INTRODUCTION
Laminins are major components of all basement membranes (BMs), the extracellular matrix that separates epithelia or endothelia from the underlying tissues.
All laminins are composed of 3 different disulfide-linked polypeptides, termed as α, β, and γ. Sixteen isoforms have been characterized with different properties, functions, and localization (for review see Simon and Bromberg1). Laminins α4 β1 γ1 (411) and α5 β1 γ1 (511) are the 2 main isoforms that have been described to modulate immune cell functions and leukocyte migration. The BM of the vascular and lymphatic systems that support immune cell trafficking is composed of these 2 isoforms.2-4 To infiltrate tissues, leukocytes must first cross a blood or lymphatic endothelial barrier and interact with laminins 411 and 511. This interaction is a key rate-limiting step in leukocyte transmigration and tissue infiltration. In laminin α4-deficient mice, the infiltration of T lymphocytes5 or neutrophils6 into inflamed tissue is reduced. These studies demonstrated that in vivo laminin α4 and laminin 411 favored leukocyte transendothelial migration. Laminin 511 also has an important role in the regulation of leukocyte transmigration. Neutrophils and T cells penetrate vascular BM regions that are characterized by low expression of laminin 511.7,8 Together, these studies suggest that T-cell transmigration through endothelial BMs occurs when laminin 411 is present and laminin 511 is weakly expressed. We recently confirmed these opposing roles of laminins on CD4+ T cell transendothelial migration.4 These data demonstrated that laminin 411 is more permissive for CD4+ T cell movement on and into the endothelial cell layer, whereas laminin 511 prevented it.
In lymph nodes (LN), which are fundamental for the initiation of immunity or tolerance,4,9 laminins 411 and 511 are also the main isoforms expressed and modulate leukocyte infiltration. We observed that these 2 isoforms were expressed predominantly in the LN cortical ridge (CR), a region that is essential for bringing T lymphocytes and antigen-presenting cells together.4 Blocking the α6 integrin-binding site on T cells to laminin α5 favored the migration of regulatory suppressive T cells (Treg) and plasmacytoid dendritic cells into the LN, while blocking laminin α4 reduced migration. Based on these observations, we hypothesized that the stromal laminins 411 and 511 could modulate not only CD4+ T-cell migration in the LN but also activation and polarization by acting as costimulatory or coinhibitory ligands.
MATERIALS AND METHODS
Cardiac Transplant and Mouse Treatment
C57BL/6 mice received heterotopic cardiac allograft transplants from sex-matched donor BALB/c mice as previously described.10 On the day of transplantation, mice were treated with anti-integrin α6 monoclonal antibody (mAb) (clone GoH3, 10 µg IV; Biolegend, San Diego, CA) or isotype control (rat IgG2a), anti-α-dystroglycan (αDG) mAb (clone IIH6C4, 10 µg IV; Merck Millipore, Darstadt, Germany), or isotype control IgM associated or not with anti-CD40L mAb (clone MR1, 0.25 mg IV; Bio X Cell, West Lebanon, NH). Anti-integrin α6 mAb or anti-αDG mAb was administered again every 3 days or once a week when associated with anti-CD40L mAb. Graft function was monitored daily by abdominal palpation.
For donor-specific transfusion (DST) treatment, C57BL/6 mice were injected intravenously with 1 × 107 BALB/c splenocytes with or without anti-CD40L mAb. DST cells were obtained from BALB/c spleens.
For complete Freund adjuvant (CFA) treatment, C57BL/6 mice were injected subcutaneously in the flank with emulsified CFA (Sigma Aldrich) complemented with 400 µg of mycobacterium tuberculosis (BD Biosciences, San Jose, CA), with or without 200 µg of Ea peptide11 (Anaspec Inc., Fremont, CA).
For the tumor model, mice were inoculated in the flank with 5 × 105 C57BL/6-derived MC38 colon carcinoma cells maintained as described.12
To induce inflammatory colitis, C57BL/6 mice were administered with 2% (w/v) dextran sulfate sodium (DSS) (molecular weight of 36 000–50 000 Da; MP Biomedicals, Santa Ana, CA) in drinking water for 24 hours or were given water only.
