reported (13), suggesting a role for β2-integrins in limiting T cell activation. We found that expression of activation markers CD25, CD44, and CD62L by splenic CD4 T cells was normal in unimmunized KI mice (Fig. 2A). Expression of CD69 was reduced significantly in KI CD4 T cells, although the levels of CD69 expression were generally very low (Fig. 2A). Proportions of naive (CD44loCD62Lhi) and effector (CD44hiCD62Llo) CD4 T cells in the spleen were equivalent in WT and KI mice (Fig. 2B). Thus, in vivo baseline activation levels of CD4 T cells are largely unaffected by the absence of LFA-1 downstream signaling.
TTT/AAA β2-integrin KI mice have normal baseline levels of T cell activation. (A) Splenic CD4 T cells from WT and KI mice were analyzed for their expression of typical T cell–activation markers CD25, CD69, CD44, and CD62L (n = 4 mice, from two independent experiments). Error bars represent SEM. (B) Representative plots showing percentages of naive CD44loCD62Lhi and effector CD44hiCD62Llo CD4 T cells in the spleen (representative of n = 4 mice). ***p < 0.001, Student t test.
TTT/AAA β2-integrin KI mice have normal baseline levels of T cell activation. (A) Splenic CD4 T cells from WT and KI mice were analyzed for their expression of typical T cell–activation markers CD25, CD69, CD44, and CD62L (n = 4 mice, from two independent experiments). Error bars represent SEM. (B) Representative plots showing percentages of naive CD44loCD62Lhi and effector CD44hiCD62Llo CD4 T cells in the spleen (representative of n = 4 mice). ***p < 0.001, Student t test.
TTT/AAA β2-integrin KI OT-II T cells show impaired activation in an in vitro coculture system
We previously reported normal activation of splenic KI T cells following i.v. transfer of WT, peptide-loaded DCs into the global KI mice (11). However, in addition to its reported role in T cell trafficking (11), the integrin/kindlin interaction plays an important role in restricting myeloid cell activation, cytokine production, and Th1 polarization in vivo (14). We wondered whether the expression of nonfunctional β2-integrins in our KI mice influenced the splenic microenvironment, potentially masking a negative effect of the mutation on splenic T cell activation in our previous experiment. Indeed, there was increased expression of IL-1β, but normal expression of IL-12 and CXCL10, in the KI spleens (Supplemental Fig. 1). Because IL-1β was reported to play a role in CD4 T cell activation (15), this may have influenced the activation of splenic T cells in vivo in our previous experiment (11). Therefore, to further investigate Ag-specific CD4 T cell activation in KI cells, we crossed TTT/AAA β2-integrin KI mice with OT-II mice, which allows for further investigations of T cell activation in vitro and in vivo. CD4 T cells from OT-II mice are TCR transgenic, specific for the OVA 323–339 peptide. Homozygous KI mice expressing the OT-II transgene were then used to assess Ag-specific T cell activation. First, KI OT-II+ T cell activation was assessed in an in vitro coculture system with Ag-presenting DCs. This experimental set-up requires the formation of an IS between T cells and DCs for efficient T cell activation, mimicking in vivo activation requirements. We generated WT BMDCs and either stimulated them with LPS to induce maturation and upregulation of costimulatory molecules or left them in an unactivated/immature state (Supplemental Fig. 2). These BMDCs were loaded with peptide OVA Ag and cultured together with CD4 T cells from WT or KI OT-II mice for up to 5 d. Our results show that KI T cells had reduced upregulation of the activation marker CD69. KI T cells also displayed reduced shedding of CD62L (Fig. 3A). This was the case in cultures with both immature and mature DCs (Fig. 3A). Expression levels of CD25 (the IL-2R) and CD44 were not significantly different between WT and KI cells cultured with LPS-activated DCs (Fig. 3A). Furthermore, KI T cells produced significantly less IL-2, the cytokine responsible for driving T cell proliferation, compared with WT T cells (Fig. 3B). Production of the typical Th1 cytokine IFN-γ was greater in the WT T cell cultures with LPS-activated DCs than with immature DCs, as expected. The KI T cell cultures showed reduced IFN-γ production compared with WT T cell cultures (Fig. 3B). Together, these data suggest that CD4 T cells lacking the β2-integrin TTT site have reduced ability to become activated in an in vitro coculture system with Ag-presenting DCs. Generally speaking, the impairment in KI T cell activation is more pronounced in cocultures with immature DCs compared with cultures with mature LPS-activated DCs. This suggests that TLR activation of DCs may provide stronger signals to T cells, thus overriding the requirement for functioning LFA-1.
