The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kandula, S.
Right arrow Articles by Abraham, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kandula, S.
Right arrow Articles by Abraham, C.
The Journal of Immunology, 2004, 173: 4443-4451.
Copyright © 2004 by The American Association of Immunologists

LFA-1 on CD4+ T Cells Is Required for Optimal Antigen-Dependent Activation In Vivo1

Sravanthi Kandula and Clara Abraham2

Department of Medicine, Section of Gastroenterology, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The leukocyte-specific integrin, LFA-1, plays a critical role in trafficking of T cells to both lymphoid and nonlymphoid tissues. However, the role of LFA-1 in T cell activation in vivo has been less well understood. Although there have been reports describing LFA-1-deficient T cell response defects in vivo, due to impaired migration to lymphoid structures and to sites of effector function in the absence of LFA-1, it has been difficult to assess whether T cells also have a specific activation defect in vivo. We examined the role of LFA-1 in CD4+ T cell activation in vivo by using a system that allows for segregation of the migration and activation defects through the adoptive transfer of LFA-1-deficient (CD18–/–) CD4+ T cells from DO11.10 Ag-specific TCR transgenic mice into wild-type BALB/c mice. We find that in addition to its role in trafficking to peripheral lymph nodes, LFA-1 is required for optimal CD4+ T cell priming in vivo upon s.c. immunization. CD18–/– DO11.10 CD4+ T cells primed in the lymph nodes demonstrate defects in IL-2 and IFN-{gamma} production. In addition, recipient mice adoptively transferred with CD18–/– DO11.10 CD4+ T cells demonstrate a defect in OVA-specific IgG2a production after s.c. immunization. The defect in priming of CD18–/– CD4+ T cells persists even in the presence of proliferating CD18+/– CD4+ T cells and in lymphoid structures to which there is no migration defect. Taken together, these results demonstrate that LFA-1 is required for optimal CD4+ T cell priming in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Accessory molecules play a role in the process of Ag recognition through two important functions: 1) enhancing adhesion of the T cell to the APC, allowing for more efficient TCR engagement; and 2) providing costimulation of T cells by transducing intracellular signals distinct from those mediated through the TCR. One of these accessory molecules is LFA-1 ({alpha}L/{beta}2 or CD11a/CD18) (1), a member of the {beta}2 integrin family. LFA-1 is involved in a number of effector functions based on the specific cell in which it is expressed. LFA-1 is expressed in an inactive form on circulating PBLs, neutrophils, monocytes, granulocytes, and NK cells, and requires activation to bind to its ligands, ICAM-1, ICAM-2, or ICAM-3 (2, 3). The {beta}2 family also consists of CD11b/CD18, CD11c/CD18, and CD11d/CD18, but LFA-1 is the only member of the {beta}2 integrin family to be expressed on peripheral T cells. The physiological importance of LFA-1 is manifest in individuals lacking the {beta}2 subunit in a disease known as leukocyte adhesion deficiency. Patients with this disease are characterized by an inability to clear pathogens, recurrent infections, and frequently, death at an early age unless treated with a bone marrow transplant (4, 5). Furthermore, interference with LFA-1:ICAM-1 interactions has reduced the pathologic features of a number of autoimmune and inflammatory diseases, both in animal models and in human disease (6, 7, 8), although it is not clear in each case whether this involves alterations in lymphocyte homing, activation, or skewing. Therefore, understanding the role of LFA-1 in vivo is critical to understanding the implications of therapeutic interventions in LFA-1 function.

The role of LFA-1 as an adhesion molecule has been well described (3, 9). LFA-1-mediated adhesion can facilitate Ag presentation to T cells (10, 11, 12, 13, 14, 15). In addition to the known role of LFA-1 in T cell adhesion, there have been a number of reports that implicate LFA-1 in costimulation of T cells (16, 17). We and others have assessed the role of LFA-1 in T cell activation using APC that were generated by gene transfer of class II and ICAM-1 into costimulation-negative cell lines (14, 15, 18, 19, 20). In these studies, it was found that Ag presentation by transfectants expressing class II and ICAM-1 can induce IL-2 secretion and proliferation in naive T cells, whereas transfectants expressing only class II cannot. In vitro, T cells from both CD11a–/– and CD18–/– mice have been reported to have defects in activation under conditions requiring cell-cell interactions (e.g., mitogens and APC, MLRs), but have normal activation in cell contact-independent conditions, such as PMA/ionomycin and cross-linked anti-CD3 (21, 22, 23). Despite in vitro evidence for the role of LFA-1 in CD4+ T cell activation, it is not yet clear whether it is required for CD4+ T cell activation in vivo. Activation in vivo occurs in the context of other costimulatory molecules and in the context of a three-dimensional matrix, both of which may combine to effectively compensate for the lack of LFA-1.

Evidence for contributions of LFA-1 to T cell function and migration in vivo has been provided through CD11a–/– and CD18–/– mice, as well as LFA-1-blocking studies. The interaction of LFA-1 with its ligands allows for improved adhesion of leukocytes to vascular endothelium, an essential step for the recruitment and migration of leukocytes into inflamed tissue (24, 25). The peripheral lymph nodes (PLN)3 of CD11a–/– mice have a substantial decrease in CD4+ and CD8+ T cells (26). The high endothelial venules of these hypocellular lymph nodes (LN) have decreased adherent lymphocytes, and the residual lymphocyte entry occurs through {alpha}4, {alpha}4{beta}7, and VCAM-1 (26). Both CD11a–/– and CD18–/– mice demonstrate defects of T cell function in vivo, including in tumor rejection, delayed-type hypersensitivity (DTH) responses, and infectious responses (21, 22, 27, 28, 29, 30, 31). Two studies attempted to better address whether there is a defect specifically in activation in vivo of LFA-1-deficient CD4+ T cells using a DTH model; however, they arrived at opposite conclusions. One study examining CD11a–/– mice concluded that T cell priming in the draining LN (DLN) was deficient (30), while the other study using CD18–/– mice concluded that T cell priming in the DLN was intact, and that the DTH response defect was secondary to a defect in T cell trafficking to the skin (31). However, in analyzing these vivo responses, there are a number of complicating issues with regard to the ability to dissect the T cell-specific activation defect: 1) LFA-1 (and other {beta}2 integrins) is expressed on multiple different hemopoietic cells; therefore, LFA-1 deficiency may be contributing to defects in the function of any of these cells, which ultimately contributes to a defect in T cell responses; 2) CD18 associates with {alpha}-chains other than CD11a, such that in the CD18–/– mice, all the {beta}2 integrins are defective, and therefore, the defects extend beyond that of LFA-1; and 3) the known dependency upon LFA-1 for T cell trafficking to LN and tissues results in a decreased number of T cells available to participate in the immune response, at the level of both priming and effector responses, which may well account for the T cell defects observed. Therefore, resolution of this issue requires a system that can restrict the LFA-1 defect to Ag-specific T cells and then track the migration and activation of these Ag-responsive T cells during an immune response.