T Cell Adoptive Transfer
At the time of CFA/Ea immunization or 24 hours after DST treatment, mice were given 5 × 106 purified CD4+ T cells intravenously with anti-integrin α6 mAb (clone GoH3, 10 µg IV; Biolegend, San Diego, CA) or isotype control, or anti-αDG mAb (clone IIH6C4, 10 µg IV; Merck Millipore, Darstadt, Germany) or isotype control IgM. CD4+ T-cell activation, proliferation, and polarization were analyzed 3 days after DST treatment or 2 days after CFA/Ea immunization.
In Vitro CD4 T-Cell Activation and Differentiation
CD4 T cells from C57BL/6 mice were activated in 96-well plates at a density of 1 × 105 in complete RPMI medium. Cells were activated with coated anti-CD3 (1 or 5 µg/mL, clone 2C11; BD Bioscience) with or without coated laminin 411 (2 µg/mL; Biolamina, Matawan, NJ) and/or laminin 511 (1 µg/mL; Biolamina). After 3 days of culture, T-cell proliferation and CD69, CD25, and CD44 expression were determined by flow cytometry. For T-cell polarization, CD4 T cells from C57BL/6 mice were cultured 5 days with coated anti-CD3 mAb (5 µg/mL) and soluble anti-CD28 (1 µg/mL, clone 37.51; eBioscience, San Diego, CA) with or without coated laminin 411 and/or laminin 511, in pro-Th1, Th2, Treg, or Th17 environments. The laminin α5 receptor was blocked with anti-α6 integrin mAb or with anti-αDG mAb. All the antibodies were preservative and endotoxin free. After 5 days of culture or after CD4 T-cell isolation from the spleen or the LNs, CD4 T cells were activated for 4 hours with phorbol 12-myristate13-acetate (PMA) (40 ng/mL; Sigma-Aldrich, Allentown, PA) and ionomycin (1 µg/mL; Sigma-Aldrich) in the presence of brefeldin A (dilution 1:1000; BD Biosciences), and cytokine expression was analyzed by flow cytometry.
Statistics
Data were analyzed with Prism 4.0c software (GraphPad Software, Inc.) using Student’s unpaired 2-tailed t tests (for single variable differences), or 1- or 2-way analysis of variance (for multiple variable differences) with Tukey’s or Bonferroni post-tests, as appropriate. Graft survival was plotted using Kaplan–Meier curves and differences between groups were estimated using the Log-rank (Mantel-Cox) test. P ≤ 0.05 were considered statistically significant.
RESULTS
Distinct Laminin α4 and α5 Expression Profiles in the LN Following the Induction of Immunity Versus Tolerance
We previously demonstrated that laminin expression changed in response to alloantigen.4 To determine whether remodeling was generalizable response, we measured laminins in other models. Differences in expression of the laminin α4 and laminin α5 were evident after induction of inflammation or immunity by subcutaneous injection of CFA or intravenously DST, or tolerance with DST+anti-CD40L mAb or anti-CD40L alone (Figure 1A; Figure S1 and Table S1, SDC, https://links.lww.com/TP/B743). The intense inflammatory reaction induced by CFA was associated with decreased laminin α4 in the CR compared to naïve LNs. Laminin α5 strongly increased in the CR and high endothelial venules (HEV). Thus, laminin α4:α5 ratios were significantly lower in the CFA group. Immunization with DST that is associated with a moderate inflammation and allogenic immune response in LNs13 increased the expression of laminin α5 in the CR. The blockade of CD40L alone or the induction of tolerance with DST+anti-CD40L significantly reduced laminin α5 expression in the CR and in the HEV leading to higher laminin α4:α5 ratios compared to the other groups. These results confirmed our previous observation4 and demonstrated that changes in laminin α4 and α5 expression in the LN occurred very rapidly after the induction of immunity or tolerance.
