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The Journal of Immunology, 2002, 168: 1579-1589.
Copyright © 2002 by The American Association of Immunologists

L-Selectin-Dependent Lymphoid Occupancy Is Required to Induce Alloantigen-Specific Tolerance1

Yalai Bai2, Jianhua Liu, Yinong Wang2, Shaun Honig, Lihui Qin, Peter Boros and Jonathan S. Bromberg3

Carl C. Icahn Institute for Gene Therapy and Molecular Medicine and Recanati/Miller Transplant Institute, Mount Sinai School of Medicine, New York, NY 10029


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Maneuvers that interfere with signals 1, 2, 3, or Ag processing can result in indefinite allograft survival. However, they are not applicable to all tissues, strains, or species, suggesting that there are additional levels of immune regulation. We hypothesized that secondary lymphoid organs are important for interactions among lymphocytes, alloantigen, and immunosuppressants that lead to tolerance. To explore this, cardiac allografts were performed with a tolerogenic immunosuppressive regimen. Concurrent administration of anti-L-selectin (CD62L) Ab, which prevents lymph node homing, prevents indefinite allograft survival and tolerance. Anti-CD62L Ab is not costimulatory, and Fab and F(ab')2 anti-CD62L have similar activities. Flow cytometry and histologic examination show that Ab shifts T cells away from lymph nodes and into spleen, peripheral blood, and graft. Tolerance is not induced in CD62L-/- mice, and adoptive transfer of CD62L-/-, but not CD62L+/+, T cells prevents tolerization in wild-type recipients. FTY720, an immunosuppressant that promotes chemokine-dependent, but CD62L-independent, lymph node homing, reverses the Ab effect. Blockade of other homing receptors also prevents tolerization. These results indicate that T lymphocytes use CD62L-dependent migration for alloantigen-specific tolerance, and suggest that lymph nodes or other lymphoid tissues are an important site for peripheral tolerization to alloantigen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Peripheral tolerance induction to alloantigen in the mature immune system remains the primary goal of transplantation. While many methods are effective in rodents, they often fail in large animal or primate experimentation, or they are not able to prevent chronic rejection (1). These findings suggest that additional mediators and levels of regulation of the immune response remain to be defined. An important consideration is the determination of the anatomic region(s) in which peripheral tolerance to alloantigen occurs. It is not currently known precisely where lymphocytes interact with alloantigen, under the cover of immunosuppression, to become tolerant. It is also not known whether lymphocyte-Ag interactions for immunity and rejection are anatomically separate from those interactions important for tolerance.

Recent studies show that the lymph node (LN)4 microenvironment is highly structured, and T-B-APC interactions occur in a defined sequence that involves both spatial and temporal variables (2, 3). Naive T cells, expressing the chemokine receptor CCR7, bind their ligands secondary lymphoid tissue chemokine (also called 6Ckine or CCL21) and EBV-induced molecule 1 ligand chemokine (CCL19), found in the T cell zone of the LN, and these receptor-ligand interactions are crucial for T cell homing to the LN (4, 5). Following initial activation of T cells, CCR7 is down-modulated and CXCR5 expression is up-regulated, promoting T cell migration to the B lymphocyte-rich follicle expressing the B lymphocyte chemoattractant ligand (CXCL13) (6). These ligands are in turn developmentally regulated by TNF and the TNF family members lymphotoxin (LT) {alpha} and LT{beta} (7). These findings suggest a highly orchestrated series of events and imply that lymphoid architecture is a critical determinant for the induction of immunity. This is supported in a recent study by Lakkis and his colleagues (8), who demonstrated that in spleen-deficient Hox11-/- mice (9) or in LN-deficient aly mice, secondary lymphoid organs are required to generate normal immunity, and that the spleen or LNs can assume similar functions, depending on the lymphatic drainage of the allograft.

These studies do not address the question of whether secondary lymphoid organs are also important for peripheral tolerization. The recent demonstration (10) that CCR7 engagement can inhibit some aspects of T cell activation suggests that the LN microenvironment could be tolerogenic. Townsend and Goodnow (11) used a TCR transgenic (Tg) model to show that T-B and T-APC interactions in the LN led to T cell activation, proliferation, and eventual disappearance, presumably by activation-induced cell death. T cell loss by activation-induced cell death did not occur in the spleen or in vitro. A number of investigators using Tg nonobese diabetic mice, constitutively expressing a variety of cytokines under the control of the insulin promoter, have demonstrated that the determination of immunity vs peripheral tolerance does not take place in the islet, but within the draining LNs of the pancreas (12). Tg expression of TNF-{alpha}, LT{alpha}, or LT{beta} in the pancreas results in lymphocyte accumulation and lymphoid neogenesis, through regulation of secondary lymphoid tissue chemokine, EBV-induced molecule 1 ligand chemokine, B lymphocyte chemoattractant, and secondary lymphoid organ development. Lymphoid neogenesis in the pancreas resulted in tolerance to islet autoantigens and inhibition of diabetes (13, 14, 15). In fact, diabetes could only be generated if additional strong costimulatory signals were supplied. This suggests a default pathway of tolerization within the LN.

