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Expression and Functional Importance of Collagen-Binding Integrins, ₁₁ and ₂₁, on Virus-Activated T Cells ¹

Susanne Ø. Andreasen^*, Allan R. Thomsen^*, Victor E. Koteliansky, Tatiana I. Novobrantseva, Andrew G. Sprague, Antonin R. de Fougerolles^2, and Jan P. Christensen^2,3,*

^* Institute of Medical Microbiology and Immunology, Panum Institute, Copenhagen, Denmark; and Biogen, Cambridge, MA 02142

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, ₁₁ and ₂₁, on activated and memory T cells. Using this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of ₁₁ and ₂₁ can be rapidly induced on acutely activated T cells, that expression of ₁₁ remains elevated on memory T cells, and that expression of ₁₁ parallels that of viral-specific effector CD8⁺ T cells (defined by tetramer and IFN- staining). In an adoptive transfer model, mAb-mediated blockade of these integrins on activated effector and memory T cells inhibited Ag-specific delayed-type hypersensitivity responses; similar decreased responses were seen upon transfer of ₁-deficient activated/memory T cells. Thus, expression of ₁₁ and ₂₁ integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect of ₁₁ blockade on the delayed-type hypersensitivity response could be bypassed by direct injection of Ag-specific T cells to inflammatory sites, demonstrating for the first time in vivo that collagen-binding integrins are involved in leukocyte migration into tissues.

	Introduction

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Adhesive interactions play a key role during inflammation by promoting leukocyte attachment to the vascular endothelium and subsequent extravasation. Among the many adhesion molecules involved in these processes are the ₄₁ (VLA-4) and _L2 (LFA-1) integrins, expressed on the leukocyte surface, and their respective ligands, VCAM-1 and ICAM-1, which are inducibly expressed on the surface of inflamed endothelium (1). Following extravasation into tissues, further positioning of leukocytes within the extracellular matrix-rich tissue environment is required. However, this latter step in the inflammatory cascade has only recently been explored (2, 3). Collagen constitutes a major part of the extracellular matrix, and mAbs against the major cell surface collagen receptors, ₁₁ (VLA-1) and ₂₁ (VLA-2) integrins, dramatically decreased effector phase inflammatory responses in a variety of in vivo animal models (2, 3). Similarly, mice lacking expression of ₁₁ also showed increased resistance to inflammatory disease (2). Although these studies demonstrated the importance in vivo of ₁₁ and ₂₁ in inflammatory disease, little is known about the mechanism by which blockade of ₁₁ and ₂₁ reduces effector inflammatory responses or the cell types involved. Using a virus infection model, we have investigated the expression and function of these collagen-binding integrins on virus-activated T cells.

Both ₁₁ and ₂₁ are heterodimeric cell surface molecules consisting of a unique -subunit (₁ or ₂) associated with the common ₁ chain (4). ₁₁ is primarily expressed on mesenchymal cells, including smooth muscle cells, fibroblasts, hepatocytes, and microvascular endothelium (5, 6). Expression of ₂₁ is found predominantly on epithelial cells (7); significant expression is also found on monocytes and platelets (4). Regarding expression on T cells, stimulation of T cell clones in vitro results in up-regulation of ₁₁ and ₂₁ (4). In addition, it has been found that certain viral infections may induce T cell expression of DX5, which following completion of this study has been revealed to be ₂₁⁺ (DX5⁺) (8, 9, 10). Also, infiltrating T cells from different chronic inflammatory exudates have been found to express ₁₁ (2, 11, 12, 13). Most information regarding the role of ₁₁ and ₂₁ as receptors for collagen has been obtained through in vitro adhesion and migration assays on collagen matrices (14, 15). In addition to its adhesive and migratory functions, cell binding to collagen through ₁₁ and ₂₁ has been shown in vitro to synergistically enhance effector T cell proliferation and cytokine secretion (16, 17).