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FIGURE 1.: Laminin α5 and laminin α4 expressions are differentially regulated in the lymph node following the induction of inflammation, immunity, or tolerance. A, Skin lymph nodes (LNs) from naïve, complete Freund adjuvant (CFA), donor-specific transfusion (DST), DST+anti-CD40L, and anti-CD40L-treated C57BL/6 mice were isolated 5 h after the treatment. B, Skin draining (dLN) and nondraining (nLN) LNs distal to the subcutaneous injection site of MC38 tumor cells were isolated 48 h after injection. C, Mesenteric LNs from untreated or dextran sulfate sodium (DSS)-treated colitic mice were isolated 24 h after treatment. All LNs were sectioned and stained for laminin α4, laminin α5, peripheral lymph node addressin, and ER-TR7. Representative images, ×100. Percentages of laminin α4 and laminin α5 positive areas, and the ratios of laminin α4:laminin α5 in the cortical ridge (CR) and high endothelial venules (HEV). Data were shown as mean ± SEM. n = at least 6 mice per condition from 3 independents experiments; 1–4 LNs/mouse, 30–100 HEVs/LN; *P < 0.05, **P < 0.01, ***P < 0.001.
We also analyzed laminin expression in draining LNs (dLN) 48 hours after subcutaneous injection of MC38 tumor cells, an adenocarcinoma known to generate local inhibitory signals.14 In the tumor dLN, the laminin α5 expression in the CR and in the HEV of the dLN was significantly reduced and the laminin α4:α5 ratio strongly increased compared to the non-dLN (Figure 1B).
We next investigated the laminin α4 and α5 expression in the DSS inflammatory colitis model.15 In the mesenteric LN of colitic C57BL/6 mice treated with DSS for 24 hours, laminin α5 expression significantly increased and the laminin α4:α5 ratio decreased compared to the control untreated group (Figure 1C).
Together, these results demonstrated that in multiple distinct models, high laminin α5 expression was associated with immunity and inflammation, while low laminin α5 expression and thus high laminin α4 expression relative to laminin α5 were associated with immune suppression and tolerance. This also suggested that the laminin α4:α5 ratios could dictate the orientation of the immune response and impact T-cell activation fate.
Laminin 411 and 511 Differentially Regulate CD4+ T-Cell Activation
To test the hypothesis that stromal laminin 411 and 511 directly modulated CD4+ T cell activation, we analyzed the proliferation and the expression of activation markers of murine CD4+ T cells activated for 3 days with anti-CD3 at 1 µg/mL (Figure 2A) or 5 µg/mL (Figure S2A, SDC, https://links.lww.com/TP/B743) in the presence or absence of coated laminin 411 and 511. Coated laminin 411 decreased in a dose-dependent manner the proliferation and the expression of CD69, CD25, and CD44. Laminin 511 had the opposite effect and strongly favored T-cell activation. Interestingly, the addition of laminin 411 to laminin 511 reduced the stimulatory effect of laminin 511 (Figure 2B; Figure S2B, SDC, https://links.lww.com/TP/B743) demonstrating that as observed in vivo, the ratio between these two laminins can dictate the level of T cell activation. We also analyzed the proliferation of human CD4+ T cells activated with anti-CD3/28 beads in the presence or absence of coated laminins (Figure 2C). As for murine CD4+ T cells, laminin 511 increased human T-cell proliferation and activation. Laminin 411 had no direct inhibitory effect on T cell activation but inhibited the immunostimulatory effect of laminin 511, confirming that the laminin 411:511 ratio dictated T cell activation fate also in human. In mice, both laminins slightly increased T cell viability (Figure S2C, SDC, https://links.lww.com/TP/B743) excluding the possibility that the inhibitory effect of the laminin 411 on T-cell proliferation was due to cell death. Together, these data demonstrated that laminin 411 and 511 are respectively coinhibitory and costimulatory ligands for CD4+ T cells.
FIGURE 2.: Stromal laminin 411 and 511 differentially modulate CD4+ T-cell activation in mice and human. Murine CD4 T lymphocytes were evaluated by flow cytometry for proliferation and CD69, CD25, CD44 expression (A, B) after 3 days of culture in plates coated with anti-CD3 (1 µg/mL) and the indicated concentrations of laminin 411 or laminin 511 (A), or with laminin 411 (2 µg/mL) and/or laminin 511 (1 µg/mL) (B). Human CD4+ T lymphocytes (C) were evaluated by flow cytometry for proliferation and CD25 expression after 3 days of culture in plates coated with laminin 411 (2 µg/mL) and/or laminin 511 (1 µg/mL) with anti-CD3/CD28 beads (T:Beads ratio = 1:1). Data were shown as representative profiles (B, C) and mean ± SEM (A–C) of 3 independent experiments with samples; *P < 0.05, **P < 0.01, ***P < 0.001.