TTT/AAA β2-integrin KI T cell activation is impaired in a coculture system. (A and B) WT BMDCs were cocultured with purified WT or KI OT-II CD4+ T cells. (A) The expression of typical T cell activation markers was measured on days 1–5 of culture. Immature BMDCs (upper panels) and LPS-matured BMDCs (lower panels). (B) The production of IL-2 and IFN-γ was measured on days 1–5 of culture. Immature BMDCs (upper panels) and LPS-matured BMDCs (lower panels). Graphs show cells from n = 4 mice, pooled from two independent experiments. All error bars represent SEM. *p < 0.05, **p < 0.01, KI versus WT, two-way ANOVA.
TTT/AAA β2-integrin KI T cell activation is impaired in a coculture system. (A and B) WT BMDCs were cocultured with purified WT or KI OT-II CD4+ T cells. (A) The expression of typical T cell activation markers was measured on days 1–5 of culture. Immature BMDCs (upper panels) and LPS-matured BMDCs (lower panels). (B) The production of IL-2 and IFN-γ was measured on days 1–5 of culture. Immature BMDCs (upper panels) and LPS-matured BMDCs (lower panels). Graphs show cells from n = 4 mice, pooled from two independent experiments. All error bars represent SEM. *p < 0.05, **p < 0.01, KI versus WT, two-way ANOVA.
TTT/AAA β2-integrin KI T cells have impaired activation in vivo
Next, we wanted to assess the role of the β2-integrin/kindlin-3 interaction in Ag-specific CD4 T cell activation in vivo. To do this, we purified and labeled CD4 T cells from either WT or TTT/AAA β2-integrin KI OT-II mice with CFSE and adoptively transferred them into WT recipient mice. T cells were transferred i.v., so they predominantly localized to the spleen. The recipient mice were immunized with OVA Ag in adjuvant i.v., and the donor T cell response in the spleen was measured. Because this experiment was done in the WT splenic environment, it avoids the potential influence of the KI splenic microenvironment on T cell activation in vivo. We found that the percentage of labeled donor cells recovered 5 d postimmunization was reduced in the KI group compared with the WT group (Fig. 4A). This reduction in KI T cell numbers reflects reduced proliferation of KI T cells compared with WT T cells, as demonstrated by the elevated expression of CFSE in KI cells (Fig. 4B). Expression of the activation marker CD44 also was reduced in KI T cells compared with WT cells (Fig. 4C), indicating that KI cells have a reduced state of activation. Together, the data presented in Fig. 4 reveal an important role for LFA-1β subunit tail interactions in the activation of CD4 T cells in vivo.
TTT/AAA β2-integrin KI T cell activation is impaired in vivo. Purified CD4+ WT or KI OT-II T cells were labeled with CFSE and injected i.v. into the tail vein of recipient WT mice. Twenty-four hours later, recipient mice were immunized i.v. with OVA peptide Ag in LPS adjuvant. Five days postimmunization, spleens were harvested. The percentage of donor CFSE+ cells (A), mean fluorescence intensity of CFSE (B), and expression of the typical T cell activation marker CD44 (C) were measured by flow cytometry (n = 5 mice). All error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, Student t test.
TTT/AAA β2-integrin KI T cell activation is impaired in vivo. Purified CD4+ WT or KI OT-II T cells were labeled with CFSE and injected i.v. into the tail vein of recipient WT mice. Twenty-four hours later, recipient mice were immunized i.v. with OVA peptide Ag in LPS adjuvant. Five days postimmunization, spleens were harvested. The percentage of donor CFSE+ cells (A), mean fluorescence intensity of CFSE (B), and expression of the typical T cell activation marker CD44 (C) were measured by flow cytometry (n = 5 mice). All error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, Student t test.