To determine whether LFA-1-deficient CD4+ T cells have an activation defect in vivo, we used the adoptive transfer of CD18–/– DO11.10 CD4+ T cells into wild-type (WT) recipients. We found that in addition to the defect in trafficking to the PLN, CD18–/– DO11.10 CD4+ T cells have a defect in priming in DLN, which results in a functional deficit in IL-2, IFN-{gamma}, and Ag-specific IgG2a production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

CD18–/– mice were generously provided by A. Beaudet (Baylor College of Medicine, Houston, TX) (22). CD18–/– mice were backcrossed onto the BALB/c background (eight generations) and then to DO11.10 TCR transgenic mice. BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and Charles River Laboratories (Wilmington, MA). T cells were phenotyped through staining of peripheral blood cells with a biotinylated mAb, KJ1-26 (Caltag Laboratories, Burlingame, CA), directed against the TCR clonotype expressed by DO11.10 T cells and by anti-CD18 (BD Pharmingen, San Diego, CA). Mice were maintained on autoclaved food and irradiated water in a specific pathogen-free facility in microisolator cages with filtered air, according to National Institutes of Health guidelines.

Cells

The transfectant into the fibrosarcoma cell line, 6132-PRO (Pro), expressing I-Ad in combination with B7-1 (ProAd-B7), has been previously described (14, 32). Cell lines were maintained in DMEM (Invitrogen Life Technologies, Gaithersburg, MD) supplemented with 10% FCS, 2 mM glutamine, 0.1 mM nonessential amino acids, 40 µg/ml gentamicin, and 50 µM 2-ME. G418 (200 µg/ml) and MXH (6 µg/ml mycophenolic acid, 250 µg/ml xanthine, and 15 µg/ml hypoxanthine) were added to the culture medium for maintenance of the transfectants.

Abs and staining reagents

Expression of transfected molecules was determined by flow cytometry using the anti-class II mAb, MKD6, and the anti-B7-1 mAb, 16-10A1 (American Type Culture Collection, Manassas, VA). The following Abs and secondary reagents used for flow cytometry were purchased from BD Pharmingen: PE-, FITC-, biotin-, and allophycocyanin-labeled anti-CD4; PE- and FITC-labeled anti-CD18; CyChrome-labeled streptavidin; PE-labeled anti-IL-2; and PE-labeled anti-IFN-{gamma}. The following Abs and secondary reagents were purchased from Caltag Laboratories: PE- and biotin-labeled KJ1-26. Alkaline phosphatase-conjugated IgG2a was purchased from Southern Biotechnology Associates (Birmingham, AL).

In vivo T cell activation and migration

Spleen-derived CD4+ T cells were isolated using CD4+ microbeads, according to manufacturer instructions (Miltenyi Biotec, Auburn, CA). The efficacy of the CD4+ T cell purification was monitored by flow cytometry and was routinely >93% pure. Freshly isolated (2.5 x 106 per mouse) CD18+/– or CD18–/– DO11.10 CD4+ T cells were stained with 2.5 µM CFSE (Molecular Probes, Eugene, OR) and adoptively transferred by i.v. injection into WT BALB/c mice. Twenty-four hours later, mice were immunized either s.c. (four injections in four quadrants of upper and lower back) or i.v. with a mixture of 25 µg of LPS, serotype Escherichia coli 026:B6 (Sigma-Aldrich, St. Louis, MO), and 50 µg of chicken OVA (Sigma-Aldrich). PLN or DLN (in the case of s.c. injection) (inguinal, axillary, brachial), mesenteric LN (MLN), and spleen were harvested at the indicated days after immunization. Cells were stained with anti-CD4 and KJ1-26 to assess either migration (at early time points before CFSE dilution) or activation (per CFSE dilution).

Intracellular cytokine staining

Freshly isolated (2.5 x 106 per mouse) CD18+/– or CD18–/– DO11.10 CD4+ T cells were adoptively transferred by i.v. injection into WT BALB/c mice. Twenty-four hours later, mice were immunized s.c. with a mixture of 25 µg of LPS/50 µg of chicken OVA. DLN were harvested 5 days after immunization, and 1 x 106 DLN cells were cocultured in 96-well round-bottom plates with 1 x 105 ProAd-B7 in the absence or presence of 2 µg/ml OVA peptide (323–339). Brefeldin A (Sigma-Aldrich) at 10 µg/ml was added after 2 h, and cells were incubated for an additional 10 h. Cells were stained with anti-CD4 and KJ1-26, fixed with 3% paraformaldehyde, permeabilized with 0.3% saponin, and stained with anti-IL-2 or anti-IFN-{gamma} for 30 min. Cytokine production by KJ1-26+/CD4+ cells was assessed, and isotype controls and stimulation with no peptide served as controls.

Measurement of chicken OVA-specific isotype Abs

Freshly isolated (2.5 x 106 per mouse) CD18+/– or CD18–/– DO11.10 CD4+ T cells were adoptively transferred by i.v. injection into WT BALB/c mice. Twenty-four hours later, mice were immunized s.c. with a mixture of 25 µg of LPS/50 µg of OVA. At weeks 1, 2, and 3 after immunization, serum was isolated from mice. Ninety-six-well plates (Costar, Cambridge, MA) were coated with OVA (50 µg/ml) in PBS, and then blocked with PBS/5% FCS. Four dilutions of serum (1/100, 1/200, 1/400, 1/800) were incubated in the wells at 37°C x 1 h. Plates were washed and incubated with IgG2a conjugated with alkaline phosphatase (Southern Biotechnology Associates). Plates were washed and incubated with phosphatase substrate (Sigma-Aldrich), and ODs were determined at 405 {lambda}.

In vitro T cell proliferation

For in vitro stimulation of freshly isolated CD4+ T cells with ProAd-B7 or splenocytes, 2.5 x 104 DO11.10 CD4+ T cells were incubated with 2.5 x 104 mitomycin C (Sigma-Aldrich)-treated ProAd-B7 or 2.5 x 105 irradiated (3000 rad) splenocytes and various concentrations of chicken OVA peptide (323–339) in a 96-well flat-bottom plate. In other experiments, freshly isolated CD18+/– or CD18–/– DO11.10 CD4+ T cells were stimulated with CD11c-isolated cells (microbeads from Miltenyi Biotec or MoFlo sorting, DakoCytomation, Carpinteria, CA) from the spleen of WT BALB/c mice after i.v. injection with 25 µg of LPS. A total of 2.5 x 104 T cells was incubated with 2.5 x 103 CD11c+ cells and various concentrations of chicken OVA peptide (323–339) in a 96-well round-bottom plate. [3H]Thymidine was added to the cultures during the last 18 h of a 72-h assay.

Statistical analyses

Statistical comparisons of cell migration, cell accumulation, and OVA-specific IgG2a between treated groups were assessed using a one-tailed Student’s t test. Values of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD18–/– DO11.10 CD4+ T cells demonstrate defects in trafficking to and priming in the PLN