To characterize the laminin receptors, antibodies against potential receptors for laminins were tested (Table 1). Among all the antibodies used, none of them were able to reverse or prevent the laminin 411 inhibitory effect. In contrast, the anti-α6 integrin blocking antibody abrogated the laminin 511 stimulatory effect on CD4+ T cell proliferation and activation (Figure 3A), while it had no effect without laminin 511, demonstrating that α6 integrin is the main T cell receptor for laminin 511, confirming previous observations.16 The α6 integrin receptor is highly expressed on naïve CD4+ T cells and upregulated upon activation (Figure 3B). Integrins favor the activation of Erk, Akt, and P38Mapk.17,18 These 3 pathways are modulated by laminins 411 and 511 (Figure 3C). Laminin 411 decreased the phosphorylation of ERK1/2, AKT, and P38MAPK in CD4+ T cells activated with anti-CD3 (Figure 3C). Laminin 511 had the opposite effect and promoted the phosphorylation of these 3 molecules via the α6 integrin (Figure 3D). Laminin 411 inhibited the stimulatory effect of laminin 511. As these pathways are essential for CD4+ T-cell proliferation and activation,19 these results highlight at the molecular level how laminin 411 and 511 regulated T-cell activation.
TABLE 1.: Potential anti-laminin receptor antibodies for in vitro use
FIGURE 3.: Laminin 511 favors CD4 T-cell activation via the receptor α6 integrin. CD4 T lymphocytes were evaluated by flow cytometry for proliferation and CD69, CD25, CD44 expression (A); for α6 integrin expression after 3 days of culture (B); or phospho-Erk1/2, phospho-Akt, and phospho-P38Mapk (C) after 30 min of culture in plates coated with anti-CD3 (1 µg/mL) and with laminin 411 (2 µg/mL) and/or laminin 511 (1 µg/mL) in the presence of anti-α6 integrin or isotype control monoclonal antibody (A, D). Data were shown as representative profiles (B, C) and mean ± SEM (A–D) of 3 independent experiments with duplicate samples. E, Donor-specific transfusion (DST) or (F) complete Freund adjuvant (CFA)/Ea-treated mice received 5 × 106 carboxyfluorescein diacetate succinimidyl ester-labeled CD45.1 TEa T-cell receptor (TCR) transgenic CD4+ T cells. Mice were euthanized 2 days after transfer, and donor lymphocytes from spleen and skin lymph nodes (LNs) were assessed for proliferation, CD69, CD25, CD44, and percentage of donor cells (CD4+ CD45.1+) in different organs by flow cytometry. Representative profiles shown together with a quantitative analysis of 3 independent experiments. Data were shown as mean ± SEM of at least 6 mice per group from 3 independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001.
We next determined whether the laminin 511 receptor α6 integrin favored CD4+ T-cell activation in vivo. C57BL/6 (H-2b) mice were immunized with BALB/c (H-2d) DST or with I-Ed peptide in CFA. These mice also were adoptively transferred with T-cell receptor transgenic (TCR Tg) CD4+ T cells from TEa mice that recognize donor I-Ed presented by recipient I-Ab (Ea antigen).11 Mice received anti-α6 integrin blocking mAb or isotype control mAb. In both models, systemic blockade of α6 integrin resulted in decreased proliferation of alloantigen-specific CD4 T cells in the LNs (Figure 3E and F) without affecting the proportion of TEa T cells in the LN (Figure S3A, SDC, https://links.lww.com/TP/B743) excluding an inhibition of T-cell recruitment in the LN. These results confirmed the immunogenic role of this laminin-specific receptor and suggested a costimulatory role for laminin 511 in vivo even under strong stimulatory conditions of CFA or fully mismatched donor alloantigen.
Laminin 411 and 511 Differentially Regulate CD4+ T-Cell Polarization
To test the modulation of polarization by laminins, we first tested the impact of laminin 411 and 511 on CD4+ T cells activated in vitro for 5 days with anti-CD3 and anti-CD28 without polarizing cytokines (Figure S2D, SDC, https://links.lww.com/TP/B743). In a neutral environment, neither laminin had an impact on Th1, Th2, and Th17 induction. In a pro-Th1 environment (Figure 4A), laminin 511 had no effect on the proportion of IFNγ+ cells but significantly increased Tbet expression (Figure 4B). The addition of laminin 411 significantly decreased IFNγ+ cells, Tbet expression, and hence Th1 induction. Addition of laminin 511 to laminin 411 reversed the laminin 411 inhibitory effect on Th1 induction. The laminin 511 effect on Tbet overexpression was mediated by the α6 integrin (Figure 4C).