B cell Ab responses are reduced in TTT/AAA β2-integrin KI mice
Baseline Ab levels in unimmunized β2-integrin knockout mice were shown to be elevated (3). Therefore, we quantified circulating Ab levels in unimmunized TTT/AAA β2-integrin KI mice. We found levels of circulating IgG, IgM, and IgA, as well as total Ig, to be equivalent in WT and KI mice (Fig. 5A).
TTT/AAA β2-integrin KI mice have impaired Ab responses in vivo. (A) Baseline circulating Ab levels were measured in unimmunized WT and KI mice (n = 4 mice). (B) WT and KI mice were immunized s.c. with OVA protein Ag in CFA. Blood samples were taken weekly for 5 wk postimmunization, and circulating Ag-specific Ab levels were measured by ELISA (n = 5 mice). All error bars represent SEM. *p < 0.05, ***p < 0.001, WT versus KI, two-way ANOVA.
TTT/AAA β2-integrin KI mice have impaired Ab responses in vivo. (A) Baseline circulating Ab levels were measured in unimmunized WT and KI mice (n = 4 mice). (B) WT and KI mice were immunized s.c. with OVA protein Ag in CFA. Blood samples were taken weekly for 5 wk postimmunization, and circulating Ag-specific Ab levels were measured by ELISA (n = 5 mice). All error bars represent SEM. *p < 0.05, ***p < 0.001, WT versus KI, two-way ANOVA.
Efficient B cell–mediated humoral immunity is dependent on B cell homing and activation, as well as the provision of “help” from CD4 T cells, a process that requires the formation of an IS between these two cell types. Therefore, we went on to analyze B cell responses in vivo by measuring serum Ab levels in WT and KI mice following immunization with protein OVA Ag in adjuvant. The results show reduced levels of OVA-specific total Ig and IgG from 2 to 5 wk postimmunization in KI mice compared with WT controls (Fig. 5B). Similarly, IgM levels were lower in KI mice from as early as 7 d postimmunization until 3 wk postimmunization, when IgM levels began to diminish (Fig. 5B). These data suggest that optimal Ab production is dependent on the β2-integrin subunit TTT site in leukocytes.
B cell development in the bone marrow is normal, but there are reduced numbers of B cells in lymph nodes from TTT/AAA β2-integrin KI mice
Because Ab responses were altered in TTT/AAA β2-integrin KI mice (Fig. 5B), we next investigated B cell development and function in these mice. KI mice had normal proportions of mature (IgDhiIgM+) and immature (IgDloIgM+) B cells in the bone marrow (Fig. 6A), indicating normal B cell development. However, KI mice displayed abnormal proportions of these cells in the blood: KI mice had less mature and more immature circulating B cells compared with WT mice (Fig. 6B). We also studied follicular (Fo) and marginal zone (MZ) B cell subsets in WT and KI spleens. Flow cytometry analyses showed that the proportions of Fo (CD21intCD23hi) and MZ (CD21hiCD23lo) B cells were normal in the TTT/AAA β2-integrin KI mouse spleens (Fig. 6C).
TTT/AAA β2-integrin KI B cells show normal development but decreased homing. Proportions of mature and immature B cells isolated from bone marrow (A) and blood (B) of WT and KI mice, as assessed by flow cytometry. Cells were gated on the B220+ population and then on IgDhiIgM+ (mature) and IgDloIgM+ (immature) B cells. Representative dot plots and pooled data from three independent experiments (n = 5 mice for bone marrow, n = 3 for blood) are shown. (C) Flow cytometry analysis of splenic Fo (CD21intCD23+) and MZ (CD21hiCD23lo) B cells from WT and KI mice. Cells were gated on the B220+ population and studied for CD21 and CD23 expression. Representative dot plots and statistical data from two independent experiments (n = 4 mice) are shown. (D) Absolute numbers and proportions of B cells (B220+) from WT and KI lymphoid organs (n = 3–4 mice). (E) The ratio of mature (IgDhiIgM+) and immature (IgDloIgM+) B cells isolated from the lymph nodes of WT and KI mice was studied as in (A) and (B). Representative dot plots and pooled data from two independent experiments (n = 3 mice) are shown. All error bars represent SEM. *p < 0.05, **p < 0.01, Student t test.