To investigate the role of LFA-1 in T cell activation in vivo, we generated CD18–/– mice x DO11.10 TCR transgenic mice that encode an OVA-specific, I-Ad-restricted TCR. In light of the marked decrease in CD4+ T cells in the PLN of LFA-1-deficient mice (26), the number of CD4+ T cells available for priming during an immune response at this site is substantially decreased. It is not clear whether the T cells that successfully traffic to the PLN activate and develop an effector response equivalent to that of WT T cells. To eliminate the contribution of defective postactivation CD18–/– T cell trafficking to nonlymphoid tissues, we focused on the priming of T cells within secondary lymphoid organs upon immunization. To avoid the influence of CD18 deficiency on non-T cell subsets, we used the adoptive transfer of CD18–/– DO11.10 CD4+ T cells into WT mice, such that only the Ag-specific T cells migrating and responding to immunization will be LFA-1 deficient. These CD18–/– CD4+ peripheral T cells are deficient only in LFA-1, as it is the only {beta}2 integrin expressed on peripheral CD4+ T cells (data not shown). We isolated CD4+ T cells from the spleen, as CD18–/– mice have very few CD4+ T cells in the PLN. To examine priming preferentially in the PLN, Ag was injected s.c. in the context of LPS as an adjuvant. To first assess whether the migration defect to PLN observed during homeostatic trafficking is still present under conditions of LPS/OVA injection, 2.5 x 106 CD18–/– CD4+ D011.10 T cells were adoptively transferred into WT BALB/c mice, and 24 h later, the mice were immunized s.c. with LPS/OVA. Three hours after immunization, absolute numbers of migrated CD4+ DO11.10 T cells to DLN were determined. Both CD18+/+ and CD18+/– mice have an identical cell surface expression of CD18 by flow cytometry, and CD18+/– mice are used as littermate controls for the CD18–/– mice. An increase in the number of CD4+ DO11.10 T cells is observed in the DLN after immunization in comparison with PBS controls (Fig. 1A). However, migration of CD18–/– DO11.10 CD4+ T cells to the DLN is decreased compared with CD18+/– CD4+ T cells (Fig. 1A), both in the absence and presence of LPS/OVA immunization. As expected, trafficking of CD18–/– CD4+ T cells is increased to the spleen compared with CD18+/– DO11.10 CD4+ T cells, under these same conditions (Fig. 1B). This is consistent with the splenic hypercellularity of CD4+ T cells in LFA-1-deficient mice under homeostatic conditions (26) and most likely secondary to a redistribution of the CD4+ T cell population due to the inability to properly traffic to LN.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 1. Migration of CD18–/– DO11.10 CD4+ T cells to DLN is impaired after s.c. immunization. Freshly isolated splenic DO11.10 CD4+ T cells from CD18+/– or CD18–/– mice were adoptively transferred to WT BALB/c mice. Twenty-four hours later, the mice were immunized s.c. with LPS/OVA or PBS control, and 3 h after immunization, DLN (A) and spleen (B) were harvested, and the number of DO11.10 (KJ1–26+) CD4+ T cells was quantified (percentage of CD4+/KJ1-26+ x number DLN or spleen cells) + SEM (n = 3 mice). The data represent at least three independent experiments. *, p < 0.05; **, p < 0.01.

 
In the context of the persistent migration defect of CD18–/– CD4+ T cells to DLN, we examined the activation upon s.c. LPS/OVA immunization of those CD4+ DO11.10 T cells that had successfully trafficked to the DLN of WT mice. Cell divisions, as indicated by CFSE dilution, 3 days after immunization were used as a measure of T cell activation. CD18–/– DO11.10 CD4+ T cells in DLN entered into cell division, but 25–40% less CD18–/– DO11.10 CD4+ T cells in DLN were able to progress to later cell divisions as compared with CD18+/– DO11.10 CD4+ T cells (Fig. 2A). To test whether the immune response was preferentially taking place in lymphoid tissues that were less impacted by the CD18–/– trafficking defect, we examined the activation of CD18–/– DO11.10 CD4+ T cells in spleen and MLN. Spleen demonstrated a higher number of CD18–/– DO11.10 CD4+ T cells in the undivided peak (Fig. 2A), again indicative of less efficient T cell activation. The activated T cells observed in the spleen are a combination of local T cell activation due to Ag that has reached the spleen and of T cells activated in the DLN that have circulated to the spleen. There were very few CD18+/– and CD18–/– T cells having undergone division in the MLN (Fig. 2A). Both CD18+/– and CD18–/– DO11.10 CD4+ T cells that entered cell division demonstrated other markers of successful T cell activation, such as up-regulation of CD44 expression (data not shown). The defect in CD18–/– DO11.10 CD4+ T cell activation in the DLN persisted at days 5 and 7 after immunization, indicating that the defect is not simply a delayed kinetic response (Fig. 2A). As expected, the total number of accumulated CD18–/– DO11.10 CD4+ T cells in the DLN at each of the time points assessed after immunization was also decreased relative to CD18+/– controls (Fig. 2B). Therefore, CD18–/– DO11.10 CD4+ T cells demonstrate a persistent defect in both migration to the DLN and proliferation in the DLN in vivo upon s.c. immunization.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 2. CD18–/– DO11.10 CD4+ T cells have a persistent activation defect upon s.c. LPS/OVA immunization. Freshly isolated spleen DO11.10 CD4+ T cells from CD18+/– or CD18–/– mice were labeled with CFSE and adoptively transferred to WT BALB/c mice. Mice were immunized 24 h later with s.c. LPS/OVA, and 3, 5, and 7 days later, A, CFSE dilution (cell divisions) of DO11.10 CD4+ T cells, and B, the number of DO11.10 CD4+ T cells in spleen, MLN (nondraining LN), and DLN were measured. The percentage of cells in the later generations of divisions is indicated. Compared with CD18+/– T cells, 25–40% less CD18–/– DO11.10 CD4+ T cells progressed into the later cell divisions in DLN on day 3. Correspondingly, CD18+/– T cell accumulation ranged from 2.5- to 7.5-fold greater than that of CD18–/– T cells on day 3, 3- to 11-fold greater on day 5, and 6- to 13-fold greater on day 7. The data are representative of three mice per condition. The data represent two independent experiments on the full time course, and at least an additional two independent experiments at day 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
CD18–/– DO11.10 CD4+ T cells demonstrate a persistent defect in T cell priming despite cotransfer with CD18+/– DO11.10 CD4+ T cells

One possibility for the CD18–/– DO11.10 CD4+ T cell activation defect upon priming in the DLN is that the severe migration defect results in the lack of critical T cell mass necessary for optimal T cell proliferation. If an adequate number of cells migrating and proliferating in the DLN is necessary for providing essential factors, such as growth factors or cell contacts, for optimal proliferation of the CD18–/– population, then the presence of proliferating CD18+/– CD4+ T cells should restore CD18–/– CD4+ DO11.10 T cell proliferation. To address this issue, we cotransferred an equal number of CD18+/– and CD18–/– DO11.10 CD4+ T cells into WT BALB/c mice. Three days after s.c. LPS/OVA immunization, CD18–/– DO11.10 CD4+ T cells revealed a similar defect in reaching the later cell divisions (Fig. 3A) compared with CD18+/– CD4+ T cells as when transferred alone (Fig. 2A). As expected, there was also a defect in accumulation of Ag-specific CD18–/– CD4+ T cells (Fig. 3B). Therefore, despite the presence of a normally dividing CD4+ DO11.10 T cell population, CD18–/– CD4+ T cells continue to demonstrate a proliferation defect.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 3. CD18–/– DO11.10 CD4+ T cells have an activation defect to s.c. LPS/OVA immunization despite cotransfer of CD18+/– DO11.10 CD4+ T cells. Freshly isolated spleen DO11.10 CD4+ T cells from CD18+/– and CD18–/– mice were labeled with CFSE and adoptively cotransferred at a 1:1 ratio into WT BALB/c mice. Mice were immunized 24 h later with s.c. LPS/OVA, and 3 days later, A, CFSE dilution (cell divisions) of DO11.10 CD4+ T cells, and B, the number of DO11.10 CD4+ T cells + SEM (n = 3 mice) in DLN were measured within the CD18+/– and CD18–/– populations. The percentage of cells in the later generations of divisions is indicated. Compared with CD18+/– T cells, 25–30% less CD18–/– DO11.10 CD4+ T cells progressed into the later cell divisions in DLN. The data are representative of two independent experiments. *, p < 0.05.