FIGURE 4.: Laminin 411 and 511 differentially regulate Th1 and Th2 polarization. CD4 T lymphocytes were evaluated by flow cytometry for intracellular expression of IFNγ (A) or IL-4 (D) after 5 days or Tbet, Gata3, and phospho-Stat6 after 16 h culture in pro-Th1 (B, C) or pro-Th2 (E, F) environments with laminin 411 (2 µg/mL) and/or laminin 511 (1 µg/mL) in the presence of functional blocking anti-α6 integrin or isotype control monoclonal antibody (C, F). Data were shown as representative profiles and mean ± SEM of at least 3 independent experiments with duplicate samples.
The addition of laminin 411 to CD4+ cells stimulated in a pro-Th2 environment also inhibited IL-4 and Th2 induction (Figure 4D). Laminin 511 had the opposite effect and increased the proportion of IL-4+ CD4+ T cells (Figure 4D) and promoted the expression of the pro-Th2 transcription factors Gata3 and phospho-Stat6 (Figure 4E). Addition of laminin 411 to 511 completely abrogated the overexpression induced by laminin 511. Each of these laminin 511-driven effects was mediated by the α6 integrin since blocking mAb prevented them (Figure 4F). We next analyzed whether the laminin 511 receptor α6 integrin also favored Th1 and Th2 induction in vivo. TEa TCR Tg CD4+ T cells were transferred, with or without anti-α6 integrin mAb, into C57BL/6 mice that received CFA/Ea, and expression of IFNγ and IL4 by TEa cells was analyzed after 3 days (Figure S3B, SDC, https://links.lww.com/TP/B743). Systemic blockade of α6 integrin resulted in a slight decreased proportion of IFNγ+ but not IL4+ alloantigen specific CD4+ T cells in the dLN.
In a pro-Th17 environment, laminin 511 increased Th17 induction by almost 4-fold (Figure 5A). Laminin 511 favored expression of the pro-Th17 factors Rorγt, phospho-Stat3, Irf4, and Batf20 and reduced the expression of the IL-6 receptor CD126 (Figure 5B). Laminin 411 had the opposite effects and significantly decreased the proportion of IL-17+ cells and the expression of the pro-Th17 transcription factors. Laminin 411 partially reversed the pro-Th17 effect of laminin 511. Addition of the anti-α6 integrin mAb in the culture was unable to inhibit the laminin 511 effect on IL17 production (Figure 5C). Among all the blocking antibodies tested (Table 1), only the blockade of the laminin receptor αDG abrogated the pro-Th17 effect of laminin 511. Surprisingly, anti-α6 integrin inhibited the effect of laminin 511 on Rorγt, phospho-Stat3, Irf4, and Batf expression while anti-αDG did not (Figure 5D). These results suggested that an unknown pro-Th17 pathway is induced by the laminin 511/αDG axis.
FIGURE 5.: Laminin 511 promotes Th17 polarization via the α-dystroglycan (αDG) receptor. CD4 T lymphocytes evaluated by flow cytometry for IL-17 after 5 days of culture (A, C), Rorγt, phopho-Stat3, Irf4, Batf, and CD126 (B, D) expression after 16 h of culture in pro-Th17 environment with laminin 411 (2 µg/mL) and/or laminin 511 (1 µg/mL) in the presence of functional blocking anti-α6 integrin monoclonal antibody, anti-αDG, or isotype control (C, D). Data were shown as representative profiles (A, B) and mean ± SEM (A–D) of at least 3 independent experiments with duplicate samples. E, complete Freund adjuvant/Ea treated mice received 5 × 106 carboxyfluorescein diacetate succinimidyl ester-labeled CD45.1 Ea T-cell receptor transgenic CD4+ T cells, with or without anti-αDG monoclonal antibody, euthanized 3 days after transfer, and donor lymphocytes from spleen and skin LNs assessed as percentage of IL-17+ donor cells (CD4+ CD45.1+) in different organs by flow cytometry. Representative profile was shown with quantitative analysis of 3 independent experiments. Data were shown as mean ± SEM of 8 mice per group from 3 independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001.
