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The Journal of Immunology, 2006, 176: 3978-3986.
Copyright © 2006 by The American Association of Immunologists

IL-7 Is a Critical Factor in Modulating Lesion Development in Skn-Directed Autoimmunity1

Pamela J. Staton*, A. Betts Carpenter{dagger} and Susan H. Jackman2,*

* Departments of Microbiology, Immunology, and Molecular Genetics and {dagger} Department of Pathology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25704


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In a murine model of autoimmunity targeted against the epidermal cell Ags, Skn, adoptive transfer of Skn-immune T cells to immunosuppressed recipients elicits skin lesions in areas of mild epidermal trauma. In this study, we examined peripheral regulation of Skn-induced autoreactivity disrupted by rendering the mice immunoincompetent. We found that regulation of Skn-directed autoimmunity was restored by cotransfer of normal syngeneic spleen cells at twice the concentration of Skn-immune cells and was evidenced by significantly reduced lesion severity by days 5–7 post-cotransfer compared with animals given injections of Skn-immune cells alone. Enrichment and depletion of normal CD4+ or CD8+ spleen cells and RT-PCR analysis of selected cytokines identified CD4+ cells as the regulatory cells in the cotransfer inoculum; however, significant reduction in lesion severity was observed only when there was a concomitant increase in levels of IL-7. The role of IL-7 was further supported in that mice cotransferred with Skn-immune cells plus normal spleen cells, but also treated with anti-IL-7 Ab, no longer exhibited reduced lesion severity. To determine whether IL-7 expression without normal spleen cell cotransfer could modulate lesion development, an IL-7-encoding plasmid (pCMV-Tag1-IL-7) was topically delivered to sites flanking the stressed skin site in Skn-induced autoimmune mice. Daily application of 15 µg of pCMV-Tag1-IL-7 significantly suppressed lesion severity. Our results support a mechanism for CD4+ T cells and IL-7 in contributing to the control of autoreactivity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Manifestation of autoimmune disease often results from various defects in immune homeostasis. Animal models have shown that T cell-mediated autoimmunity can be induced by disproportionate production of self-reactive T cells in the thymus, by inappropriate activation of these cells to self Ags, and/or by defective control of their proliferation in peripheral sites (1).

We have described previously an autoimmune response directed to murine skin that targets the epidermal cell Ags, Skn (2). Skn Ags are expressed by epidermal cells (3, 4, 5) and neuronal cells (3, 6). They are encoded by two unlinked genes, Skn 1 and Skn 2 (7), each with alternative alleles recognized as alloantigens in various mouse strains (8).

The Skn model of autoimmunity is based on the adoptive transfer of anti-Skn-specific lymphocytes (Skn-immune cells). However, to demonstrate clinical disease, two additional conditions must be met in the recipient. The target organ, the skin, must experience mild trauma, and the recipient’s immunocompetency must be impaired. These conditions can be produced by shaving and by a single injection of cyclophosphamide, respectively, before the introduction of Skn-immune lymphocytes. When all three components are provided, skin lesions invariably form at the shaved skin site within 5–10 days postadoptive transfer of Skn-immune cells (2).

In this study, we investigated the immunosuppression component for initiation of the Skn-autoimmune response. As has been often demonstrated, interference with immune competency results in impaired regulation of autoreactive lymphocytes introduced into the periphery. There is substantial evidence that CD4+ T cells have critical regulatory function in controlling autoimmunity and maintaining tolerance to self Ags (9, 10). Several subsets of regulatory cells have been described. Some subsets have been defined by selective secretion of certain cytokines, such that those producing high levels of TGF-beta or IL-10 are referred to as Th3 (11, 12) or T regulatory 1 (13) cells, respectively. Alternatively, regulatory cells also have been identified by expression of cell surface markers. The naturally occurring CD4+ T regulatory cell coexpresses CD25 (14) and Foxp3 (15) and functions through cell-cell contact rather than from cytokine-mediated suppression (16, 17).

In this study, we show that a population of CD4+ normal spleen cells, when cotransferred with autoreactive Skn-immune cells, can suppress Skn-directed autoreactivity. However, this suppression is apparent only with concurrent elevation of IL-7 gene expression in the skin. Furthermore, topical gene therapy with IL-7-expressing plasmid DNA can replace the requirement for cotransfer of CD4+ normal spleen cells for reducing the clinical manifestation of skin lesions in recipients of Skn-immune autoreactive T cells. These data suggest that IL-7 can have a contributing role in the control of autoreactivity.