Given the importance of collagen-integrin interactions in modulating effector inflammatory responses in vivo and in affecting T cell function in vitro, we sought to investigate the expression and function of ₁₁ and ₂₁ integrins on virus-activated T cells. The murine lymphocytic choriomeningitis virus (LCMV) ⁴ infection has been extensively used as a model system to study T cell migration in the context of an antiviral response (18, 19, 20). Although this virus is a natural mouse pathogen that causes little nonspecific inflammation, a strong Ag-specific inflammatory response is induced, which is dominated by entry of IFN-⁺CD8⁺ T effector cells into the infected tissues (18, 21, 22). Furthermore, we have previously established the role of integrins ₄₁, _L2, and _M2 (Mac-1), and their ligands in the process of extravasation by blocking these molecules (23, 24, 25). Thus, LCMV infection constitutes a well-established model for studying the mechanisms underlying virus-induced, Tc1 cell-dependent inflammation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Female BALB/cA and C57BL/6 mice (6–7 wk old) were purchased from Bomholtgaard (Ry, Denmark), and were acclimatized for at least 1 wk before experimentation. VLA-1^-/- mice were produced locally from breeding pairs originating from Biogen (Cambridge, MA). Animals were housed under specific pathogen-free conditions, as validated by testing of sentinels for unwanted infections according to Federation of European Laboratory Animal Science Association standards.

Virus infection

LCMV Traub strain (LCMV-Traub) and vesicular stomatitis virus (VSV) Indiana strain were produced, stored, and quantified, as described previously (26, 27).

Infections

In most experiments, mice were infected with 10³ LD₅₀ of LCMV-Traub i.v. An i.v. injection of LCMV into immunocompetent mice normally results in transient immunizing infection (18, 26, 28). For infection with VSV, mice were inoculated with 10⁶ PFU i.v. (29).

mAbs for flow cytometry

The following mAbs were purchased from BD PharMingen (San Diego, CA) as rat anti-mouse Abs: PE-conjugated anti-CD49b (integrin ₂ chain) (clone HM2); FITC-conjugated anti-CD49b (clone Ha1/29); FITC-conjugated anti-CD49d (integrin ₄ chain) (clone R1-2); FITC-, PE-, and CyChrome-conjugated anti-CD8a (clone 53-6.7); FITC- and PE-conjugated anti-CD4 (clone H129.19); FITC and biotinylated anti-L-selectin (clone MEL-14); and FITC- and PE-conjugated anti-IFN- (clone XMG1.2). Purified hamster anti-mouse CD49a (integrin ₁ chain) (clone Ha31/8), FITC-conjugated anti-hamster IgG (clones G70-204 and G94-56), and PE-conjugated anti-hamster IgG (clones G70-204 and G94-90.5) were obtained from the same source. mAb to anti-CD29 (integrin ₁ chain) (clone HM1-1) was obtained from BD PharMingen and biotinylated, as recommended by the manufacturer (Pierce, Rockford, IL). Furthermore, anti-CD49a (clone Ha31/8) and hamster group II control mAb (hamster anti-keyhole limpet hemocyanin) (clone Ha4/8) were fluorescently labeled using the Alexa 488 protein-labeling kit, as recommended by the manufacturer (Molecular Probes, Eugene, OR).

MHC/peptide tetramer for flow cytometry

H-2D^b/gp_33–41 tetramer was obtained through the National Institute of Allergy and Infectious Diseases tetramer facility and the National Institutes of Health AIDS Research and Reference Reagent Program.

Ab treatment

For in vivo blocking of ₁₁ and ₂₁, 250 µg of mAb Ha31/8 (hamster anti-₁) and Ha1/29 (hamster anti-₂) was injected i.v. or i.p. on the indicated days. Hamster group II mAb Ha4/8 (hamster anti-keyhole limpet hemocyanin) was used as control. Abs were produced and purified, as previously described (30). Flow cytometric analysis did not reveal any depletion of activated CD8⁺ from the spleen of mAb-treated mice (data not shown).