Lymphocytes recirculate from blood into lymphoid organs and express homing receptors for endothelial cells lining vascular and lymphatic vessels. One of these receptors, L-selectin (CD62L), mediates the homing of leukocytes to LNs through binding to its carbohydrate ligands on the high endothelial venules. L-selectin is constitutively expressed by most leukocytes and does not require external, activating signals for function, although CD62L expression can be increased by certain activating factors (16). Unlike any other leukocyte adhesion proteins, CD62L is rapidly down-modulated from the cell surface following cell activation. Treatment of leukocytes with specific anti-L-selectin Abs also leads to rapid down-modulation of CD62L expression (17), and in vivo the mAbs cause rapid egress of T cells from the LN (18, 19). L-selectin-deficient or knockout mice have been independently generated (20) and have a general failure of T cells to home to the LNs. Nonetheless, these animals have intact immune responses and are capable of rejecting allografts (21, 22, 23). The effects of acquired or genetic depletion of CD62L on peripheral tolerization, however, have not been previously reported.

Given the above considerations, we hypothesized that T cells utilize CD62L to migrate to LNs, and that under the influence of systemic immunosuppression, the LN becomes a site integral for tolerization. The results in this study demonstrate that the MEL-14 anti-CD62L mAb inhibits T cell homing to the LN and inhibits peripheral tolerance induction. Likewise, CD62L-/- mice cannot be tolerized, and adoptive transfer of their cells to wild-type recipients also prevents tolerance. Flow cytometric and histologic analysis of secondary lymphoid organs and graft-infiltrating lymphocytes reveals changes in the distribution of T lymphocyte between the LNs and periphery, a change in the ratio of CD4:CD8 cells, and also a change in structure of secondary lymphoid organs according to whether recipients have received tolerogenic or nontolerogenic treatments. These results suggest that LNs or other secondary lymphoid organs participate in peripheral tolerization to alloantigen.


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

CBA/J (H-2k), BALB/c (H-2d) and C57BL/6 (H-2b), and C57BL/6 CD62L-/- mice 8–10 wk of age were purchased from The Jackson Laboratory (Bar Harbor, ME). Timed pregnant C57BL/6 (H-2b) and C3H (H-2k) mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). The TCR Tg DES strain on a CBA background (24) was a kind gift of A. Mellor (MCG, Augusta, GA). All mice were housed in a specific pathogen-free facility in microisolator cages. All experiments were performed with age- and sex-matched mice in accordance with IUCAC-approved criteria.

Reagents

The 12-15 rat IgG1 anti-murine CD2 hybridoma was a gift of P. Altevogt (Immunology and Genetics Institute, Heidelberg, Germany); the 145-2C11 hamster anti-murine CD3{epsilon} hybridoma was a gift of J. A. Bluestone (University of California, San Francisco, CA); and the KM201 rat IgG1 anti-murine CD44 hybridoma was a gift of P. Kincaide (Oklahoma City, OK). The 3C7 rat IgG2b anti-murine CD25{alpha}, PS/2 rat IgG2b anti-murine VLA-4{alpha}, R1-2 rat IgG2b anti-murine VLA-4{alpha}, IM7 rat IgG2b anti-murine CD44, and the MEL-14 rat IgG2a anti-murine CD62L hybridomas were purchased from the American Type Culture Collection (Manassas, VA). All hybridomas were grown in culture, and supernatants were purified over protein G or A columns (Amersham Pharmacia Biotech, Piscataway, NJ). FITC-conjugated rat anti-mouse CD8{alpha} mAb, R-PE-conjugated rat anti-mouse CD4 mAb, rat anti-mouse CD16/CD32 mAb, R-PE-conjugated rat IgG2a,{kappa} isotype standard, and rat IgG2a,{kappa} mAb isotype standard were purchased from PharMingen (San Diego, CA). FTY720, a kind gift of V. Brinkman (Novartis Pharma AG, Basel, Switzerland), was dissolved in water and used at a concentration of 0.1 mg/ml. Two Ab-capture ELISA for IFN-{gamma} were used according to the manufacturer’s instructions (PharMingen).

Nonvascularized cardiac transplantation

Donor neonatal (P1-2) C3H or C57BL/6 mice were sacrificed, and whole hearts were removed and placed in a s.c. pocket in the ear pinnae of recipients (25). The survival of the allografts was followed by EKG (Powerlab Software Installer, AD Instruments, Mountain View, CA) every other day. Cessation of cardiac electrical activity for three consecutive readings was the determinant of rejection. Recipients received i.v. injections of different Abs in 0.5 ml PBS at the indicated times. For tolerization, recipients received 100 µg anti-CD2 mAb on days 0 and 1, and 100 µg anti-CD3 mAb on days 2, 3, 4, 5, and 10 with respect to transplantation. Anti-CD62L was administered at 100 µg on days 0 and 1, or other indicated days. FTY720 was administered by gavage daily.

Vascularized cardiac transplantation

Cardiac grafts from BALB/c donors were collected by dividing and excising the aorta and the pulmonary artery. The cardiac grafts were then transplanted into C57BL/6 recipients by suturing donor aorta and donor pulmonary artery end-to-side to the recipient abdominal aorta and inferior vena cava, respectively (26). Graft function was monitored every other day by abdominal palpation. Rejection was defined as complete cessation of a palpable beat and was confirmed by direct visualization after laparotomy.