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

Female C57BL/6J (B6), A/J (A), and B6AF1/J (B6AF1) mice were purchased from The Jackson Laboratory. Mice were housed in a conventional animal facility. Sentinel mice were used to screen for various pathogens and were consistently negative. The Institutional Animal Care and Use Committee of Marshall University approved all experiments.

Anti-Skn lymphocytes

Because Skn congenic mice are not available, hemopoietic radiation chimeras were produced and immunized, as previously described (2), to induce a specific response to Skn alleles found on A/J strain mouse skin (Skn 1.1, Skn 2.1) and to avoid a response to the alternative Skn alleles (Skn 1.2, Skn 2.2), to other minor histocompatability Ags found on leukocytes, and to major histocompatability complex Ags. Spleen cells from these chimeras were used as the source of donor anti-Skn lymphocytes (Skn-immune cells) for adoptive transfer into B6AF1 recipients.

Pretreatment of recipients before adoptive cell transfer

As previously described (2), B6AF1 recipients (4–8 mo of age) were pretreated with mild epidermal trauma by shaving with a razor blade at a site on the dorsal aspect and with immunosuppression by an i.v. injection of cyclophosphamide (NEOSAR; Adria Laboratories) at a dose of 200 mg/kg body weight. Epidermal trauma was performed 1 day (day –1), and immunosuppression was delivered 4 h before adoptive cell transfer (day 0).

Adoptive cell transfer and evaluation of lesions

As previously described (2), spleen cells from immunized chimeras (Skn-immune cells) were dispersed into a single cell suspension, washed, and adjusted to the appropriate cell number in PBS. B6AF1 recipients received the designated number of Skn-immune cells delivered i.v. in 0.2 ml of PBS (day 0). Some recipients received concomitant transfer of unfractionated or fractionated normal spleen cells from syngeneic B6AF1 donors, as designated in Results.

Clinical scoring. All recipients were examined daily for gross cutaneous lesions, and a clinical score was given on a scale of 0–5, as follows: 0 = normal; 1 = scaling or flaking; 2 = erythema without pitting or crusts; 3 = erythema with pitting and/or crusts over <50% of shaved area; 4 = similar to grade 3, area affected >50%, but less than total shaved area; 5 = similar to grade 3, area affected covering total shaved area. Two individuals, one of whom was unaware of specific treatment, examined mice for lesions.

Histological examination. At various times after cell transfer, mice were killed, and biopsies of the shaved skin site were fixed in 10% neutral-buffered formalin. Paraffin-embedded sections were stained with H&E. Histopathology was assessed based on the gross lesion scale of 0–5, with the following findings: 0 = normal; 1 = mild acanthosis (2x normal) and hyperkeratosis; 2 = pronounced acanthosis (>2x normal), hyperkeratosis, parakeratosis, and dermal thickening (≥2x normal); 3 = grade 2 changes plus focal pustular crusts and a dermal cellular infiltrate; 4 = grade 3 changes with focal ulceration and cellular infiltrate extending into the s.c. fat; 5 = changes as described for grade 4 with extensive ulceration and cellular infiltration. Histopathology was analyzed by two individuals in a blinded fashion. Lesion grades reported in Results were based on combined gross and histological evaluation.

RNA extraction and RT-PCR

Tissues were homogenized with a Tekmar SDT Tissumizer (Tekman) or with a Mini BeadBeater using 2.5-mm zirconia beads (BioSpec Products), and total cellular RNA was extracted with TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. RNA was stored at –70°C. cDNA was synthesized from 1 µg of total RNA in a 30 µl reaction using Moloney murine leukemia virus reverse transcriptase and oligo d(T)16. Five-microliter aliquots of the cDNA reaction products were amplified by PCR at 95°C for 45 s, 60°C for 45 s, and 72°C for 1 min for 35 cycles, with a final extension at 72°C for 7 min. PCR products were analyzed by electrophoresis on 3% NuSieve/1% SeaKem (FMC Bioproducts) agarose gels and visualized by ethidium bromide staining. ODs of the bands were obtained using the EDAS 120 System (Eastman Kodak), and the relative level for each cytokine was calculated as OD260 for the cytokine band ÷ OD260 for a beta-actin band. Primers were originally purchased from BD Clontech and/or synthesized at the Marshall University DNA Core Facility.