 
CD18–/– DO11.10 CD4+ T cells demonstrate defects in production of IL-2 and IFN-{gamma} upon priming in the LN

In light of the defect in proliferation observed in CD18–/– DO11.10 CD4+ T cells upon LN priming, we assessed whether there was a defect in IL-2 production. WT mice were adoptively transferred with CD18+/– or CD18–/– DO11.10 CD4+ T cells and immunized s.c. 24 h later with LPS/OVA. DLN were harvested 5 days after immunization and cocultured in vitro with OVA peptide and APC (ProAd-B7) that provide for Ag-specific, LFA-1-independent activation (see Fig. 7C). The APC were generated from a costimulatory-negative fibrosarcoma cell line (6132- PRO) that was transfected with the class II Ag, I-Ad, in combination with the well-documented costimulatory molecule, B7-1 (ProAd-B7) (14). The decreased number of CD18–/– DO11.10 CD4+ T cells present in the DLN would be expected to result in a decreased level of cytokine secretion into the supernatant of whole LN preparations. Therefore, we used intracellular cytokine staining to determine the percentage of DO11.10 CD4+ T cells producing IL-2. A decreased percentage of CD18–/– DO11.10 CD4+ T cells produced IL-2 (~33%) in comparison with CD18+/– T cells (~75%) (Fig. 4). Costimulation through LFA-1 has been shown to result in Th1 skewing (33, 34, 35, 36). In addition, T cell production of IFN-{gamma} is known to require adequate progression into cell division (37). We therefore assessed IFN-{gamma} production by CD18–/– DO11.10 CD4+ T cells after priming in LN. Consistent with previously published reports, ~6% of CD18+/– DO11.10 CD4+ T cells secrete IFN-{gamma} in the DLN (38). However, a decreased percentage of CD18–/– DO11.10 CD4+ T cells produced IFN-{gamma} (~1%) in comparison with CD18+/– T cells (~6%) (Fig. 4). Therefore, the defect in priming of CD18–/– DO11.10 CD4+ T cells in LN results in defects in production of IL-2 and IFN-{gamma} upon restimulation.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 7. Decreased proliferation of CD18–/– DO11.10 CD4+ T cells upon Ag presentation with splenocytes or CD11c-isolated DC. Irradiated splenocytes (A), spleen-derived CD11c DC (18 h after i.v. LPS injection) (B), or ProAd-B7 (C) were cocultured with splenocyte CD4+ T cells purified from CD18+/– or CD18–/– DO11.10 TCR transgenic mice in the presence of increasing concentrations of OVA peptide. Thymidine incorporation was measured during the last 18 h of a 72-h assay. In vivo LPS-activated spleen-derived DC demonstrated functional maturation by less efficient processing of whole Ag in comparison with PBS-injected mice (data not shown). Note the potency of DC as the ratio of T cells to DC in B is 10:1 in comparison with a T cell:APC ratio of 1:10 and 1:1 in A and C, respectively.

 


View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 4. CD18–/– DO11.10 CD4+ T cells have a defect in IL-2 and IFN-{gamma} production after s.c. LPS/OVA immunization. Freshly isolated spleen DO11.10 CD4+ T cells from CD18+/– or CD18–/– mice were adoptively transferred into WT BALB/c mice. Mice were immunized 24 h later with s.c. LPS/OVA, and 3 days later, DLN were harvested and restimulated in vitro with ProAd-B7 in the absence or presence of OVA peptide. The percentage of DO11.10 CD4+ T cells producing IL-2 and IFN-{gamma} is indicated. The data are representative of three mice per condition. The data represent two independent experiments.

 
CD18–/– DO11.10 CD4+ T cells demonstrate a defect in T cell-dependent IgG2a isotype switching upon s.c. immunization

OVA-specific IgG2a production upon s.c. immunization of BALB/c mice with LPS/OVA requires the presence of adoptively transferred DO11.10 CD4+ T cells that secrete IFN-{gamma} in the DLN (39). For the production of other OVA-specific isotype Abs, such as IgG1, the presence of endogenous BALB/c T cells is sufficient (39). In light of the defect in CD18–/– DO11.10 CD4+ T cell proliferation and IFN-{gamma} production upon s.c. LPS/OVA immunization, we sought to determine whether this had a functional effect on T cell-dependent OVA-specific IgG2a isotype switching. WT mice were adoptively transferred with CD18–/– DO11.10 CD4+ T cells and immunized 24 h later with s.c. LPS/OVA, and OVA-specific IgG2a was assessed at 1-wk intervals for 3 wk. In comparison with CD18+/– controls, mice receiving CD18–/– DO11.10 CD4+ T cells demonstrated a persistent significant defect in isotype switching to OVA-specific IgG2a (Fig. 5).



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 5. CD18–/– DO11.10 CD4+ T cells have a persistent defect in serum OVA-specific IgG2a upon s.c. immunization with LPS/OVA. Freshly isolated spleen DO11.10 CD4+ T cells from CD18+/– or CD18–/– mice were adoptively transferred into WT BALB/c mice. Mice were immunized 24 h later with s.c. LPS/OVA, and 1, 2, and 3 wk later serum was harvested and OVA-specific IgG2a was measured by ELISA. The data are represented as relative units = OD + SEM (n = 4 mice). **, p < 0.01; ***, p < 0.001.

 
CD18–/– DO11.10 CD4+ T cells demonstrate a severe defect in T cell priming upon i.v. immunization

We considered the possibility that the activation defect observed by CD18–/– DO11.10 CD4+ T cells upon LN priming might still be related to the migration defect to LN. One consideration would be that the activation defect might reflect a selection bias in the subset of CD18–/– DO11.10 CD4+ T cells that do successfully traffic to the PLN. For example, the 10–20% subset of CD18–/– CD4+ T cells successfully trafficking to the PLN do so through {alpha}4, {alpha}4{beta}7, and VCAM-1 (26). As demonstrated in the migration studies above, CD18–/– DO11.10 CD4+ T cells do not have a defect in migration to the spleen, and in fact, have a relative increase in the trafficking to spleen (Fig. 1B). Therefore, examining the priming of CD18–/– DO11.10 CD4+ T cells in the spleen eliminates potential secondary consequences of the trafficking defect contributing to the T cell activation defect observed upon priming in LN, such as the possibility of a subset bias effect as well as the critical mass effect addressed in the cotransfer experiments (Fig. 3). The spleen is the primary site of T cell activation when Ag is administered i.v. (i.e., hematogenous). CD18–/– DO11.10 CD4+ T cells adoptively transferred into WT BALB/c mice demonstrate the expected increase in migration of T cells to spleen upon i.v. immunization with LPS/OVA in comparison with PBS, along with an increased migration relative to CD18+/– CD4+ T cells under both control and immunized conditions (data not shown). To examine splenic priming of adoptively transferred CD18–/– DO11.10 CD4+ T cells, recipient mice were immunized 24 h after transfer with i.v. LPS/OVA. Three days after immunization, CD18–/– DO11.10 CD4+ T cells in the spleen demonstrate a severe defect in cell division relative to CD18+/– controls (Fig. 6A). Less CD18–/– DO11.10 CD4+ T cells enter cell division in comparison with CD18+/– DO11.10 CD4+ T cells. In addition, 45–60% less CD18–/– DO11.10 CD4+ T cells primed in the spleen progress into the later cell divisions compared with CD18+/– T cells. Again, this defect persists over 7 days, such that it is not merely one of delayed kinetics (Fig. 6A). Furthermore, compensatory T cell activation does not take place in other secondary lymphoid structures (Fig. 6A). CD18–/– DO11.10 CD4+ T cells that undergo cell divisions also up-regulate CD44 as a marker of T cell activation (data not shown). The defect in cell division correlates to a substantial decrease in the absolute number of CD18–/– DO11.10 CD4+ T cells present in the spleen at each of the time points assessed (Fig. 6B). This is observed even at the early 3-day time point, despite the increased number of CD18–/– CD4+ T cells present at the onset of activation in light of the enhanced migration to spleen (Fig. 1B). Therefore, despite an initial migration advantage to the spleen, CD18–/– DO11.10 CD4+ T cells demonstrate a severe persistent defect in entry and progression into cell division upon splenic priming that is associated with a significant decrease in accumulation of Ag-specific CD4+ T cells.