Cell preparations and adoptive transfer

Mice were sacrificed, and the spleens and LNs were removed and gently dissociated into single-cell suspensions. RBCs were removed by Tris-NH4Cl lysis. If indicated, cell suspensions were passed through a nylon wool column to enrich for T cells; these cells were routinely 80–90% T cells. For cultures, cells were placed in complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 1x nonessential amino acids, and 2 x 10-5 M 2-ME). Lymphocytes were isolated from peripheral blood using density separation medium (Cedarlane, Hornby, Canada). For adoptive transfer, 2 x 106 splenocytes were injected i.v. into the tail vein in 0.5 ml vol of PBS. For adoptive transfer of TCR Tg T cells from DES mice, single-cell suspensions of spleen were made and enriched for T cells with nylon wool, and CD8+ T cells were isolated by negative immunomagnetic selection using Dynabeads Mouse CD4 (L3T4) and Dynabeads Mouse Pan B (B220) (Dynal, Lake Success, NY). The enriched population of CD8+ T cells (1 x 107 cells/ml) was labeled with CFSE (Molecular Probes, Eugene, OR) by incubating the cells in an 8 µM solution of CFSE in RPMI 1640 at 37°C for 10 min, and labeling was quenched with cold PBS. A total of 2 x 106 CD8+ DES Tg T cells was adoptively transferred to recipient mice.

Proliferation assay

A total of 1 x 105 splenocytes was placed in 200 µl triplicate cultures in complete medium for 72 h at 37°C in a 5% CO2/95% air atmosphere. Abs were added at the initiation of cultures. Eighteen hours before the termination of the culture, the wells were pulsed with 0.5 µCi [3H]thymidine (NEN Life Science Products, Pittsburgh, PA) and then harvested on glass fiber filters. Incorporation was quantified with a scintillation counter (Wallac, Gaithersburg, MD). The results are expressed as the mean of triplicate determinations ± SEM.

Mixed leukocyte reaction (MLR)

A total of 2 x 105 responder splenocytes was cocultured in triplicate with 2 x 105 1500 rad gamma-irradiated stimulator cells for 5 days. Abs were added at the initiation of culture. Eighteen hours before the termination of the culture, the wells were pulsed with 0.5 µCi [3H]thymidine and incorporation was quantified with a scintillation counter. Results are expressed as mean of triplicate determinations ± SEM.

Fragmentation of Fab and F(ab')2 Abs

Purified anti-CD62L mAb was mixed with immobilized papain or immobilized pepsin (Pierce, Rockford, IL), and incubated for 5 h with agitation at 37°C. The Fab, F(ab')2, and Fc fragments and undigested IgG were recovered from immobilized gels using the separator tube supplied by the manufacturer, and further purified using a protein G column. Purity of the preparations was confirmed with SDS-PAGE analysis.

Flow cytometry

Cell washes and Ab dilutions were performed in PBS plus 1% BSA at 4°C. Flow cytometric analysis was performed on FACScan flow cytometer (BD Biosciences, San Jose, CA). Results are expressed as percentage of cells staining above background. mAbs were titered at regular intervals during the course of these studies to ensure that saturating concentrations were used.

Confocal microscopy

Frozen spleen and LN samples, embedded in OCT compound, were sectioned at 8 µm, fixed, and mounted with Gel/Mount (Biomeda, Foster City, CA). Photographs of fluorescent sections were made on a Zeiss (Oberkochen, Germany) laser-scanning confocal microscope 410 with Zeiss plan-neofluor objectives. Images were captured by optimizing the contrast and brightness settings on the confocal microscope for fluorescent signal, scanning the image with the four-line average function, and then pseudocoloring the images. The confocal images were imported into Adobe (Mountain View, CA) Photoshop 4.0, and then sized and labeled.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-CD62L mAb prevents tolerance induction and has no costimulatory effect

We hypothesized that lymphoid tissues are important for induction of peripheral, alloantigen-specific tolerance, and therefore LN homing and localization are required for tolerization. Corollaries of the hypothesis are that localization of T lymphocytes to other compartments, or LN depletion of T lymphocytes will prevent peripheral tolerance induction. To test this, we performed cardiac allografting with the anti-CD2 plus anti-CD3 mAb tolerization protocol. We previously showed that the combination of anti-CD2 plus anti-CD3 mAbs caused indefinite allograft survival and alloantigen-specific tolerance in the nonvascularized cardiac transplant model (27). To test the hypothesis, allograft recipients also received the MEL-14 anti-CD62L mAb, which is known to alter the distribution of T cells among secondary lymphoid organs by causing T cells to leave the LN (17, 18). The administration of anti-CD2 plus anti-CD3 mAbs resulted in prolonged allograft survival for all recipients and tolerance (Fig. 1GoA, and data not shown) (25). If anti-CD62L mAbs were added to the tolerogenic regimen of anti-CD2 plus anti-CD3 mAbs, mean survival time was only 37 ± 4.1 days, showing that prolonged graft survival and tolerance were prevented. Isotype control mAbs or anti-IL-2R mAbs did not inhibit tolerance (Fig. 1GoB). Animals receiving anti-CD62L mAb alone had graft survival of 20 ± 2.2 days, and those receiving anti-CD62L mAb in combination with either anti-CD2 mAb only, or anti-CD3 only, had graft survivals of 16.4 ± 0.9 days and 32 ± 4.4 days, respectively. This was similar to animals receiving anti-CD2 mAb alone or anti-CD3 mAb alone, which rejected allografts by 18.6 ± 2.3 days and 33.8 ± 1.7 days, respectively. These results indicate that anti-CD62L mAb does not have costimulatory or proinflammatory effects that cause earlier graft rejection. To determine the time at which anti-CD62L mAb administration is important for inhibiting tolerance, the mAb was administered on days 0 and 1, 4 and 5, 10 and 11, or 20 and 21 after transplantation to recipients that also received the tolerogen. The results (Fig. 1GoB) show that anti-CD62L mAb prevents tolerance when administered as late as 3 wk after allografting. This suggests that LN occupancy by T cells is required for more than a brief time after grafting and administration of tolerogen.