TTT/AAA β2-integrin KI B cells show normal development but decreased homing. Proportions of mature and immature B cells isolated from bone marrow (A) and blood (B) of WT and KI mice, as assessed by flow cytometry. Cells were gated on the B220+ population and then on IgDhiIgM+ (mature) and IgDloIgM+ (immature) B cells. Representative dot plots and pooled data from three independent experiments (n = 5 mice for bone marrow, n = 3 for blood) are shown. (C) Flow cytometry analysis of splenic Fo (CD21intCD23+) and MZ (CD21hiCD23lo) B cells from WT and KI mice. Cells were gated on the B220+ population and studied for CD21 and CD23 expression. Representative dot plots and statistical data from two independent experiments (n = 4 mice) are shown. (D) Absolute numbers and proportions of B cells (B220+) from WT and KI lymphoid organs (n = 3–4 mice). (E) The ratio of mature (IgDhiIgM+) and immature (IgDloIgM+) B cells isolated from the lymph nodes of WT and KI mice was studied as in (A) and (B). Representative dot plots and pooled data from two independent experiments (n = 3 mice) are shown. All error bars represent SEM. *p < 0.05, **p < 0.01, Student t test.
We reported previously that KI mice have enlarged spleens, but smaller inguinal and mesenteric lymph nodes, compared with WT mice (11). In this study, we found that the absolute number of B220+ B cells was lower in KI lymph nodes than in WT lymph nodes (Fig. 6D), similar to what we reported previously for CD4+ T cells (11) and likely reflecting a problem with lymphocyte homing to the lymph nodes in KI mice. B cell numbers in the spleen were slightly increased in KI mice (Fig. 6D) due to splenomegaly. However, the proportions of B cells, as assessed by flow cytometry of isolated cells from spleen and other lymphoid organs (inguinal and mesenteric lymph nodes, Peyer’s patches, and bone marrow), were largely normal in TTT/AAA β2-integrin KI mice (Fig. 6D). The maturation state of WT and KI B cells in the lymph nodes also was assessed by flow cytometry; despite the reduced cellularity of the lymph nodes, the proportions of mature and immature B cells were normal in KI mice (Fig. 6E).
Together, these data implicate that B cell development in the bone marrow and subsequent maturation in secondary lymphoid organs are not disturbed by the TTT/AAA mutation of β2-integrin, but problems in homing to lymph nodes result in lower numbers of B220+ cells in lymph nodes and an increased proportion of immature B cells in the bloodstream of TTT/AAA β2-integrin KI mice.
B cell activation in vitro is normal in TTT/AAA β2-integrin KI mice
β2-integrins were shown to lower the threshold for B cell activation (16), but it is unknown whether LFA-1/kindlin-3 interactions affect B cell activation. To investigate whether the lowered Ag-specific Ab response of KI mice is solely due to impaired T cell activation or whether the mutation also causes changes in B cell activation, we studied the functions of WT and KI B cells in more detail. Splenic B cells were characterized for their integrin expression and expression of activation markers. As reported previously for other cell types, there were reduced levels of CD11a and CD18 in KI B cells compared with WT cells, but activation markers were expressed at similar levels in both populations (Supplemental Fig. 2). Static adhesion assays confirmed that TTT/AAA β2-integrin KI B cells were deficient in binding to ICAM-1 (Fig. 7A), as we reported previously for CD4+ T cells (11). Furthermore, KI B cells show less aggregation in response to BCR or LPS stimulus (Fig. 7B). In contrast, KI B cell adhesion to the β1 integrin ligands fibronectin and VCAM was normal (Supplemental Fig. 3). We next studied the proliferation and activation of splenic B cells in vitro. B cells isolated from WT and KI spleens were grown for 48 h in the presence of anti-IgM or LPS to trigger activation through the BCR and TLR4, respectively. No changes in B cell proliferation (Fig. 7C) or the expression of CD69, CD86, or MHC Class II after 48 h of stimulation were detected (Fig. 7D). Early BCR signaling (p-Syk, p-Akt, p-Erk) also appeared normal in KI cells compared with WT cells (Fig. 7E).