Cell preparations

Spleen single-cell suspensions were obtained by pressing the organ through a fine steel mesh. When used for analysis by flow cytometry, erythrocytes were lysed by 0.83% NH₄Cl treatment (31).

Fluorescence staining and flow cytometric analysis

Staining for flow cytometry was performed, as described previously (32, 33, 34). Briefly, 1 x 10⁶ cells were stained with directly labeled mAbs in FACS medium (PBS containing 10% rat serum, 1% BSA, and 0.1% NaN₃) for 20 min in the dark at 4°C and subsequently washed. When using unconjugated Abs, cells were incubated with primary Abs, followed by incubation with secondary Abs before additional surface staining with directly labeled Abs was performed. Cells incubated with biotin-conjugated Abs were further incubated with streptavidin-Tri-Color (Caltag Laboratories, Burlingame, CA). After washing twice, cells were fixed with 1% paraformaldehyde. In case of tetramer staining, cells were incubated with H-2D^b/gp_33–41 tetramer for 1 h at 20°C before surface labeling. To detect intracellular IFN-, splenocytes (10⁷/ml) were incubated with an immunodominant MHC-class I-restricted epitope (LCMV gp_33–41, 0.1 µg/ml), monensin (3 µM), and IL-2 (50 IU/ml) for 5 h, after which cells were surface labeled, fixed, and permeabilized using PBS/saponin (0.05%), and anti-IFN- was added. After incubation for 20 min in the dark, cells were washed twice in PBS/saponin. Samples were acquired on a FACSCalibur instrument (BD Biosciences, San Jose, CA), and at least 10⁴ mononuclear cells were gated using a combination of forward angle and side scatter to exclude dead cells and debris. Data were analyzed using CellQuest software (BD Biosciences).

Staining with CFSE was performed by resuspending 20 x 10⁶ spleen cells/ml in HBSS/0.1% BSA (warm) and adding CFSE to a final concentration of 5 µM. Cells were incubated for 15 min at 37°C, and the reaction was stopped by adding ice-cold HBSS. Cells were washed twice in HBSS, counted, and injected into animals.

Immunohistochemistry

Spleen cells from animals infected with LCMV 8 days earlier were depleted of plastic-adherent cells by incubation for 1 h at 37°C in tissue culture flasks. The remaining cells were labeled with CFSE, and 50 x 10⁶ cells were injected i.v. into recipients that had been infected with LCMV 4 h earlier with 10⁷ LD₅₀ in the right footpad. The next day, both left and right footpads were excised, for immunohistochemical staining, and frozen in dry ice using OCT compound (Sakura, Torrance, CA). Acetone-fixed frozen sections (10 µm thick) were blocked in a 3% BSA/PBS solution for 30 min at room temperature. Slides were washed and sections were incubated with 5 µg/ml PE-conjugated anti-CD31 mAb (BD PharMingen) in 3% BSA/PBS for 1 h at room temperature. Slides were then washed in PBS and mounted in Citifluor (Ted Pella, Redding, CA). The stained sections were examined by dual immunofluorescent microscopy (Leica, Wetzlar, Germany). Adoptively transferred CFSE-labeled leukocytes are shown in green, and CD31-expressing vascular endothelium is shown in red.

LCMV-specific delayed-type hypersensitivity (DTH)

For a primary response, mice were infected locally in the right hind footpad with 10³ LD₅₀ of LCMV-Traub, and the local swelling reaction was followed between days 6 and 13 postinfection (p.i.); footpad thickness was measured using a dial caliper (Mitutoyo 7309; Mitutoyo, Tokyo, Japan), and virus-specific swelling was determined as the difference in thickness of the injected right and the untreated left footpad (31, 35).