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  		FIGURE 1. Interfering with LN-homing molecules prevents indefinite allograft survival and tolerance. A and B, Cardiac allograft survival after administration of anti-CD2, anti-CD3, and/or anti-CD62L or control mAbs. Transplanted mice received mAbs i.v. at the times and doses indicated. A, Anti-CD62L mAb was given on days (d) 0 and 1 after transplantation. B, Anti-CD62L mAb was given on the days indicated. C, Anti-CD44 or anti-VLA-4{alpha} mAbs were given on days 0 and 1.

 
Since nonvascularized allografts may utilize secondary lymphoid organs in a fashion distinct from vascularized allografts (8, 28), these experiments were repeated using the vascularized cardiac allograft model. In this model, anti-CD2 plus anti-CD3 mAbs also induced prolonged graft survival, and alloantigen-specific tolerance was demonstrated by placement of a second cardiac allograft (Table IGo). On the other hand, addition of anti-CD62L mAb to the tolerogenic regimen prevented prolonged allograft survival and tolerance (median survival time, 26.7 ± 6.7 days). Thus, the primarily vascularized and nonvascularized cardiac allografts behave in a similar fashion.


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  		Table I. Anti-CD62L mAb prevents tolerance in vascularized transplants1

 
Additional controls were performed to rule out other costimulatory activities of the anti-CD62L mAb. Naive splenic lymphocytes were stimulated in culture with anti-CD3, anti-CD2, anti-CD62L, or control mAbs singly or in combination. Anti-CD3 mAbs (1 or 0.1 µg/ml) induced substantial proliferation that could be partly inhibited by anti-CD2 mAb (25, 27, 29) (Fig. 2GoA). When anti-CD62L mAb was given in combination either with anti-CD3 or with anti-CD3 plus anti-CD2, there was no additional effect on T cell proliferation. Assay of these culture supernatants for IFN-{gamma} showed no significant difference between anti-CD3 mAb stimulated and cultures stimulated with combinations of anti-CD3 plus anti-CD2 and/or anti-CD62L mAbs (Fig. 2GoB). Thus, anti-CD62L mAb neither inhibits nor drives polyclonal T cell activation. In a similar experiment (Fig. 2GoC), CBA/J splenocytes were cultured in MLR with histoincompatible C57BL/6 stimulators, and various combinations of anti-CD2 and/or anti-CD62L mAbs were added at the initiation of culture. As expected, anti-CD2 mAb had a small inhibitory effect on the MLR response (25, 27). Anti-CD62L mAb alone, or in combination with anti-CD2 mAb, had no additional effect on the MLR. Taken together, these results show that anti-CD62L does not costimulate T cell responses.



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  		FIGURE 2. Anti-CD62L mAb is not costimulatory. A, CBA/J splenic lymphocytes were stimulated with anti-CD3 mAb (0.1–1 µg/ml) along with anti-CD2 (10 µg/ml), anti-CD62L (10 µg/ml), or isotype control mAbs, and proliferation responses were measured after 3 days. B, Supernatants from A were assayed for IFN-{gamma} content by ELISA. C, Responder CBA/J splenic lymphocytes were incubated with C57BL/6 stimulators plus anti-CD2 (10 µg/ml) and anti-CD62L (1–10 µg/ml) mAbs and MLR measured after 5 days. D, Cardiac allograft survival with different fragments of anti-CD62L mAbs combined with the anti-CD2 plus anti-CD3 mAb tolerogenic regimen. Fab and F(ab')2 anti-CD62L mAbs were administered as equal Ag-binding equivalents compared with intact mAb. Doses and regimen as in Fig. 1GoA.

 
To rule out the possibility that the activity of anti-CD62L mAb is due to some other effect of the Ab, Fab and F(ab')2 MEL-14 were administered along with the tolerogenic regimen (Fig. 2GoD). The results demonstrate that both Fab and F(ab')2 MEL-14 prevented indefinite graft survival and tolerance. Thus, Fc{gamma} and Fc{gamma}Rs are not required for the inhibition of graft survival and tolerance. Likewise, CD62L cross-linking by mAb is also not a requisite for inhibition of tolerance.

Since the migration and localization of T cells to the LN is a multistep process requiring the interaction of many different receptor-ligand pairs, our hypothesis predicts that interfering with other elements involved in lymphocyte homing and trafficking may also prevent tolerization. To determine this, recipients received the tolerogenic regimen along with mAbs to the VLA-4{alpha} (CD49d) integrin, responsible for homing to vascular and lymphatic endothelium, through binding to its ligands fibronectin, VCAM-1, and mucosal addressin cell adhesion molecule-1 (30, 31), or mAbs to CD44, the mucin receptor, responsible for homing to endothelium (32). The results (Fig. 1GoC) demonstrate that two different anti-VLA-4{alpha} mAbs (PS/2, R1-2) and two different anti-CD44 mAbs (IM7, KM81) are able to prevent tolerization when administered with the tolerogenic regimen.