TTT/AAA β2-integrin KI B cells show impaired adhesion but normal activation and proliferation. (A) Static adhesion assays of splenic B cells purified from WT and KI mice to ICAM-1. Data are from two/three independent experiments (n = 3 mice). (B) B cell aggregation in response to proliferation stimuli. Photomicrographs were taken after 48 h of stimulation with anti-IgM and LPS and are a representative of four independent experiments (n = 6 mice). Scale bar, 400 μm. (C and D) Proliferation and activation of WT and KI splenic B cells in vitro in response to anti-IgM and LPS. CFSE-labeled B cells were grown in the presence of different concentrations of anti-IgM or LPS and subjected to flow cytometry for proliferation (C) and activation (D) after 48 h. Result of three independent experiments (n = 3–5 mice). (E) B cell signaling. Phosphorylation of Syk, Akt, and Erk in WT and KI B cells stimulated through the BCR, with anti-IgM for the indicated times, was determined by Western blot analysis. All error bars represent SEM. *p < 0.05, Student t test.
TTT/AAA β2-integrin KI B cells show impaired adhesion but normal activation and proliferation. (A) Static adhesion assays of splenic B cells purified from WT and KI mice to ICAM-1. Data are from two/three independent experiments (n = 3 mice). (B) B cell aggregation in response to proliferation stimuli. Photomicrographs were taken after 48 h of stimulation with anti-IgM and LPS and are a representative of four independent experiments (n = 6 mice). Scale bar, 400 μm. (C and D) Proliferation and activation of WT and KI splenic B cells in vitro in response to anti-IgM and LPS. CFSE-labeled B cells were grown in the presence of different concentrations of anti-IgM or LPS and subjected to flow cytometry for proliferation (C) and activation (D) after 48 h. Result of three independent experiments (n = 3–5 mice). (E) B cell signaling. Phosphorylation of Syk, Akt, and Erk in WT and KI B cells stimulated through the BCR, with anti-IgM for the indicated times, was determined by Western blot analysis. All error bars represent SEM. *p < 0.05, Student t test.
In conclusion, our results show that, although present in normal proportions in peripheral lymphoid organs, B cells lacking the β2-integrin TTT site have problems in homing to the appropriate lymphoid organs and/or out of them. In addition, KI B cells display reduced adhesion in vitro, as well as impaired T cell–dependent and T cell–independent Ab production in vivo. Therefore, the integrin/kindlin-3 interaction is required for optimal T cell activation, B cell homing, and Ab responses in vivo.
Discussion
We showed previously that the TTT site in the β2-integrin subunit is only necessary for T cell activation with soluble anti-CD3 and not with plate-bound anti-CD3 in vitro (11). We now show that this site is essential for the Ag-specific activation and proliferation of T cells in vitro by DCs and in vivo in the spleen following immunization of mice, as well as for optimal Ab responses in vivo. The results from this study highlight that lymphocyte LFA-1 downstream signaling mediated by the TTT site on the β2-integrin subunit is necessary for optimal immune responses in vivo.
This TTT site in the β2-integrin cytoplasmic domain regulates integrin function in cells by mediating binding to cytoplasmic proteins, including kindlin-3 (11). Kindlins provide a physical link between the integrin and the cortical actin cytoskeleton and are thought to be involved in integrin conformational change to the active, fully open state, as well as contributing to downstream integrin signaling (17). In lymphocytes, kindlin-3 binds LFA-1 and is essential for firm adhesion of both T and B cells (18–20), and it stabilizes integrin/ligand interactions in T cells following TCR engagement in vitro (21). Meanwhile, 14-3-3 proteins bind to the TTT site when one or more of the T residues are phosphorylated (e.g., after T cell activation by phorbol esters) and initiate downstream integrin signaling (22). We showed that mutation of the TTT site in T cells results in the loss of actin reorganization, cell spreading, and adhesion to ICAM-1 (11, 22, 23). Sorting nexin family members also bind this region of the β2-integrin tail, specifically in the endocytic compartment during the integrin-recycling process (24), and loss of this association in TTT/AAA KI cells results in reduced expression of integrins on the cell surface, although this reduced expression is unlikely to contribute to the impairments in lymphocyte activation seen in this study (11).