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 6. CD18–/– DO11.10 CD4+ T cells have a persistent defect in activation upon i.v. LPS/OVA immunization. Freshly isolated spleen DO11.10 CD4+ T cells from CD18+/– or CD18–/– mice were labeled with CFSE and adoptively transferred to WT BALB/c mice. Mice were immunized 24 h later with i.v. LPS/OVA, and 3, 5, and 7 days later, A, CFSE dilution of DO11.10 CD4+ T cells, and B, the number of DO11.10 CD4+ T cells in spleen, MLN (nondraining LN), and PLN were measured. The percentage of cells in the later generations of divisions is indicated. Compared with CD18+/– T cells, 45–60% less CD18–/– DO11.10 CD4+ T cells progressed into the later cell divisions in spleen on day 3. Correspondingly, CD18+/– T cell accumulation was 3.5-fold greater than that of CD18–/– T cells on day 3, 5-fold greater on day 5, and 11-fold greater on day 7. The low number of CD18–/– DO11.10 CD4+ T cells in the spleen by day 7 limits the equivalent collection of T cells. The data are reflective of three mice per condition, and at least two additional independent experiments on day 3. **, p < 0.01; ***, p < 0.001.

 
DO11.10 CD4+ T cells from CD18–/– mice demonstrate defects in Ag-specific activation with WT spleen-derived APC

One possible mechanism for the CD18–/– DO11.10 CD4+ T cell activation defect is at the level of T cell:APC interactions. Ag-dependent activation of LFA-1-deficient CD4+ TCR transgenic T cells has not been reported. To determine whether CD18–/– DO11.10 CD4+ T cells were defective in activation upon Ag presentation with spleen-derived APC, we assessed their activation upon coculture with WT irradiated splenocytes. CD18–/– DO11.10 CD4+ T cells had a substantial defect in activation when Ag was presented by splenocytes (Fig. 7A). As dendritic cells (DC) are highly effective APC, and are most likely the APC involved in priming CD4+ T cells in the secondary lymphoid organs, we examined whether purified DC harvested from spleen demonstrated a dependency upon LFA-1:ICAM-1 for efficient Ag-dependent activation of T cells. DC were harvested from the spleen of mice treated in vivo with LPS to simulate the source and stimulation conditions of the DC involved in the in vivo i.v. priming experiments. CD18–/– DO11.10 CD4+ T cells demonstrate a significant defect in activation upon stimulation with Ag by spleen-derived CD11c-purified DC (Fig. 7B). As expected, the DC were much more effective at activating CD4+ T cells on a per cell basis than was whole spleen (Fig. 7, A and B). The activation defect upon Ag presentation using splenocyte-derived APC may be an intrinsic activation defect in CD18–/– DO11.10 CD4+ T cells due to abnormal development, or an LFA-1-specific defect. We addressed this issue by using ProAd-B7 as APC, thereby providing for a system that is CD18 independent, but Ag and cell contact dependent. We find that CD18–/– DO11.10 CD4+ T cells do not have an intrinsic defect in T cell activation, with activation equal to that of WT T cells upon presentation with Ag by ProAd-B7 (Fig. 7C). Therefore, CD18–/– DO11.10 CD4+ T cells exhibit a substantial defect in activation upon Ag presentation by WT splenocytes and DC, thereby accounting for at least one of the mechanisms contributing to the deficient proliferation of CD18–/– DO11.10 CD4+ T cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ability to dissect whether LFA-1 on CD4+ T cells plays a role in priming in vivo has been confounded by: 1) the importance of LFA-1 for T cell trafficking to PLN, and 2) the presence of LFA-1 on other hemopoietic cells, including the APC involved in T cell priming. Using an Ag-specific T cell activation system with transfer of CD4+ DO11.10 responder CD18+/– or CD18–/– T cells into WT mice, we have defined that in addition to the role LFA-1 plays in migration of CD4+ T cells to LN, it is required for optimal priming of CD4+ DO11.10 T cells. This defect in CD4+ T cell priming is not secondary to a defect in critical mass, as it occurs even when initial T cell activation takes place in the context of adequate CD4+ responder T cells. In addition, the defect in priming is not due to a population bias secondary to selective trafficking, as it also occurs in secondary lymphoid structures where there is no trafficking deficit. Consequences of the impaired CD18–/– DO11.10 CD4+ T cell trafficking and priming in the LN include a defect in IL-2 and IFN-{gamma} production along with a persistent defect in serum OVA-specific IgG2a. One mechanism contributing to the Ag-specific priming defect is at the level of T cell:APC interactions.

The defect in progression of CD18–/– DO11.10 CD4+ T cells to later cell divisions can result in certain defects in effector outcome. One such outcome is IFN-{gamma} production (37). A number of studies have shown in vitro that stimulation of T cells in the context of LFA-1 results in enhanced Th1 skewing (33, 34, 35, 36). Other reports have argued that this outcome depends upon the dose of Ag (40). Reports in vivo demonstrating decreased cytokine secretion in whole ex vivo LN preparations are confounded by the decreased number of T cells present (28), with an inability to identify cytokine secretion of Ag-specific T cells on a per cell basis. Our study demonstrates that CD18–/– DO11.10 CD4+ T cells are defective in IFN-{gamma} production upon priming in LN. Furthermore, in the DO11.10 CD4+ T cell adoptive transfer model, it has been shown that adequate IFN-{gamma}-secreting DO11.10 CD4+ T cells in the PLN are required for OVA-specific IgG2a production (39). Upon s.c. immunization, mice adoptively transferred with CD18–/– DO11.10 CD4+ T cells demonstrated a persistent defect in the production of OVA-specific IgG2a. The defect in IgG2a production in our study, therefore, argues for a decrease in the IFN-{gamma} available after priming in the DLN. This decreased IFN-{gamma} available in the LN may be secondary to a combination of the decrease in the number of CD18–/– DO11.10 CD4+ T cells in the DLN and in the percentage of CD18–/– CD4+ T cells producing IFN-{gamma}.