Adoptive transfer of LCMV-specific DTH

Two approaches were used to assess adoptively transferred LCMV-specific DTH: systemic transfer and local transfer. Systemic transfer: mice were infected in their right hind footpad with 10⁷ LD₅₀ of LCMV-Traub. Four hours later, they were injected i.v. with 5 x 10⁷ splenocytes from B6 donors that had been infected 8 days previously with 10³ LD₅₀ of LCMV-Traub i.v. Local transfer: mice were infected in both hind footpads with 10⁷ LD₅₀ of LCMV-Traub. Four hours later, they were injected in the right footpad with 3 x 10⁶ day 8 primed splenocytes and in the left footpad with 3 x 10⁶ splenocytes from naive animals. For both types of transfer, footpad thickness was measured 16, 24, 48, and 72 h after injection, and virus-specific swelling was calculated as the difference in thickness of the right and left footpad. Donor cells were routinely treated with mitomycin C (25 µg/ml, to prevent secondary expansion in the recipients) and depleted of plastic-adherent cells by incubation for 1 h at 37°C in tissue culture flasks. Previous analysis has established that Ag-primed donor CD8⁺ T cells are essential for induction of the swelling reaction in both experimental situations (25, 31, 36).

	Results

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Kinetics of ₁₁ and ₂₁ expression following LCMV infection

Initial experiments were conducted to examine the kinetics of ₁₁ and ₂₁ expression on CD4⁺ and CD8⁺ T cells following LCMV infection. A minor percentage (10%) of splenic CD8⁺ T cells in naive mice express either ₁₁ or ₂₁. Following infection, a rapid increase in the fraction of cells expressing these molecules is observed, and at the peak of the response (day 8–10) about half of splenic CD8⁺ cells express ₁₁ and slightly fewer express ₂₁ (Fig. 1A). Coinciding with the decline in viral titers (37), a gradual decrease in the fraction of cells expressing these adhesion molecules is noted, and 2–3 mo after infection 35% of CD8⁺ T cells express ₁₁ and 20% express ₂₁. Although less pronounced, some increase in the fraction of cells expressing ₁₁ and ₂₁ molecules is also observed within the CD4⁺ T cell subset, reaching peak values day 12 p.i. (20–30% for both molecules, Fig. 1B), and then expression rapidly returns to a level indistinguishable from that seen in naive mice.

View larger version (37K): [in this window] [in a new window]   FIGURE 1. Kinetics of 11 and 21 expression on CD4+ and CD8+ cells during infection with LCMV. C57BL/6 mice were infected with LCMV i.v., and on the indicated days spleen cells were stained with anti-CD8 (A and C) or anti-CD4 (B and D) and anti-1 or anti-2 Abs. A and B, Representative histograms for gated CD8+ (A) or CD4+ (B) cells from infected (thick line) mice analyzed at the time of maximal expression; analysis of naive (thin line) animals is shown for comparison, and numbers in parentheses represent percentage of positive cells in naive mice. C and D, Total numbers of splenic CD8+ (C) and CD4+ (D) T cells together with absolute numbers of cells expressing 11 or 21. Medians and ranges of 3–10 mice are depicted.

Finally, to determine whether an increase in ₁₁⁺ and ₂₁⁺ CD8⁺ T cells is specific to LCMV infection or more generally associated with viral infection, C57BL/6 mice were infected i.v. with VSV, and expression of ₁₁ and ₂₁ on splenic CD8⁺ T cells was examined. Although the induced expansion of the CD8⁺ T subset is less pronounced during this infection (29), a significant increase in the fraction and number of CD8⁺ cells expressing these integrins was also observed following VSV infection (data not shown).

Expression of ₁₁ and ₂₁ is restricted to T cells with a preactivated phenotype

Because previous studies have shown that virus-activated T cells are ₄₁^high (34) together with _L2^high, CD44^high, and L-selectin^low (23), we examined the coexpression of ₁ and the three -subunits on splenic CD8⁺ T cells from day 8 LCMV-infected mice. As shown in Fig. 2A, all ₁⁺, ₂⁺, and ₄⁺ cells are also expressing ₁, confirming expression of ₁₁, ₂₁, and ₄₁ on a large subset of CD8⁺ T cells during acute infection. Subsequently, we determined the degree of coexpression of these molecules. As can be seen in Fig. 2B, expression of ₁₁ and ₂₁ correlated on virus-activated CD8⁺ T cells. Furthermore, all CD8⁺ T cells expressing either ₁₁ or ₂₁ were to be found exclusively in the ₄₁^high (Fig. 2C) and L-selectin^low subset (data not shown).