LFA-1 is a key component of the IS that forms between CD4 T cells and DCs or B cells, contributing to the stability of the contacts (7). A loss of LFA-1 function in T cells may reduce the duration of cellular contacts and, thus, have a negative impact on T cell activation. Indeed T cell–DC contact duration, frequency, and intensity have a significant impact on the magnitude and polarization of the T cell response (25). LFA-1 is localized to the peripheral supramolecular activation cluster of the IS and initiates “outside–in” integrin signaling following adhesion to ICAM-1, which, in turn, triggers a range of downstream effects, including further LFA-1 clustering, actin cytoskeleton rearrangement, and orientation of the cell toward the synapse (reviewed in Ref. 26). Thus, it seems likely that integrin signaling relies on TTT-dependent interactions with kindlin-3 and/or 14-3-3 proteins to drive IS formation and subsequent T or B cell activation. Further studies are required to determine the phosphorylation status of the TTT residues under these activation conditions, the specific roles of kindlin-3 and 14-3-3 proteins in this process, and to directly visualize the effects of the TTT/AAA mutation in T cells on IS formation with DCs and B cells. In addition to its role in IS formation, LFA-1 is thought to contribute directly to T cell activation via adhesion-independent mechanisms. For example, previous findings support a role for LFA-1 in initiating and/or maintaining T cell IL-2 production (27) by affecting IL-2 expression at the transcription level (28, 29). LFA-1 also has the potential to impact on the Th1/Th2 balance of the CD4 T cell response (30, 31). In this study, we show that baseline expression of activation markers by splenic T cells was normal in TTT/AAA KI mice and that the proportions of naive and effector/memory T cells in the spleen were unaltered, indicating that the development of these effector/memory responses is not dependent on high-affinity LFA-1.
The specific role for LFA-1 in the lymphocyte-activation process has proved controversial because of the difficulty in dissecting out the involvement of this molecule in lymphocyte homing compared with a direct role in cellular activation. To overcome this, we made use of in vitro–activation techniques. Previously, we showed largely normal activation of KI CD4 T cells in vitro under conditions of polyclonal stimulation with plate-bound anti-CD3 (11). However, when cells are stimulated with soluble anti-CD3, which, in contrast to plate-bound anti-CD3 promotes LFA-1–mediated T cell–T cell synapse formation, T cell activation is significantly reduced (11). In this study, we show a reduction in KI T cell activation in a coculture system with DCs, indicating that in circumstances in which T cell activation requires IS formation with APCs (or other T cells), T cell LFA-1 is required for optimal activation (32).
Many previous in vivo studies in β2- or αL-knockout mice reported reduced lymphocyte activation in immune reactions at peripheral sites, such as delayed-type hypersensitivity responses in the footpad (4), contact sensitivity reactions in the ear (3, 4), and peripheral antitumor responses (6), in which LFA-1 likely plays a major role in cellular homing. β2-Integrin involvement in systemic immunity remains disputed, with reports of normal β2-deficient T cell responses to systemic Listeria infection (33) but impaired systemic responses to Streptococcus (3). In this study, to more thoroughly investigate the involvement of the β2-integrin (and specifically the cytoplasmic domain TTT site) in cellular activation rather than migration, we specifically analyzed the T cell response in the spleen, because T cell migration to this organ is unaffected by the TTT/AAA mutation (11).
Analysis of β2-deficient T cell responses in an Ag-specific manner was performed previously using TCR-transgenic β2-knockout mice. However, these studies focused on the gut immune responses for which a defect in T cell homing was also shown (34, 35), although the investigators also reported a defect in systemic T cell responses to model Ag (34). In this study, we provide firm evidence to indicate an essential role for fully functioning LFA-1 and, specifically, for β2-integrin tail interactions, in splenic CD4 T cell activation and proliferation in vivo under conditions in which T cell homing does not have an impact. These data build on our previous study in which we showed normal TTT/AAA β2-integrin KI T cell activation in the spleen of KI mice following the adoptive transfer of Ag-loaded DCs (11). The discrepancy between these findings is likely due to the influence of β2-integrin deficiency in other leukocyte populations on the resulting T cell response in our previous study in the global TTT/AAA β2-integrin KI mouse (11) (Supplemental Table I). In addition to their adhesive functions, it is well established that β2-integrins perform regulatory functions, restricting various inflammatory responses of DCs and macrophages (14, 36–38) and limiting subsequent T cell activation (14, 39, 40). In support of this, we show in this study that the splenic environment of KI mice is more inflammatory and suggest that the higher levels of IL-1β in the KI spleen may have contributed to the T cell–activation response observed in our previous study (11, 15).