We next analyzed whether the laminin 511 receptor αDG also favored Th17 induction in vivo. TEa TCR Tg CD4+ T cells were transferred, with or without anti-αDG mAb, into C57BL/6 mice that received CFA/Ea, and expression of IL-17 by TEa cells was analyzed after 3 days (Figure 5E). Systemic blockade of αDG resulted in a decreased proportion of IL-17+ alloantigen specific CD4+ T cells in the dLN. The percentage of proliferating cells and the absolute number of the TEa CD4+ T cells were unchanged, excluding the possibility that the decreased proportion was due to antibody-mediated T-cell depletion or a migration defect of the T cells to the LN. Blockade of α6 integrin had no effect on the proportion of IL-17+ cells (Figure S3B, SDC, https://links.lww.com/TP/B743). These results demonstrated, for the first time, a major role for the laminin-specific receptor αDG in Th17 induction in vivo and suggested a costimulatory role for laminin 511.
In a pro-Treg environment, laminin 411 significantly increased the proportion of CD25+ FoxP3+ CD4+ T cells (Figure 6A). Laminin 511 had the opposite effect and reduced the proportion of Treg cells by 2-fold. To explore why laminin 511 inhibited Treg induction in the presence of transforming growth factor β, the expression of pro-Th17 factors was analyzed (Figure 6B). Laminin 511 favored the expression of Rorγt, Irf4, and Batf, and increased Stat3 phosphorylation, demonstrating that in a pro-Treg environment, laminin 511 promoted the induction of Th17 pathways and so decreased Treg polarization. Laminin 411 almost completely inhibited the expression of these pro-Th17 molecules and thus favored Treg induction. The inhibitory effect of laminin 511 on Treg induction was abrogated by anti-α6 integrin mAb (Figure 6C) and not by anti-αDG (data not shown). Induction of pro-Th17 factors by laminin 511 was also inhibited by anti-α6 integrin (Figure 6D).
FIGURE 6.: Laminin 411 and 511 differentially regulate regulatory T cell polarization. CD4 T lymphocytes evaluated by flow cytometry for CD25, FoxP3 expression after 5 days of culture (A, C), Rorγt, phopho-Stat3, Irf4, and Batf (B, D) expression after 16 h of culture in pro-regulatory T cell environment, with laminin 411 (2 µg/mL) and/or laminin 511 (1 µg/mL) in the presence of functional blocking anti-α6 integrin or isotype control monoclonal antibody (C, D). Data were shown as representative profiles (A–C) and mean ± SEM (A–D) of at least 3 independent experiments with duplicate samples; *P < 0.05, **P < 0.01, ***P < 0.001.
Together, these results demonstrated that laminins 411 and 511 were important regulators for CD4+ T cell polarization and had divergent effects.
Targeting the Laminin 511 Receptors α6 Integrin and αDG Interferes With the Induction of Alloimmunity and Cardiac Allograft Rejection
Cardiac allograft rejection is associated with a large decrease in the laminin α4:α5 ratio around post capillary venules, suggesting that the costimulatory effect of laminin 511 could also be present in the transplant in addition to the LN (Figure S4, SDC, https://links.lww.com/TP/B743). We hypothesized that blocking the main receptors for laminin 511, the α6 integrin and the αDG, would interfere with immunity and favor the generation of immune suppression and tolerance in the LN and in the cardiac transplant. To test this hypothesis, otherwise untreated allograft recipients received anti-α6 integrin mAb or anti-αDG intravenously. No side effects or infections have been observed in the treated animals. Anti-α6 integrin resulted in a small but significant prolongation of graft survival (Figure 7A) similar to the protection induced by antilaminin α5 antibody treatment that we observed previously.4 In comparison to potent, widely used immunosuppressive agents such as CTLA4-Ig21 or rapamycin,22 the protection induced by anti-α6 integrin mAb alone was moderate. Anti-αDG alone had no effect on graft survival (Figure 7B). We further hypothesized that increasing the laminin α4:α5 ratio with anti-CD40L mAb as demonstrated in Figure 1A would improve the efficacy of anti-α6 integrin or anti-αDG treatment for immune protection. Thus, mice were injected once on the day of transplantation with anti-CD40L and treated with or without anti-α6 integrin or anti-αDG (Figure 7C). The addition of anti-α6 integrin or anti-αDG to anti-CD40L treatment resulted in long-term protection. These data suggested that increasing the laminin α4:α5 ratio in the LN CR synergized with the inhibition of the laminin 511-α6 integrin or αDG interactions on CD4+ T cells to promote immune suppression and tolerance.