View larger version (60K): [in this window] [in a new window]   FIGURE 2. Coexpression of 1, 2, 4, and 1 chains on CD8+ T cells. A, Day 8 spleen cells from LCMV-infected C57BL/6 animals were stained with anti-CD8; anti-1; and anti-1, anti-2, or anti-4 Abs. Dot plot depicts coexpression of the 1 chain and various -chains on gated CD8+ T cells. B, Coexpression of 1 and 2 chains on naive and day 8 LCMV-infected spleen cells is shown for gated CD8+ T cells. C, Coexpression of 1 or 2 and 4 chains on naive and day 8 LCMV-infected spleen cells is shown for gated CD8+ T cells. All plots shown are representative for at least three animals, and numbers shown refer to the percentage of CD8+ T cells that are positive for the markers used.

View larger version (51K): [in this window] [in a new window]   FIGURE 3. Expression of 11 and 21 on splenic CD8+ cells in immune mice. A, C57BL/6 animals were infected with LCMV, and 90 days later spleen cells were stained with anti-CD8, anti-4, and anti-1 or anti-2 Abs. Dot plots for gated CD8+ T cells are depicted. B, Either naive (41low, R1) or primed (41high, R2) cells were gated out and compared in a histogram. A and B, Numbers refer to the percentage of CD8+ T cells that are positive for the markers used. All plots are representative of at least three animals.

Expression of ₁₁ and ₂₁ on viral Ag-specific T cells

Although LCMV infection results in marked up-regulation of ₁₁ and ₂₁ expression on a large number of activated CD8⁺ T cells, it is of interest to determine whether expression of these integrins is induced on virus-specific T cells. Therefore, to study expression of ₁₁ and ₂₁ on virus-specific cells during the acute and memory phase, CD8⁺ splenocytes were analyzed for coexpression of ₁₁ or ₂₁ and MHC/peptide tetramers. As evident from Fig. 4A, virus-specific T effector cells (defined as tetramer⁺ cells) showed increased levels of ₁₁ and ₂₁ expression compared with naive cells. Regarding expression of ₁₁ and ₂₁ on virus-specific T cells during the memory phase, expression of ₁₁ appears to be retained on the vast majority (>80%) of virus-specific CD8⁺ T cells, whereas expression of ₂₁ differs little from that on naive cells. A similar expression pattern was found when virus-specific CD8⁺ cells were defined by detection of IFN- in peptide-stimulated cells (Fig. 4B).

View larger version (40K): [in this window] [in a new window]   FIGURE 4. 11 and 21 expression on Ag-specific CD8+ T cells. A, Spleen cells from naive, acutely infected, and immune animals were stained with anti-CD8, H-2Db/gp33–41 tetramer, and anti-1, anti-2 Abs, or isotype control Ab. Plots depicting gated CD8+ T cells are presented. Numbers refer to the percentage of CD8+ T cells that are positive for the markers used. All plots are representative of at least three animals. B, Spleen cells from naive, acutely infected, and immune mice were incubated with gp33–41 peptide for 5 h, surface stained with anti-CD8 and anti-1 or anti-2 Abs, permeabilized, and stained for IFN-. CD8+IFN-+ T cells were gated, and 11 and 21 expression on gated cells is depicted (thick lines). For comparison, 11 and 21 expression on naive CD8+ T cells is included (thin lines). Numbers refer to percentage of gated cells positive for integrin expression in infected mice, while numbers in parentheses refer to CD8+ cells in naive mice. Plots are representative of at least three animals.