Anti-CD62L alters the distribution of T cells

Since the anti-CD62L mAb inhibited tolerance induction by anti-CD2 plus anti-CD3 mAbs, and since previous studies (17, 18, 19) demonstrated that this mAb caused shedding of CD62L with the displacement of T cells out of LNs to the spleen and peripheral blood, we next determined the distribution of CD4+ and CD8+ T cells in the treated allograft recipients to ascertain whether similar lymphocyte shifts occurred here. Recipients received nonvascularized cardiac allografts and were either left untreated, received the tolerogenic anti-CD2/anti-CD3 mAb tolerogenic regimen, or received the nontolerogenic anti-CD2/anti-CD3/anti-CD62L mAb combination. Groups of five recipients were sacrificed 0, 5, 10, 30, or 40 days after transplantation, and spleen, peripheral blood (200 µl/mouse), allograft-draining cervical LNs (1 LN/mouse), and nondraining axillary, iliac, and mesenteric LNs (six LN/mouse) were harvested, made into pooled single-cell suspensions, and analyzed by counting total viable mononuclear cells, and quantitating CD4 and CD8 populations by fluorescent flow cytometry.

The results of these experiments (Fig. 3Go) show that the total mononuclear cell number in the spleen and peripheral blood is far less in the tolerant group than in the nontolerized group receiving anti-CD62L mAb, while there are similar numbers of cells in the LNs of these two groups. This means that the tolerant group has a higher percentage of total lymphocytes in the LNs compared with the nontolerant group. It should also be noted that the untreated group, which is undergoing unmodified rejection of the allograft, has an early and sustained increase in LN cell numbers, particularly the draining LNs, as expected. Additional controls (not shown) demonstrated that mAb treatments alone without transplantation resulted in similar changes, but alloantigen stimulation accentuated the differences.



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  		FIGURE 3. Anti-CD62L mAb changes the distribution of lymphocytes. Total mononuclear cells; CD4/CD8 ratios; total CD4+ T cells; and total CD8+ T cells. Five mice per group were sacrificed on days 0, 5, 10, 30, and 40 after nonvascularized cardiac transplantation, and splenocytes, peripheral blood (200 µl/mouse), draining cervical LNs (one LN/mouse), and nondraining LNs (six LNs/mouse) were harvested and analyzed by cell counts and flow cytometry. Separate treatment groups are noted in the figure; doses and regimen are as in Fig. 1GoA.

 
The CD4:CD8 ratios of the tolerant and nontolerized groups are different in the spleen and peripheral blood vs draining and nondraining LNs (Fig. 3Go). The tolerant group has higher ratios in the LNs, while the nontolerized group has higher ratios in the central compartments. These results have been repeated and confirmed in three separate experiments. Thus, the nontolerized group has a greater number and percentage of lymphocytes and a higher CD4:CD8 ratio in the spleen and peripheral blood in comparison with the tolerant group, which has greater cell numbers, a higher percentage of total lymphocytes, and a higher CD4:CD8 ratio in the LNs. Additional experiments showed that anti-CD2 and/or anti-CD3 mAbs did not alter the expression of CD62L on CD4+ or CD8+ lymphocytes in the LN or spleen, while anti-CD62L caused complete shedding (not shown). Taken together, the data suggest that CD4+CD62L+ T cells must remain in the LN during tolerance induction.

Our hypothesis predicts that the occupancy of LNs or other lymphoid organs by specific T cells is essential during tolerogenesis. The flow cytometric results (Fig. 3Go) support this notion, but characterize entire CD4+ and CD8+ lymphoid populations. The analysis would be facilitated if Ag-specific T lymphocytes could be separately visualized and enumerated (33). To accomplish this, we used lymphocytes from DES mice, which possess a TCR Tg specific for H-2Kb on the CBA background (24, 33) and are compatible with the C57BL/6 into CBA-nonvascularized transplant model. Initial studies demonstrated that TCR Tg CD8+ splenic T cells could be adoptively transferred into wild-type CBA recipients, at the time of allografting and treatment with anti-CD2 plus anti-CD3 mAbs, and that prolonged graft survival and tolerance could still be induced, while untreated recipients receiving adoptive transfer still rejected their grafts at a normal time (data not shown). Next, allograft recipients received the tolerogenic or nontolerogenic mAb regimens along with 2 x 106 purified, CD8+, CFSE-labeled, splenic DES TCR Tg T cells. The recipients were sacrificed 5 days later, and 8-µm frozen sections of the grafts, spleens, and draining LN were made. The results demonstrate that the anti-CD2 plus anti-CD3 mAb tolerogenic regimen partially decreases CFSE+ cells in the LN and spleen in comparison with rejecting controls not treated with mAbs (Fig. 4Go, F and J vs G and K). In contrast, the anti-CD2/anti-CD3/anti-CD62L mAb treatment almost completely depleted CFSE+ cells from the LN (Fig. 4GoH), while significantly increasing their density in the spleen (Fig. 4GoL). These cell distributions and numbers are commensurate with the flow cytometric data (Fig. 3Go). Examination of the allografts demonstrated no CFSE+ lymphocytic infiltration in the tolerogen-treated group, while treatment with the anti-CD2/anti-CD3/anti-CD62L mAbs had an easily discernible infiltrate. Therefore, the alterations in cell surface expression of CD62L by the mAb, and subsequent changes in trafficking permitted these CD8+ cells to migrate to the graft early after transplantation, despite the continued presence of anti-CD2 and anti-CD3 mAbs in the serum. This result also demonstrates that the tolerogenic protocol does not deplete Ag-specific T cells.