FIGURE 7.: Blocking laminin 511 receptor α6 integrin and α-dystroglycan (αDG) prolongs allogenic cardiac graft survival. C57BL/6 mice received BALB/c heterotopic cardiac allografts on day 0 along with anti-α6 integrin or isotype control monoclonal antibody alone (10 µg IV, day 0 and every 3 days) (A), anti-αDG or isotype control monoclonal antibody alone (B), anti-α6 integrin or anti-αDG (10 µg IV, day 0 and once a week) with anti-CD40L (250 µg, IV day 0 only) (C). Data were presented as percent survival; n = at least 5 mice per group; *P < 0.05, **P < 0.01, ***P < 0.001.
DISCUSSION
In this study, we demonstrated that induction of immunity or tolerance modulated very rapidly the expression of laminin α4 and laminin α5 (Figure 1) in the LN. Inflammation and allogenic responses (Figure 1A and C) increased laminin α5 expression rapidly in the CR and HEV of the dLN. Induction of tolerance with anti-CD40L treatment (Figure 1A) or the presence of immunosuppressive environment (Figure 1B) reduced significantly the expression of laminin α5. For laminin α4 expression, only the major inflammation associated with CFA treatment was able to decrease its expression in the CR. It has been clearly demonstrated that the expression of the laminins is differentially controlled by numerous signals such as growth factors, cytokines, and proteases.16,23,24 The regulation of laminin expression in the LN cortical ride is a complex process that could involve either laminin polymerization at the cell surface, de novo protein production, laminin degradation, or migration of laminin positive cells. These different mechanisms may contribute to the fact that the laminin 411 and 511 composition in the LN is variable and dynamic. However, the rapid overexpression of laminin α5 observed only 5 hours after induction seems to be inconsistent with de novo protein synthesis.
During adaptive immune responses, LNs undergo important modifications in size and structure.25-28 Infection induces transcriptional changes in LN stromal cells resulting in expanded fibroblastic reticular cell networks that persist for months, a process termed as “immunological scarring.”25 In our study, we demonstrated that the laminin α4:α5 ratio in the LN was one of the first main modifications associated with LN remodeling and was directly correlated to the state of the immune response. Moreover, these changes in laminin α4 and α5 expression in the LN were persistent for at least 72 hours after induction of immunity or tolerance.4 Based on these observations, we hypothesized that laminin isoforms 411 and 511 directly impacted T lymphocyte activation. In vitro studies demonstrated that addition of laminin 411 to anti-CD3 stimulated cultures reduced proliferation and activation (Figure 2A and B). In contrast, addition of laminin 511 increased proliferation and activation by up to 8-fold. Previous studies described the modulation of T cell activation by laminins, but with conflicting results.16,29,30 Discrepancies among reports may be explained by the use of poorly characterized laminin preparations isolated from human placenta versus recombinant laminin proteins, variation in laminin concentrations, and use of unpurified lymphocyte populations or T cell lines. Interestingly, the in vivo proliferation analysis in LN of CD4+ T cells in lama4−/− full knock out mice showed no difference compared to wild type.5 The discrepancy between this study and ours, which showed laminin 411 decreased proliferation, may be related to developmental compensatory mechanisms in germline knock out mice. Indeed, in these mice, laminin α5 distribution in endothelial BM was altered and associated with substantial modifications of myeloid and T-cell migration. These changes could affect T cell activation and proliferation compared to wild type and compensate for the loss of the inhibitory effect of laminin 411.