Functional role of ₁₁ and ₂₁ on virus-specific CD8⁺ T cells

To examine the functional role of ₁₁ and ₂₁ on T cells, LCMV-induced T cell-mediated inflammation in a collagen-rich environment, the footpad, was studied (38). This is a primarily CD8⁺ T cell-mediated inflammatory reaction generated in response to virus replicating locally in the footpad (25, 31, 36). First, we studied the effect ₁₁ and ₂₁ blockade had on LCMV-induced inflammation in intact animals. Naive mice were infected with LCMV in their right hind footpad and given either anti-₁ mAb, anti-₂ mAb, a combination of both, or isotype control mAb on days 3, 4, and 6 p.i., and footpad swelling was measured on days 6–13. Blocking either ₁₁ alone or in combination with ₂₁ resulted in a slight delay in the primary footpad-swelling reaction, as none of the mice in these two groups presented any significant swelling on day 7 p.i., in contrast to the control mAb or anti-₂ mAb-treated mice. However, by day 8 p.i., a substantial inflammatory response was subsequently induced in the two anti-₁ mAb-treated groups, and the magnitude of this response was comparable to that seen in the other groups (Fig. 5A). Although this result indicated that there is no absolute requirement for ₁₁ and ₂₁, these molecules could still play an important role alongside other adhesion molecules with parallel function. In the intact mouse, the extremely high number of virus-specific T cells present might simply obscure their importance.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. Effect of anti-1 and anti-2 treatment on primary response and adoptive DTH reaction in LCMV-infected animals. A, C57BL/6 animals were infected with LCMV in their right hind footpad and received 250 µg of the indicated Abs i.v. on days 3, 4, and 6 p.i. Controls received isotype-matched Ab. Medians and ranges of five animals per group are shown; no response was observed before day 6 p.i. B, Recipients were infected with LCMV in the footpad and at the same time received 250 µg of the indicated Ab i.v.; 4 h later, they received 5 x 107 adherent cell-depleted, LCMV-primed donor cells. Medians and ranges of five animals are shown; one representative experiment of three is depicted. C, BALB/cA recipients were infected with LCMV in their right hind footpad; 4 h later, they received 5 x 107 adherent cell-depleted, LCMV-primed donor cells from either VLA-1-/- or BALB/cA mice. Medians and ranges of five animals are shown; one representative experiment of three is depicted. *, p < 0.05, observed vs control using the Mann-Whitney U test.

To show that the reduced DTH reaction was not a result of less activated VLA-1^-/- cells, but rather a reduction in numbers of cells present in the inflammatory site, we repeated the DTH experiment, this time with CFSE-labeled spleen cells. When footpads from these animals were excised for immunohistochemical staining, it was evident that less CFSE⁺ cells were found in the inflammatory site with VLA-1^-/- donor cells compared with wild-type cells (Fig. 6). Individual bright fields were quantitated, and a significant reduction (p < 0.01) in median numbers of cells/bright field was found, 26 with BALB/cA donor cells and 9 with VLA-1^-/- donor cells, respectively (Fig. 6D); no CFSE⁺ cells were found in the uninfected footpads, confirming the homing specificity to sites of infection (Fig. 6B).

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Reduced inflammation during adoptive DTH reaction in LCMV-infected animals when VLA-1-/- mice are used as donors. Recipients (BALB/cA) were infected with LCMV in their right hind footpad, and 4 h later they received 5 x 107 adherent cell-depleted, LCMV-primed donor cells that had been CFSE labeled. Twenty-four hours later, both left and right footpads were excised for immunohistochemical staining and stained with PE-conjugated anti-CD31. A, Infected footpad from animal that received BALB/cA cells. B, Uninfected footpad from animal that received BALB/cA cells. C, Infected footpad from animal that received VLA-1-/- cells. CFSE-labeled leukocytes are shown in green, and CD31-expressing vascular endothelium is shown in red. Pictures are one representative set of footpads from four animals. D, Quantitative analysis of groups A and C; cells were counted at x20 magnification; each point represents one bright field counted. *, p < 0.05, BALB/cA donor cells vs VLA-1-/- donor cells using the Mann-Whitney U test.