T cell enrichment and depletion

The StemSep procedure (StemCell Technologies) was used as described by the manufacturer. Briefly, a single cell suspension of normal B6AF1 spleens was prepared and incubated in the appropriate Ab mixture. After washing, the cell suspension was treated with anti-biotin tetrameric Ab complexes (StemCell Technologies), followed by the addition of magnetic colloid (StemCell Technologies). The mixture was then positioned in a rare earth magnet and cells were allowed to flow through the column. The flow-through fraction plus three washes with PBS were collected. In some experiments, the EasySep procedure (StemCell Technologies) was used, as described by the manufacturer, in place of the StemSep procedure. To evaluate the success of the enrichment or depletion procedures, the flow-through cell populations were stained with PE-labeled anti-CD4 or FITC-labeled anti-CD8 Ab (BD Pharmingen) and analyzed by flow cytometry using a FACScan flow cytometer and CellQuest software (FACScan; BD Biosciences). The Ab mixtures were purchased from StemCell Technologies. The CD4+ T cell enrichment mixture contained biotinylated Abs to CD11b, CD45R, CD8, myeloid differentiation Ag (GR-1), and erythroid cells (TER119); some mixtures also contained biotinylated Abs to CD49b and CD19, but not GR-1. The CD8+ T cell enrichment mixture consisted of biotinylated Abs to CD4, CD45R, CD11b, GR-1, TER119, and NK cells; some mixtures also contained biotinylated Ab to CD49b. The CD4+, CD8+, or CD3+ T cell depletion mixtures consisted of biotinylated Ab to CD4, CD8, or CD3, respectively. The number of fractionated spleen cells cotransferred after enrichment or depletion was adjusted to reflect the approximate number of CD4+ or CD8+ cells found in the normal murine spleen, i.e., 24 and 12%, respectively. This resulted in the cotransfer of 76 x 106 CD4-depleted, 24 x 106 CD4-enriched, 88 x 106 CD8-depleted, or 12 x 106 CD8-enriched normal spleen cells.

In vivo anti-IL-7 treatment

The hybridoma cell line producing hu/mu (human/murine) IL-7 M25 Ab was obtained from Amgen (formerly Immunex). Culture supernatants were concentrated with Centricon-Plus-80 units (Fisher Scientific), purified with protein A affinity columns (Sigma-Aldrich), and dialyzed against sterile PBS.

Mice were prepared, as described above, and received injections i.v. with either Skn-immune cells alone or a cotransfer of Skn-immune plus normal spleen cells. Some mice receiving the cotransfer of Skn-immune plus normal cells were also given injections i.p. of 1 mg of M25 mAb on days 1, 3, 5, 7, 9, and 10 postcell transfer (one experiment) or with 3 mg of M25 mAb on days 3, 5, and 7 postcell transfer (two experiments). Both dose regimens provided similar results, and the data have been combined. Control mice received the cotransfer of Skn-immune plus normal cells and were given injections i.p. of an equivalent volume of the diluent, PBS.

Expression vector construction and plasmid DNA preparation

A murine IL-7 cDNA in a BlueScript KS vector was a gift from B. Rich (Brigham and Women’s Hospital, Division of Dermatology, Harvard Institutes of Medicine, Boston, MA). It was subsequently inserted into the pCMV-Tag1 mammalian expression vector that contained an N terminus FLAG coding sequence (Stratagene). This preparation was sequenced by the Marshall University DNA Core Facility, which verified the presence and correct orientation of the IL-7 cDNA. Control plasmid consisted of the pCMV-Tag1 vector without the IL-7 insertion. Adherent C2 mouse muscle cells (courtesy of T. Green, Marshall University, Huntington, WV) were transfected with Lipofectin reagent (Invitrogen Life Technologies), following the manufacturer’s directions. Supernatant from the transfected C2 cells was used in a bioactivity assay with IL-7-dependent 2E8 cells (TIB-239 FL; American Type Culture Collection) to verify the biologic activity of the pCMV-Tag1-IL-7 construct.

Plasmid preparations were produced using the Endofree Plasmid Maxi Kit (Qiagen), following the manufacturer’s instructions. All plasmid preparations for topical application were resuspended in TE buffer.