The different routes of immunization in our study demonstrate varying degrees of CD18–/– T cell proliferation defects. Specifically, the proliferation defect of CD18–/– DO11.10 CD4+ T cells in spleen (where priming first occurs) upon i.v. immunization is more severe than that observed in DLN upon s.c. immunization. Upon s.c. immunization, CD18–/– DO11.10 CD4+ T cells in the DLN enter cell division, but the majority of the cells do not progress beyond five cell divisions. Upon i.v. immunization, fewer CD18–/– CD4+ T cells enter into cell division in the spleen, and the progression through cell division is further decreased such that the majority of cells do not progress beyond three cell divisions. The contrast in the severity of the s.c. and i.v. immunization CD18–/– T cell proliferation defect relative to CD18+/– T cells is also observed in the PLN, arguing that the effect is not simply secondary to the lymphoid structure in which the activation is taking place, but rather the route of immunization. One possibility for this differential outcome is that the DC presenting Ag under i.v. immunization conditions have increased requirements for LFA-1:ICAM-1 interactions during T cell activation in comparison with the tissue-derived DC recruited upon s.c. immunization. ICAM-1 expression increases with DC maturation, and the expression varies based on the DC maturation stimulus (36). ICAM-1 levels are known to vary on different DC subsets (41). We found that ICAM-1 expression on CD11c+ cells isolated from PLN under i.v. and s.c. immunization conditions was similar (on the total CD11c+ population, as well as within each CD11c+CD11b– and CD11c+CD11b+ subpopulation under the two immunization conditions; data not shown). Therefore, there may be other compensatory mechanisms for the lack of LFA-1 on responder CD4+ T cells during s.c. immunization relative to i.v. immunization. Despite the differences in the severity of defects, CD18–/– DO11.10 CD4+ T cells demonstrate priming defects upon both routes of immunization assessed.

CD18–/– DO11.10 CD4+ T cells demonstrate a decrease in accumulation of Ag-specific T cells after priming both in LN and in the spleen. This may be due to one or more of the following: 1) decreased initial T cell migration; 2) decreased proliferation; 3) increased apoptosis; or 4) increased egress. Decreased proliferation most likely contributes to a decreased number of antigenic-specific CD18–/– T cells upon priming in both LN and spleen. In contrast, decreased migration contributes to the decrease in the number of Ag-specific CD18–/– CD4+ T cells only in the LN, as there is actually enhanced migration of CD18–/– CD4+ T cells to the spleen. Whether costimulation through LFA-1 also contributes to rescue from cell death and/or a prevention of T cell egress upon Ag activation in vivo is not known. Depending on the conditions, stimulation through LFA-1 has been reported to be both proapoptotic (42, 43) and antiapoptotic (44) in T cells. Stimulation of T cells through the TCR in the absence of a costimulatory signal often results in cell death. However, in vitro, costimulation through LFA-1 alone is not sufficient to rescue from the cell death (18, 45). The role of LFA-1 in T cell apoptosis under conditions of Ag activation in vivo is not known. LFA-1 has been shown to contribute to the retention of lymphocytes in the lung (46); however, whether LFA-1 contributes to lymphocyte retention in the LN, through, for example, the down-regulation of sphingosine-1-phosphate receptor on lymphocytes in the LN (47), is not known. Therefore, the absence of LFA-1 on DO11.10 CD4+ T cells during i.v. and s.c. immunization results in a decreased accumulation of Ag-specific T cells, which is most likely a combination of defects in migration (s.c. immunization) and proliferation (i.v. and s.c. immunization), as well as possibly in cell death and lymphoid organ egress.

Considerations of mechanisms accounting for the CD18–/– CD4+ T cell activation defect may include both adhesive and costimulatory functions of LFA-1. First, the absence of adhesion provided by LFA-1 may result in a failure of CD4+ T cells: 1) to properly localize to the appropriate region of secondary lymphoid structures; or 2) to form T cell:APC conjugates. Second, in the absence of costimulation provided by LFA-1, CD4+ T cells may not: 1) appropriately initiate distinct LFA-1-dependent signaling pathways; or 2) initiate necessary structural rearrangements at the level of intracellular scaffolding, enabling efficient assembly of signaling molecules, or at the level of the organization of the immunological synapse (48, 49). The improper localization of LFA-1-deficient T cells is a less likely factor, as previous studies examining localization of cells by immunohistochemistry within the white pulp of the spleen demonstrate a minimal inhibition in localization of B and T cells upon blocking LFA-1 interactions or in the absence of CD18, unless trafficking was also blocked by anti-{alpha}4{beta}1 (50, 51). In addition, H&E sections of the spleen and LN in our mice demonstrate intact architecture with proper T cell zone segregation, despite the hypocellularity of the PLN and histiocytic expansion of the sinusoidal regions (data not shown). Furthermore, the in vitro studies eliminate the issue of proper T cell localization as a sole mechanism for the T cell activation defect by directly coculturing T cells and APC. These studies show a clear CD18–/– DO11.10 CD4+ T cell activation defect upon Ag presentation by irradiated splenocytes (Fig. 7A) and spleen-derived CD11c-purified DC (Fig. 7B). Therefore, despite the potency of DC as APC, they clearly depend upon LFA-1:ICAM-1 interactions for optimal DO11.10 CD4+ T cell activation (Fig. 7B).

In conclusion, these studies demonstrate that in addition to the migration defect to PLN upon s.c. immunization, CD18–/– DO11.10 CD4+ T cells have a defect in progression through cell division and are defective in production of IL-2 and IFN-{gamma} as well as OVA-specific IgG2a.


   Acknowledgments
 
We thank Alexander Khoruts, Marisa Alegre, and Yang-Xin Fu for helpful discussions; Ellen Chuang, Anita Chong, Emma Masteller, and Lisa Sevilla for critical reading of the manuscript; and Arthur Beaudet for providing reagents.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 C.A. was supported by National Institutes of Health Grant DK02905. Back

2 Address correspondence and reprint requests to Dr. Clara Abraham, Department of Medicine, Section of Gastroenterology, The University of Chicago, 5841 South Maryland Avenue, MC 6084, Chicago, IL 60637. E-mail address: cabraham{at}medicine.bsd.uchicago.edu Back

3 Abbreviations used in this paper: PLN, peripheral LN; DC, dendritic cell; DLN, draining LN; DTH, delayed-type hypersensitivity; LN, lymph node; MLN, mesenteric LN; WT, wild type. Back