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  		FIGURE 4. Distribution of Ag-specific CD8+ T cell changes in response to tolerogenic or nontolerogenic treatments. CFSE-labeled, CD8+, DES TCR Tg splenic T cells were adoptively transferred (2 x 106) on the day of transplantation. Grafts (A–D), spleens (E–H), and draining LNs (I–L) were harvested 4 days after transplantation and treatment with various mAb regimens, as indicated. mAb dosing and regimen as in Fig. 1GoA. Txp, transplant.

 
The flow cytometric and histologic data (Figs. 3Go and 4Go) demonstrate significant shifts in lymphocyte populations. To determine how this affects secondary lymphoid organ architecture, spleens and draining LNs were harvested from allograft recipients 5 days after the various mAb treatments and sections stained by H&E. The results (Fig. 5Go) show that the untreated rejecting controls have increases in the splenic white pulp and LN paracortex in comparison with untransplanted controls. The group treated with the anti-CD2 plus anti-CD3 mAb tolerogenic regimen has a decrease in splenic white pulp in comparison with the rejecting control, and a significant decrease in LN lymphocytes along with nuclear fragmentation within the follicles. The group treated with the nontolerogenic anti-CD2/anti-CD3/anti-CD62L mAb combination has a very large increase in the splenic white pulp with incursion of leukocytes into the red pulp. The LNs in this group have massive lymphocyte depletion with apoptosis. These results are commensurate with the data in Figs. 3Go and 4Go, demonstrating a shift in lymphoid populations dependent on treatment with tolerogenic or nontolerogenic regimens.



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  		FIGURE 5. Architecture of spleen and LN changes in response to tolerogenic and nontolerogenic treatments. Mice treated as in Fig. 1Goa; LNs (A–D) and spleens (E–H) harvested after 5 days; and tissue stained for H&E. Txp, transplant.

 
CD62L deficiency prevents tolerance

The studies show that anti-CD62L mAb alters T cell trafficking and prevents tolerization. A number of controls showed that anti-CD62L mAb is not costimulatory for T cells. As an alternative approach to rule out other effects of the mAbs, we used L-selectin knockout mice. The hypothesis predicts that because CD62L-deficient T cells do not migrate to LNs (20), these recipients should not be tolerized. To test this, C57BL/6 CD62L-/- mice were directly used as recipients of C3H-nonvascularized cardiac allografts. These animals have small LNs with a general failure of T cells to localize to the LN (20, 21, 22, 23). These recipients were grafted, and then treated or untreated with the anti-CD2 plus anti-CD3 mAb tolerogenic regimen. All CD62L-/- recipients rejected their grafts within 18 days, even if they received anti-CD2 plus anti-CD3 mAbs (Table IIGo, groups V and VI). It is striking that the combination of the potent immunosuppressants anti-CD2 and anti-CD3 mAbs was unable to prolong graft survival beyond that of controls.


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  		Table II. CD62L deficiency prevents tolerance1

 
In a similar experiment, C3H donor-nonvascularized cardiac allografts were transplanted to four groups of C57BL/6 recipients. Two groups were left untreated, and two received the tolerogenic mAb regimen. In addition, the recipients were adoptively transferred with 2 x 106 nylon wool T cell-enriched splenic lymphocytes from wild-type or CD62L-/- C57BL/6 donors. All mAb-untreated controls rejected their allografts within 18 days, regardless of which cell type was transferred (Table IIGo, groups I and III). The tolerogen-treated group, receiving wild-type cells, had all grafts survive beyond 50 days (Table IIGo, group IV). However, the tolerogen-treated group, adoptively transferred with CD62L-/- cells, rejected all grafts (Table IIGo, group II). These results demonstrate that a genetic approach results in findings similar to the mAb approach, and obviates concerns that the MEL-14 anti-CD62L mAb may have some other, unknown mechanisms of action. These results are equally important because the adoptive transfer technique demonstrates that it is most likely the trafficking of CD62L-/- or CD62L+ T cells, but not non-T cells, that determines graft survival, confirming more definitively the hypothesis. This result also shows that the adoptive transfer of CD62L-/- T cells prevents tolerance by the wild-type recipient T cells already resident in the LNs. Thus, T cell LN occupancy is required, but not wholly sufficient for tolerance. This finding implies that the CD62L-/- T cells become primed to alloantigen, yet are not subject to tolerogenic influences by the LN, because of their inability to home there.