The role of talin1, another integrin activator that binds to the β2 cytoplasmic domain, also was studied in T cells (32, 41). Talin1 is essential for homing to lymph nodes, as well as in contact-dependent, but not in contact-independent (CD3/CD28- or PMA/ionomycin-induced), T cell proliferation. Talin1 was also critical for prolonged T cell–DC contacts, but the formation of transient contacts was unaffected. The clustering of LFA-1 at the IS was not affected by talin1 deficiency, but the polarization of vinculin and F-actin at the IS was talin1 dependent (32). These and other studies (42) show that kindlin-3 and talin have distinct, although somewhat overlapping, functions in integrin-dependent leukocyte activation, proliferation, and homing to lymphoid organs. It is possible that the integrin/kindlin interaction regulates, for example, integrin clustering at the IS, because kindlin was reported to regulate β3-integrin clustering on the cell surface (43). Our findings are in accordance with these studies. However, both kindlin-3 and talin bind to and activate several integrin families (β1, β2, and β3) (28, 44), whereas our results show the importance of specific β2-integrin interactions with kindlin-3. Furthermore, talin1 does not bind to the TTT/AAA sequence (11).
Previous studies of the Ab levels in unimmunized β2-integrin–knockout mice showed a substantial increase in total Ig and especially in IgG levels present in the bloodstream (3). However, our results show that TTT/AAA β2-integrin KI mice have normal serum Ab levels and that, in response to immunization, Ab production in KI mice is significantly reduced compared with WT mice. B cell functions were studied earlier in mice in whom talin1 was selectively depleted (CD19Tln1−/− mice) (45), revealing the role of active VLA-4 and LFA-1 integrins in B cell functions. Interestingly, the talin1-null B cells show similar Ab-production profile as those from TTT/AAA β2-integrin KI mice, with normal levels of serum Igs in unimmunized mice and severely attenuated levels of IgG and IgM in response to immunization (45). Also, B cell numbers in the lymph nodes were decreased, reflecting the difficulty of talin1-null B cells to home to lymph nodes (45). Because talin1 regulates both LFA-1 and VLA-4 integrins, which are known to have overlapping functions in B cell homing and other functions (46, 47), it is important to note that the TTT/AAA β2-integrin KI mutation alone causes a severe reduction in the Ab response, showing the importance of β2-integrins and their intracellular interactions with kindlin and other cytoplasmic-binding partners in Ab production. Specifically, KI mice had a severe impairment in the initial production of IgM, whereas the reduction in IgG levels was more modest, suggesting that the T-independent IgM response might be more dependent on high-affinity LFA-1 than the subsequent T-dependent isotype switch and affinity-maturation steps in B lymphocytes.
Although the defect in Ab production following immunization in KI mice may not be due to attenuated B cell proliferation or proximal BCR signaling, there are several other possible explanations for this result. First, as discussed earlier, KI CD4 T cells show an attenuated ability to form an IS with DCs and to be activated. Because the subsequent formation of an IS between a CD4 T cell and a B cell is vital for the Ab production by the latter, the lack of an appropriate T cell stimulus most likely affects full activation of B cells and optimal Ab production. Second, lymphocyte homing to lymphoid organs, especially lymph nodes, and egress from the circulation are impaired in KI mice, likely affecting B cell interactions with other leukocytes that are required for optimal B cell activation and Ig production. Thus, integrin-mediated interactions with endothelial cells and other leukocytes requiring high-affinity LFA-1 are essential for optimal B cell responses in vivo.
Therefore, our current findings provide novel evidence for an important role of LFA-1–mediated adhesion and/or downstream signaling via the β2 subunit TTT site in optimal systemic T and B cell responses in vivo.
Acknowledgements
We thank Lyndsey Robertson for practical help with experiments and Colin Watts for providing the OT-II mice.
Footnotes
This work was supported by the Academy of Finland, The Ella and Georg Ehrnrooth Foundation, The Sigrid Juselius Foundation, Biocentrum Helsinki, the Biotechnology and Biological Sciences Research Council, the Liv och Hälsa Foundation, the Magnus Ehrnrooth Foundation, and Tenovus Scotland.
The online version of this article contains supplemental material.
References
Disclosures
The authors have no financial conflicts of interest.