Received for publication May 17, 2004. Accepted for publication July 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kurzinger, K., T. Reynolds, R. N. Germain, D. Davignon, E. Martz, T. A. Springer. 1981. A novel lymphocyte function-associated antigen (LFA-1): cellular distribution, quantitative expression, and structure. J. Immunol. 127:596.[Abstract]
  2. Springer, T. A.. 1990. Adhesion receptors of the immune system. Nature 346:425.[Medline]
  3. Lub, M., Y. van Kooyk, C. G. Figdor. 1995. Ins and outs of LFA-1. Immunol. Today 16:479.[Medline]
  4. Larson, R. S., T. A. Springer. 1990. Structure and function of leukocyte integrins. Immunol. Rev. 114:180.
  5. Bunting, M., E. S. Harris, T. M. McIntyre, S. M. Prescott, G. A. Zimmerman. 2002. Leukocyte adhesion deficiency syndromes: adhesion and tethering defects involving {beta}2 integrins and selectin ligands. Curr. Opin. Hematol. 9:30.[Medline]
  6. Moriyama, H., K. Yokono, K. Amano, M. Nagata, Y. Hasegawa, N. Okamoto, K. Tsukamoto, M. Miki, R. Yoneda, N. Yagi, et al 1996. Induction of tolerance in murine autoimmune diabetes by transient blockade of leukocyte function-associated antigen-1/intercellular adhesion molecule-1 pathway. J. Immunol. 157:3737.[Abstract]
  7. Kato, Y., A. Yamataka, H. Yagita, H. Bashuda, K. Okumura, T. Miyano. 1995. Prevention of fetal bowel allograft rejection by combined treatment with anti-ICAM-1 and anti-LFA-1 antibodies. J. Pediat. Surg. 30:1093.[Medline]
  8. Yacyshyn, B. R., M. B. Bowen-Yacyshyn, L. Jewell, J. A. Tami, C. F. Bennett, D. L. Kisner, W. R. Shanahan, Jr. 1998. A placebo-controlled trial of ICAM-1 antisense oligonucleotide in the treatment of Crohn’s disease. Gastroenterology 114:1133.[Medline]
  9. Dustin, M. L., T. A. Springer. 1989. T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature 341:619.[Medline]
  10. Kuhlman, P., V. T. Moy, B. A. Lollo, A. A. Brian. 1991. The accessory function of murine intercellular adhesion molecule-1 in T lymphocyte activation: contributions of adhesion and co-activation. J. Immunol. 146:1773.[Abstract]
  11. Ybarrondo, B., A. M. O’Rourke, A. A. Brian, M. F. Mescher. 1994. Contribution of lymphocyte function associated-1/intercellular adhesion molecule-1 binding to the adhesion/signaling cascade of cytotoxic T lymphocyte activation. J. Exp. Med. 179:359.[Abstract/Free Full Text]
  12. Bachmann, M. F., K. McKall-Faienza, R. Schmits, D. Bouchard, J. Beach, D. E. Speiser, T. W. Mak, P. S. Ohashi. 1997. Distinct roles for LFA-1 and CD28 during activation of naive T cells: adhesion versus costimulation. Immunity 7:549.[Medline]
  13. Brunmark, A., A. M. O’Rourke. 1997. Augmentation of mature CD4+ T cell responses to isolated antigenic class II proteins by fibronectin and intercellular adhesion molecule-1. J. Immunol. 159:1676.[Abstract]
  14. Abraham, C., J. Griffith, J. Miller. 1999. The dependence for leukocyte function-associated antigen-1/ICAM-1 interactions in T cell activation cannot be overcome by expression of high density TCR ligand. J. Immunol. 162:4399.[Abstract/Free Full Text]
  15. Abraham, C., J. Miller. 2001. Molecular mechanisms of IL-2 gene regulation following costimulation through LFA-1. J. Immunol. 167:5193.[Abstract/Free Full Text]
  16. Van Seventer, G. A., Y. Shimizu, K. J. Horgan, S. Shaw. 1990. The LFA-1 ligand ICAM-1 provides an important costimulatory signal for T cell receptor-mediated activation of resting T cells. J. Immunol. 144:4579.[Abstract]
  17. Van Seventer, G. A., W. Newman, Y. Shimizu, T. B. Nutman, Y. Tanaka, K. J. Horgan, T. V. Gopal, E. Ennis, D. O’Sullivan, H. Grey, S. Shaw. 1991. Analysis of T cell stimulation by superantigen plus major histocompatibility complex class II molecules or by CD3 monoclonal antibody: costimulation by purified adhesion ligands VCAM-1, ICAM-1, but not ELAM-1. J. Exp. Med. 174:901.[Abstract/Free Full Text]
  18. Zuckerman, L. A., L. Pullen, J. Miller. 1998. Functional consequences of costimulation by ICAM-1 on IL-2 gene expression and T cell activation. J. Immunol. 160:3259.[Abstract/Free Full Text]
  19. Dubey, C., M. Croft, S. L. Swain. 1995. Costimulatory requirements of naive CD4+ T cells: ICAM-1 or B7-1 can costimulate naive CD4 T cell activation but both are required for optimum response. J. Immunol. 155:45.[Abstract]
  20. Cai, Z., H. Kishimoto, A. Brunmark, M. R. Jackson, P. A. Peterson, J. Sprent. 1997. Requirements for peptide-induced T cell receptor down-regulation on naive CD8+ T cells. J. Exp. Med. 185:641.[Abstract/Free Full Text]
  21. Scharffetter-Kochanek, K., K. Norman, D. C. Bullard, I. Lorenzo, N. P. van Nood, S. Rich, W. Smith, A. L. Beaudet. 1996. Generation and characterization of a CD18 complete knock-out mouse: new insights in the function of the CD18 molecule?. J. Invest. Dermatol. 106:809.
  22. Scharffetter-Kochanek, K., H. Lu, K. Norman, N. van Nood, F. Munoz, S. Grabbe, M. McArthur, I. Lorenzo, S. Kaplan, K. Ley, et al 1998. Spontaneous skin ulceration and defective T cell function in CD18 null mice. J. Exp. Med. 188:119.[Abstract/Free Full Text]
  23. Shier, P., G. Otulakowski, K. Ngo, J. Panakos, E. Chourmaouzis, L. Christjansen, C. Y. Lau, W.-P. Fung-Leung. 1996. Impaired immune responses toward alloantigens and tumor cells but normal thymic selection in mice deficient in the {beta}2 integrin leukocyte function-associated antigen-1. J. Immunol. 157:5375.[Abstract]
  24. Randolph, G., M. Furie. 1996. Mononuclear phagocytes egress from an in vitro model of the vascular wall by migrating across endothelium in the basal to apical direction: role of intercellular adhesion molecule 1 and the CD11/CD18 integrins. J. Exp. Med. 183:451.[Abstract/Free Full Text]
  25. Warnock, R., S. Askari, E. Butcher, U. von Andrian. 1998. Molecular mechanisms of lymphocyte homing to peripheral lymph nodes. J. Exp. Med. 187:205.[Abstract/Free Full Text]
  26. Berlin-Rufenach, C., F. Otto, M. Mathies, J. Westermann, M. J. Owen, A. Hamann, N. Hogg. 1999. Lymphocytes migration in lymphocyte function-associated antigen (LFA)-1-deficient mice. J. Exp. Med. 189:1467.[Abstract/Free Full Text]
  27. Schmits, R., T. M. Kundig, D. M. Baker, G. Shumaker, J. J. Simard, G. Duncan, A. Wakeham, A. Shahinian, A. van der Heiden, M. F. Bachmann, et al 1996. LFA-1-deficient mice show normal CTL responses to virus but fail to reject immunogenic tumor. J. Exp. Med. 183:1415.[Abstract/Free Full Text]
  28. Schonlau, F., K. Scharffetter-Kochanek, S. Grabbe, B. Pietz, C. Sorg, C. Sunderkotter. 2000. In experimental leishmaniasis deficiency of CD18 results in parasite dissemination associated with altered macrophage functions and incomplete Th1 cell response. Eur. J. Immunol. 30:2729.[Medline]