FTY720 can reverse the effect of anti-CD62L mAb

FTY720 is a novel sphingosine-derived immunosuppressant that prolongs allograft survival in a number of models (34, 35). Notably, FTY720 causes lymphopenia and promotes LN homing of T lymphocytes (36), and its molecular mechanism most likely relates to chemokine receptor signal transduction (37). This suggests that FTY720 may act through a mechanism that is independent of the expression of CD62L, and may be able to reverse the inhibitory effect of anti-CD62L mAb on T cell trafficking and graft survival. To test this, FTY720 was administered along with anti-CD62L mAb to untransplanted, normal animals. Spleens, LNs, and peripheral blood were harvested after 5 days, and the distribution of CD4+ and CD8+ T cells was determined. FTY720 prevented both the depletion of T cells from the LN and the increase of T cells in the spleen and peripheral blood by anti-CD62L mAb (Fig. 6Go, B–D). Using mAb-untreated CD62L-/- mice, FTY720 administration likewise resulted in T cell homing to the LN from the spleen and peripheral blood (Fig. 6Go, B–D). Thus, FTY720 acts independently of CD62L. Next, allograft recipients received anti-CD2/anti-CD3, anti-CD2/anti-CD3/FTY720, anti-CD2/anti-CD3/anti-CD62L, or anti-CD2/anti-CD3/anti-CD62L/FTY720. The results in Fig. 6GoA demonstrate that a 50-day course of FTY720 is able to reverse the anti-CD62L mAb effect and restore prolonged allograft survival to the treated group. Important controls show that anti-CD2/anti-CD3/FTY720 is no different from the tolerogenic mAb regimen alone, and FTY720 alone at this dose has no effect on graft survival, demonstrating that is not tolerogenic on its own in the transplant model. In additional experiments (not shown), a short course of FTY720, administered for 14 days starting at the time of transplantation, is unable to restore graft survival in anti-CD62L mAb-treated recipients. This result is consistent with the requirement for sustained LN occupancy and the prolonged effect of anti-CD62L mAb in vivo (Fig. 1GoB).



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  		FIGURE 6. FTY720 reverses the effect of anti-CD62L on T cell distribution and tolerance inhibition. A, Cardiac allograft survival with different combinations of FTY720 (0.1 mg/kg/mouse/day), anti-CD2, anti-CD3, and anti-CD62L mAbs. mAb dosing and regimen as in Fig. 1GoA. B–D, Total CD4+ and CD8+ T cell populations from spleen (B); peripheral blood (C); and LNs (D) of wild-type or L-selectin-deficient mice 5 days after treatment with FTY720 (0.1 mg/kg/mouse/day) and/or anti-CD62L mAb (100 µg i.v. x 2 doses). Tissues were pooled from five animals per group; the counts represent the cells from one spleen, 200 µl peripheral blood, and three LNs (mesenteric, axillary, and iliac nodes) per mouse. KO, Knockout; wt, wild type.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results show that tolerance induced by anti-CD2 plus anti-CD3 mAbs, a regimen that affects signal 1 and signal 2 of T cell activation, for vascularized and nonvascularized allografts can be prevented by the MEL-14 anti-CD62L mAb. Anti-CD62L mAb is not proinflammatory or costimulatory, is not functioning by an Fc{gamma}- or Fc{gamma}R-mediated mechanism, and is functioning through a mechanism available over at least a 3-wk period. The adoptive transfer of CD62L-/- T cells and the use of CD62L-/- recipients provide complementary genetic evidence and confirm the importance of CD62L and LN occupancy by T cells in tolerization. The results in this study and the work of others (17, 18, 19) suggest that anti-CD62L causes rapid shedding of cell surface CD62L and subsequent redistribution of T cells from the LN to other compartments, particularly the peripheral blood and the spleen. The results demonstrate that tolerant recipients have a greater number and percentage of their lymphocytes, and a higher CD4:CD8 ratio, in LNs compared with nontolerant recipients, which have higher numbers, percentages, and ratios in the spleen and peripheral blood. These results support the notion that the differential distribution of T cells between LNs and other compartments is an essential feature for the successful induction of peripheral tolerance to alloantigen, using conventional systemic immunosuppressive modalities. The results suggest that T cells must be in the LNs or other lymphoid tissues at the time of tolerization, and remain in these lymphoid tissues for a period of time.

L-selectin is generally considered to be a receptor important for proinflammatory events and proper trafficking of leukocytes during positive immune responses (38). Nonetheless, previous studies show that anti-CD62L mAbs or CD62L deficiency are not generally immunosuppressive, but result in altered leukocyte distribution and kinetics of immune responses (20, 21, 22, 23). As shown in this study and previously (19, 39), anti-CD62L mAbs do not prolong allograft survival, and CD62L-/- recipients are fully capable of vigorously rejecting allografts. Instead, interfering with CD62L function inhibits tolerization, and this is not due to costimulatory influences (Fig. 2Go). It is unlikely that anti-CD62L acts only by increasing the peripheral pool of lymphocytes to cause rejection, since that would be expected to cause rejection soon after mAb administration, which did not occur (Fig. 1Go). Furthermore, adoptive transfer of only a small number of CD62L-/- T cells causes rejection in wild-type recipients (Tables IGo and IIGo), and this should not result in a large increase in the peripheral pool of Ag-specific T cells. We favor the interpretation that in the setting of conventional systemic immunosuppression, the LN or other lymphoid organs are tolerogenic environments for T cells, and inhibiting CD62L function prevents T cells from reaching lymphoid organs. It is noteworthy that interfering with CD62L with mAb can convert a chronic, parasitic infection with an ineffective Th2 response and nodal hypertrophy into a Th1 response that clears both infection and LN hyperplasia (40). CD4+CD25+CD62L+ regulatory T cells have been shown to play a role in preventing graft-vs-host disease (41), and CD4+CD62L+ T cells can prevent autoimmune diabetic in nonobese diabetic mice (42). Thus, CD62L+ T cells and lymphoid migration may play diverse roles in negative immune regulation.