View larger version (38K): [in this window] [in a new window]   FIGURE 7. Effect of anti-1 and anti-2 treatment on the capacity to adoptively transfer an LCMV-specific DTH reaction. Recipients were infected with LCMV in the footpad, and 4 h later they received LCMV-primed donor cells that had been pretreated with Ab and washed before injection. Donor cells were given either i.v. (5 x 107 cells, systemic) (A) or in the footpad (3 x 106 cells, local) (B). Medians and ranges of five animals are shown. *, p < 0.05, observed vs control using the Mann-Whitney U test.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Upon infection with LCMV, a rapid increase in expression of ₁₁ and ₂₁ was observed on CD8⁺ T cells, reaching its peak on day 8–10 p.i., when 50% of splenic CD8⁺ T cells expressed either ₁₁ or ₂₁. The percentage of ₁₁⁺ and ₂₁⁺ T cells reached its maximum at the same time, as did the total expansion of the T cell population. As a result, the number of ₁₁⁺ or ₂₁⁺ T cells was expanded 40-fold over the course of an acute 8-day LCMV infection. Further analyses revealed that expression of the two molecules defined more or less the same subset of CD8⁺ cells as expression of ₁₁ and ₂₁ correlated on CD8⁺ splenocytes. Thus, the expression of the two molecules is almost identical in the acute phase of the immune response. Regarding the CD4⁺ subset, much less dramatic changes were observed, and furthermore, only a 2-fold increase in the total number of splenocytes with this phenotype was observed at the peak time point. Direct visualization of virus-specific CD8⁺ T cells (defined by tetramer staining or demonstration of intracellular cytokine) revealed an elevated expression of both molecules on cells with known antiviral specificity. Similar up-regulation of ₁₁ and ₂₁ was also observed for CD8⁺ cells in LCMV-infected BALB/c mice and in C57BL/6 mice infected with VSV, suggesting that systemic viral infections could be more generally associated with up-regulation of ₁₁ and ₂₁ on virus-specific T cells. However, this should be tested in other viral models.

Contrary to the acute phase, during which the expression of ₁₁ and ₂₁ did not differ substantially, a significant difference was observed in the memory phase. A subset of primed (₄₁^high) CD8⁺ cells still expressed ₁₁, whereas the expression of ₂₁ returned to near background levels. Focusing on CD8⁺ T cells of known virus specificity (tetramer⁺), it was observed that most (>80%) express ₁₁ in immune animals. Nevertheless, it is clear that a significant subset of primed ₄₁^high ₁₁^- cells exists in immune animals. Because the ₄₁^high ₁₁^- subset is much expanded in virus-infected mice, there can be little doubt that most of the cells with this phenotype have been activated as a result of virus infection. This leaves two major explanations as to its presence in immune mice: either these cells represent cells of low avidity for Ag (i.e., below the cutoff of the methods used to visualize Ag-specific cells) that have not differentiated to the same extent as have cells of higher avidity, or alternatively, this subset is made up of cells of totally unrelated specificity expanded as a result of bystander activation (33, 39). Recently, evidence has been presented indicating that under certain conditions naive cells may differentiate and acquire a phenotype overlapping with that of Ag-primed cells (40, 41).