  29. Prince, J. E., C. F. Brayton, M. C. Fossett, J. A. Durand, S. L. Kaplan, C. W. Smith, C. M. Ballantyne. 2001. The differential roles of LFA-1 and Mac-1 in host defense against systemic infection with Streptococcus pneumoniae. J. Immunol. 166:7362.[Abstract/Free Full Text]
  30. Andrew, D. P., J. P. Spellberg, H. Takimoto, R. Schmits, T. W. Mak, M. M. Zukowski. 1998. Transendothelial migration and trafficking of leukocytes in LFA-1-deficient mice. Eur. J. Immunol. 28:1959.[Medline]
  31. Grabbe, S., G. Varga, S. Beissert, M. Steinert, G. Pendl, S. Seeliger, W. Bloch, T. Peters, T. Schwarz, C. Sunderkotter, K. Scharffetter-Kochanek. 2002. {beta}2 integrins are required for skin homing of primed T cells but not for priming naive T cells. J. Clin. Invest. 109:183.[Medline]
  32. Zuckerman, L. A., A. J. Sant, J. Miller. 1995. Identification of a unique costimulatory activity for murine T helper 1 T cell clones. J. Immunol. 154:4503.[Abstract]
  33. Salomon, B., J. A. Bluestone. 1998. Cutting edge: LFA-1 interaction with ICAM-1 and ICAM-2 regulates Th2 cytokine production. J. Immunol. 161:5138.[Abstract/Free Full Text]
  34. Jenks, S. A., J. Miller. 1999. Inhibition of IL-4 responses after T cell priming in the context of LFA-1 costimulation is not reversed by restimulation in the presence of CD28 costimulation. J. Immunol. 164:72.
  35. Camacho, S. A., W. R. Heath, F. R. Carbone, N. Sarvetnick, A. LeBon, L. Karlsson, P. A. Peterson, S. R. Webb. 2001. A key role for ICAM-1 in generating effector cells mediating inflammatory responses. Nat. Immunol. 2:523.[Medline]
  36. Smits, H. H., E. C. de Jong, J. H. N. Schuitemaker, T. B. H. Geijtenbeek, Y. van Kooyk, M. L. Kapsenberg, E. A. Wierenga. 2002. Intercellular adhesion molecule-1/LFA-1 ligation favors human Th1 development. J. Immunol. 168:1710.[Abstract/Free Full Text]
  37. Bird, J. J., D. R. Brown, A. C. Mullen, N. H. Moskowitz, M. A. Mahowald, J. R. Sider, T. F. Gajewski, C.-R. Wang, S. L. Reiner. 1998. Helper T cell differentiation is controlled by the cell cycle. Immunity 9:229.[Medline]
  38. Reinhardt, R. L., D. C. Bullard, C. T. Weaver, M. K. Jenkins. 2003. Preferential accumulation of antigen-specific effector CD4 T cells at an antigen injection site involves CD62E-dependent migration but not local proliferation. J. Exp. Med. 197:751.[Abstract/Free Full Text]
  39. Pape, K. A., A. Khoruts, A. Mondino, M. K. Jenkins. 1997. Inflammatory cytokines enhance the in vivo clonal expansion and differentiation of antigen-activated CD4+ T cells. J. Immunol. 159:591.[Abstract]
  40. Rogers, P. R., M. Croft. 2000. CD28, Ox-40, LFA-1, and CD4 modulation of Th1/Th2 differentiation is directly dependent on the dose of antigen. J. Immunol. 164:2955.[Abstract/Free Full Text]
  41. Straw, A. D., A. S. MacDonald, E. Y. Denkers, E. J. Pearce. 2003. CD154 plays a central role in regulating dendritic cell activation during infections that induce Th1 or Th2 responses. J. Immunol. 170:727.[Abstract/Free Full Text]
  42. Wang, L., J. J. Y. Chen, B. B. Gelman, R. Konig, M. W. Cloyd. 1999. A novel mechanism of CD4 lymphocyte depletion involves effects of HIV on resting lymphocytes: induction of lymph node homing and apoptosis upon secondary signaling through homing receptors. J. Immunol. 162:268.[Abstract/Free Full Text]
  43. Ropke, C., P. Gladstone, M. Nielsen, N. Borregaard, J. A. Ledbetter, A. Svejgaard, N. Odum. 1996. Apoptosis following interleukin-2 withdrawal from T cells: evidence for a regulatory role of CD18 ({beta}2-integrin) molecules. Tissue Antigens 48:127.[Medline]
  44. Winter, S. S., J. J. Sweatman, M. B. Lawrence, T. H. Rhoades, A. L. Hart, R. S. Larson. 2001. Enhanced T-lineage acute lymphoblastic leukemia cell survival on bone marrow stroma requires involvement of LFA-1 and ICAM-1. Br. J. Hematol. 115:862.[Medline]
  45. Palmer, E. M., L. Farrokh-Siar, J. M. van Seventer, G. A. van Seventer. 2001. IL-12 decreases activation-induced cell death in human naive Th cells costimulated by intercellular adhesion molecule-1. I. IL-12 alters caspase processing and inhibits enzyme function. J. Immunol. 167:749.[Abstract/Free Full Text]
  46. Thatte, J., V. Dabak, M. B. Williams, T. J. Braciale, K. Ley. 2003. LFA-1 is required for retention of effector CD8 T cells in mouse lungs. Blood 101:4916.[Abstract/Free Full Text]
  47. Matloubian, M., C. G. Lo, G. Cinamon, M. J. Lesneski, Y. Xu, V. Brinkmann, M. L. Allende, R. L. Proia, J. G. Cyster. 2004. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 427:355.[Medline]
  48. Monks, C. R. F., B. A. Freiberg, H. Kupfer, N. Sciaky, A. Kupfer. 1998. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395:82.[Medline]
  49. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285:221.[Abstract/Free Full Text]
  50. Lo, C. G., T. T. Lu, J. G. Cyster. 2003. Integrin-dependence of lymphocyte entry into the splenic white pulp. J. Exp. Med. 197:353.[Abstract/Free Full Text]
  51. Lu, T. T., J. G. Cyster. 2002. Integrin-mediated long-term B cell retention in the splenic marginal zone. Science 297:409.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Int ImmunolHome page
Y. Wang, K. Shibuya, Y. Yamashita, J. Shirakawa, K. Shibata, H. Kai, T. Yokosuka, T. Saito, S.-i. Honda, S. Tahara-Hanaoka, et al.
LFA-1 decreases the antigen dose for T cell activation in vivo
Int. Immunol., September 1, 2008; 20(9): 1119 - 1127.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. L. Mueller, M. S. Thomas, B. J. Burbach, E. J. Peterson, and Y. Shimizu
Adhesion and Degranulation-Promoting Adapter Protein (ADAP) Positively Regulates T Cell Sensitivity to Antigen and T Cell Survival
J. Immunol., September 15, 2007; 179(6): 3559 - 3569.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
B. Graf, T. Bushnell, and J. Miller
LFA-1-Mediated T Cell Costimulation through Increased Localization of TCR/Class II Complexes to the Central Supramolecular Activation Cluster and Exclusion of CD45 from the Immunological Synapse
J. Immunol., August 1, 2007; 179(3): 1616 - 1624.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
R. Castriconi, A. Dondero, C. Cantoni, M. Della Chiesa, C. Prato, M. Nanni, M. Fiorini, L. Notarangelo, S. Parolini, L. Moretta, et al.
Functional characterization of natural killer cells in type I leukocyte adhesion deficiency
Blood, June 1, 2007; 109(11): 4873 - 4881.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Marski, A. L. Ye, and C. Abraham
CD18 Is Required for Intestinal T Cell Responses at Multiple Immune Checkpoints
J. Immunol., February 15, 2007; 178(4): 2104 - 2112.
[Abstract] [Full Text] [PDF]

Home page