The effect of laminin 511 is mainly mediated by integrin α6 (Figure 3A). The molecular pathways activated by laminin 511, such as ERK1/2, AKT, and P38MAPK, were inhibited by anti-α6 integrin antibody. Blocking α6 integrin also reduced CD4 T cell proliferation in different models in vivo (Figure 3E and F), confirming that α6 integrin was a costimulatory receptor for CD4 T cells. Blocking α6 integrin prolonged allograft survival and resulted in long-term survival when combined with anti-CD40L mAb (Figure 7). Thus, the laminin 511-α6 integrin axis seems to be a promising target in transplantation.
The receptor for laminin 411 that mediated T cell inhibition is still unknown despite using multiple different antibodies. This means that the antibodies were unable to block efficiently laminin 411 interaction with its receptor or the receptor is an unknown nonintegrin receptor. Laminin 411 was described to bind multiple receptors such as integrins α6- or α7- and αDG with poor affinity.31,32 This may explain why blocking only 1 receptor did not produce any effect. More investigations are needed to characterize laminin 411 receptors that mediated T-cell inhibition, which can be an interesting therapeutic target.
We analyzed the impact of laminin 411 and 511 on CD4 T-cell polarization toward the well-described Th1, Th2, Th17, and Treg paths.33 Laminin 511 had little impact on Th1 polarization, slightly increased Th2 CD4 T-cell induction, and greatly promoted Th17 induction (Figures 4A, C and 5A). The main pathways20 involved in Th17 polarization were upregulated in the presence of laminin 511 (Figure 5B). The prototypic Th17 transcription factor Rorγt was also highly upregulated by laminin 511. As for proliferation, the laminin 511 effects on Rorγt expression and phosphorylation of pro-Th17 pathways were mediated by α6 integrin. However, only the blocking of αDG34 inhibited IL-17 induced by laminin 511 (Figure 5C). The pro-Th17 effect of αDG was confirmed in vivo in CFA/antigen immunized mice (Figure 5E). We demonstrated for the first time that αDG promotes Th17 generation and could be a promising therapeutic target. Indeed, the blocking of αDG prolonged cardiac allograft survival and resulted in long-term survival when combined with anti-CD40L mAb (Figure 7B)
Likely because of activation of pro-Th17 pathways, laminin 511 inhibited induction of FoxP3 regulatory CD4 T cells (Figure 6) via α6-integrin. Interestingly, laminin 411 showed an opposite effect compared to laminin 511. Laminin 411 inhibited Th1, Th2 (Figure 4A and C), and Th17 polarization (Figure 5A), but favored Treg induction (Figure 6A). Both Th17 and Treg cells can develop from naïve CD4+ T cell precursors under the influence of the same cytokine, transforming growth factor β1.33 We demonstrated here that laminins 411 and 511 are important factors driving Treg and Th17 plasticity. Th1, Th2, and Th17 have been described to play an important role in allograft rejection. Th1 cells are considered as 1 of the main actors in acute allograft rejection,35 while Th2 seems to be more involved in chronic rejection.36 The active role of Th17 cells in transplant rejection has been described more recently and is linked to neutrophil recruitment into the graft.37 Due to the central role of CD40L in Th1 activation, anti-CD40L inhibits Th1 induction and acute rejection38 but favors Th2 induction that could be deleterious for transplant outcome as described previously.36 Moreover, it has been demonstrated that in the absence of a Th1 alloimmune response, aggressive Th17 lymphocytes come into play and favor allograft rejection in a model of chronic rejection.39 The inhibition of Th2 and Th17 induction associated with anti-α6 integrin and anti-αDG that we observed may explain the long-term graft protection when these 2 antibodies are used in association with anti-CD40L treatment (Figure 7C). Thus, targeting the laminin 511 receptors α6 integrin and αDG to reduce CD4 T-cell proliferation and Th1, Th2, and Th17 polarization in favor of Treg cells induction appear as a promising therapeutic strategy for transplantation.
All together, these data demonstrated that laminin 411 and 511 expressed in the LN are coinhibitory and costimulatory ligands, respectively, for CD4 T cells. Laminin 411 and 511 had opposite effects that countered each other. These findings demonstrated that laminins are not only passive LN structural molecules, but also act as one of the molecular switches for tolerance and immunity by directly influencing T-cell activation and functions.
ACKNOWLEDGMENTS
This work was prepared while Terez Shea-Donohue was employed at University of Maryland School of Medicine. The opinions expressed in this article are the author’s own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States Government.
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