Several studies have been performed that describe changes in expression of integrins during viral infections, and from these studies there appears to be a hierarchy among the different integrins. Following activation of CD8⁺ T cells, most integrins become up-regulated (19). However, the degree of persistence varies significantly. _M2 (42) and ₂₁ (present study) rapidly become down-regulated following elimination of Ag and appear to be markers of recent activation. ₁₁ persists on the majority of memory CD8⁺ T cells, and the level of expression appears to be relatively stable throughout the observation period (up to 120 days). ₄₁ and _L2 are also up-regulated on activated cells, and an increased expression is maintained on the majority of memory CD8⁺ cells, although at a level lower (3-fold) than that on recently activated cells (23, 43). However, this level of expression appears to be stable for months. Both ₄₁ and _L2 bind to ligands found on endothelium (44, 45, 46, 47), thus probably ensuring that memory cells are still capable of extravasating from the vasculature and patrol the peripheral tissues. Expression of ₁₁ by the same subset of cells could further promote their migration by allowing adhesive interactions with the extracellular matrix.

With regard to a functional role, we studied a well-defined model of LCMV-induced T cell-mediated inflammation: the footpad DTH reaction. This model of CD8⁺ T cell-mediated inflammation requires migration of cells in extracellular matrix to induce measurable footpad swelling. Furthermore, in a recently published study by de Fougerolles et al. (2), blocking ₁₁ or ₂₁ led to diminished footpad swelling in the SRBC-induced DTH model. Blocking ₁₁ and ₂₁ was also found to have an inhibitory effect on the LCMV-induced footpad-swelling reaction. Although anti-₁ or anti-₂ mAb treatment had little effect on primary LCMV-induced footpad responses, adoptive transfer experiments readily revealed an effect of both Abs. This pattern has been observed before (19), and is likely to reflect the different conditions of the analysis. In the adoptive transfer, a rather limited number of effector cells is offered to the recipient. This would make this approach more sensitive (less cells to block) in revealing all molecules of importance, because even small changes in the efficiency of the donor cells are likely to be exposed. Treatment of mice with either anti-₁ or anti-₂ mAb was found to significantly inhibit a footpad DTH response in mice adoptively transferred with recently activated effector T cells, a result that was confirmed using VLA-1^-/- donor cells instead of Ab treatment. One proposed mechanism of action for why disruption of ₁₁ and ₂₁ interaction with collagen can decrease inflammatory responses is that these interactions are critical for migration of immune cells within the inflamed tissue (2). Using the adoptive transfer method, we directly showed that the inhibitory effect of anti-₁ mAb treatment on the DTH response could be bypassed by direct injection of effector T cells into the inflammatory site. This result indicates that ₁₁ plays a minor role once the cells are in position (please note that the number of injected cells is likely to markedly exceed what is the case following natural migration), and strongly suggests a role for ₁₁ in focusing of CD8⁺ Ag-specific T cells at sites of inflammation.

In conclusion, these results highlight the inducible and rapid expression of ₁₁ and ₂₁ on effector CD8⁺ T cells, the selective maintenance of ₁₁ on memory CD8⁺ T cells, and the functional importance of these two integrins for Ag-specific T cell responses. Given that ₁₁ is most likely important for migration of Ag-specific T cells within the extracellular matrix, this molecule is an obvious target for therapeutic treatment.

	Acknowledgments

 
We thank Grethe Thørner Andersen for her skillful technical assistance. The National Institute of Allergy and Infectious Diseases Tetramer Facility and the National Institutes of Health AIDS Research and Reference Reagent Program are acknowledged for supplying MHC-peptide tetramers.

	Footnotes

 
¹ This study was supported in part by the Danish Rheumatism Association, the Danish Medical Research Council, the Biotechnology Center for Cellular Communication, the Beckett Foundation, and the Novo Nordisk Foundation. J.P.C. is the recipient of a research fellowship from the Weimanns Foundation, Denmark.

² A.R.d.F. and J.P.C. contributed equally to this study.

³ Address correspondence and reprint requests to Dr. Jan Pravsgaard Christensen, Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, 3C Blegdamsvej, DK-2200 Copenhagen N, Denmark. E-mail address: J.Pravsgaard{at}immi.ku.dk

⁴ Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; DTH, delayed-type hypersensitivity; p.i., postinfection; VSV, vesicular stomatitis virus.

Received for publication March 3, 2003. Accepted for publication July 10, 2003.

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