The studies of Lakkis and colleagues (8) elegantly demonstrate that secondary lymphoid organs are critical for priming T cells to alloantigen and subsequent allograft rejection. These findings complement older reports that graft-derived dendritic cells (DC) traffic to both draining and systemic lymphoid organs to present alloantigen (43, 44). Our studies suggest that secondary lymphoid organs are equally important for the induction of tolerance. The results imply that alloantigen is presented in the LNs to CD62L+ T cells, and that under the influence of systemic immunosuppression the process of tolerance can ensue. Unlike priming, which can clearly take place in the spleen, tolerance in our model did not seem to occur in that location. Indeed, in preliminary experiments, Hox11-/- mice lacking a spleen had prolonged allograft survival, and anti-CD62L prevented graft survival (Y. Bai and J. S. Bromberg, unpublished results). Why the LN may be more tolerogenic than the spleen is not certain, but it is interesting to note that CCR7 engagement in the LN may specifically be immunosuppressive and prevent formation of the immunological synapse (10). Thus, there may by distinct secondary lymphoid organs with tolerogenic or nontolerogenic potentials. These conclusions may also depend on the type of immunosuppression administered. In particular, other tolerogenic regimens could rely on alternative secondary lymphoid organs or homing receptors, and experiments in those systems could reach conclusions different from ours.

The results suggest a complex interplay among CD62L+ T cells that remain in the LN or recirculate, and CD62L- T cells that generally cannot enter the LN. It is not clear whether the CD62L+ T cells continue to recirculate, or if a subpopulation must remain in the LN to support the tolerant state. It is also not clear how recirculating CD62L+ T cells interact with CD62L- (memory or activated) T cells in the spleen or elsewhere in the periphery. Since both anti-VLA-4{alpha} and anti-CD44 mAbs prevented tolerance, and since these cell surface receptors are generally associated with memory and/or activated T cells (30, 31, 32, 45), the data suggest that CD62L+ T cells may interact with these other T cell subsets in secondary lymphoid tissues to induce tolerance in those subsets.

The results (Figs. 4Go and 5Go) suggest not only that the location of T cells in a particular secondary lymphoid organ is important, but also that the structure of the organ and the precise microanatomic domains in which T cells localize are critical determinants of priming vs tolerization. The results imply that changes in lymphoid organ structure are an important aspect of the mechanism and efficacy of immunosuppressive regimens, and that the microanatomic interaction of T cells with APC will be altered depending on the regimen administered to an allograft recipient. In support of this concept, preliminary work with confocal microscopy shows diminished interactions between TCR Tg T cells and DC in the LNs of tolerized recipients (Y. Bai and J. S. Bromberg, unpublished results). Conversely, there is a marked increase in splenic TCR Tg T-DC interactions in recipients treated with the nontolerogenic anti-CD62L mAb regimen. These domain interactions suggest other potential targets for probing mechanisms of tolerization. In particular, the CCL19/CCL21-CCR7 and CXCL13-CXCR5 receptor ligand interactions may be relevant.

These studies indicate that the definition of domains in which alloantigen is presented and in which lymphocytes interact with Ag are critical determinants of the tolerization process. Other approaches to alloantigen-specific tolerance, such as costimulatory blockade, should be analyzed with reference to their effects on secondary lymphoid organ structure and the microanatomic arrangements of cellular interactions. For example, anti-CD40L mAb causes significant and prolonged changes in splenic germinal center architecture (46). More durable approaches to tolerance, such as microchimerism, may also require analysis of lymphoid organ structure (44). Our hypothesis suggests that chimeric interactions in the LN may be germane for tolerance, or that donor CD62L-/- hemopoietic cells may be unable to induce tolerance. Nonspecific inflammation and infectious insults are known to impede tolerance and to interfere with host adaptation to the graft in clinical transplantation (47). Since these events supply activational signals that cause shedding of CD62L (17), it may be appropriate to examine secondary lymphoid organ structure in these settings. Finally, there may be implications for other types of immunity. In HIV infection, it is well known that there is a persistent reservoir of virus in the LNs accompanied by ineffective immune responses (48). In concert with the immunomodulatory effects of viral genes (48), the LN microenvironment may further impede the development of a productive and protective immune response.


   Acknowledgments
 
We thank Dr. Gwen Randolph for help with the confocal microscopy and analysis, and Drs. Altevogt, Bluestone, Brinkman, Kincaide, and Mellor for reagents. We acknowledge the technical contributions of D. Chen, M. Mao, and Y. Zhang.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant R01 AI41428 (to J.S.B.). Back

2 Current address: Department of Surgery, Yale University, New Haven, CT. Back

3 Address correspondence and reprint requests to Dr. Jonathan S. Bromberg, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1104, New York, NY 10029-6574. E-mail address: jon.bromberg{at}mountsinai.org Back

4 Abbreviations used in this paper: LN, lymph node; DC, dendritic cell; LT, lymphotoxin; MLR, mixed leukocyte reaction; Tg, transgenic. Back

Received for publication October 10, 2001. Accepted for publication December 5, 2001.


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