Topical application of plasmid DNA

Animals were anesthetized by i.p. injection with ketamine and/or brief inhalation of ether. On day –1, all superficial hair was shaved with a razor blade at a site on the dorsal aspect, as described above. In addition, dorsal hair was clipped using Oster animal shears at sites adjacent to the shaved area. The following day, the mice were given injections i.v. of cyclophosphamide and 4 h later with 50 x 106 Skn-immune cells. The clipped site, designated as the plasmid application site, was then gently brushed with 100 strokes using a nylon, round-bristled child’s toothbrush. A 20 µl sample containing pCMV-Tag1-IL-7 in TE buffer (10 mM Tris, 1 mM EDTA) was placed in the center of the clipped site and distributed over the entire clipped area using the side of the pipette tip. Control applications consisted of either pCMV-Tag1-null vector or vehicle alone; results were comparable, and these control applications were used interchangeably. Animals remained anesthetized for ~10 min following plasmid application or until no liquid was observed on the skin surface.

mRNA produced from the pCMV-Tag1-IL-7 vector was detected by RT-PCR, as described above, using the following primers: 5'-TCGGGCAATTACTATCAGTTC-3' and 3'-GATTACAAGGATGACGACGAT-5'. To nullify each tissue for the presence of plasmid, cDNA reactions without reverse transcriptase were also prepared. Following PCR, the OD260 for this reaction band, if any, was subtracted from the cytokine band OD260 measurement before use in the formula described above.

Statistical analysis

Statistical analysis was conducted using the Mann-Whitney rank sum test for evaluating lesions grades, and the t test was used to evaluate cytokine:beta-actin ratios. Significance was defined as p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Cotransfer of CD4+ normal spleen cells is associated with diminished severity of skin lesions

Reports from other models of autoimmunity show that transfer of sufficient numbers of peripheral T cells from normal donors can inhibit the development of disease or control disease severity in recipients who would otherwise exhibit immune pathology (9). To evaluate whether normal spleen cells contain such a population for limiting Skn-directed autoreactivity, B6AF1 mice were shaved and cyclophosphamide treated, as previously described (2). Four hours postcyclophosphamide treatment, animals received injections of 50 x 106 Skn-immune cells alone or with concomitant cotransfer of 100 x 106 normal spleen cells, defined in this work as cells obtained from B6AF1 mice that were never immunized against Skn Ags. Lesions were observed daily over a 7-day period. Animals receiving cotransfer of normal spleen cells demonstrated progressive reduction in lesion severity on days 5, 6, and 7 postcell transfer when compared with those given injections of Skn-immune cells alone (Fig. 1a). This was shown with both histological (Fig. 1, c–e) and gross (Fig. 1, f and g) evaluation of lesion sites. Although the transfer of normal spleen cells at twice the number of Skn-immune cells reduced the severity of lesions, transfer of normal spleen cells at numbers equivalent to the Skn-immune dose (50 x 106) was not able to diminish lesion development (data not shown).


Figure 1
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  		FIGURE 1. Time course evaluation of lesion moderation by cotransfer of normal spleen cells. a, Mice were shaved, cyclophosphamide pretreated, and given injections of 50 x 106 Skn-immune cells alone (•) or with the cotransfer of 100 x 106 normal spleen cells ({circ}). Points represent mean lesion grade ± SE for 14 or 17 animals from five or four different experiments for • and {circ}, respectively. *, p = 0.025; **, p ≤ 0.001. b–e, H&E staining of normal mouse skin (b) and from lesional skin representative of grade 1 (c), grade 3 (d), and grade 4 (e), demonstrating H, hyperkeratosis (thickening of the stratum corneum); P, parakeratosis (persistence of nuclei in the stratum corneum); A, acanthosis (epidermal thickening); C, pustular crusts; U, ulceration, thickened dermis, and/or dermal infiltrates. Scale bar, 100 µm. f and g, Representative examples of gross lesions of grade 1 (f) and grade 3 (g).

 
There is convincing evidence that CD4+ T cells are the prominent regulatory cells for protection against immune pathology (18), although this does not preclude other cell types from participating in the regulatory process. To determine the phenotype of the cell(s) suppressing Skn-directed autoreactivity, normal spleen cells were either depleted or enriched for CD4+ or CD8+ populations before cotransfer. On day 7 postcell transfer, the shaved skin site was then graded for lesion development (Fig. 2). As previously observed, animals receiving only Skn-immune cells formed lesions at a mean grade of 3.4 ± 0.9, while animals receiving cotransfer of unfractionated normal spleen cells had a significantly reduced lesion grade of 1.3 ± 0.2 (p < 0.005). For mice given fractionated normal spleen cells, those receiving a cotransfer of 24 x 106 CD4-enriched cells demonstrated reduced lesion severity (mean lesion grade of 1.5 ± 0.5), while depletion of CD4+ cells eliminated the ability of the remaining cells to do so. Furthermore, moderation of lesion grade was dependent on the number of CD4-enriched cells cotransferred, in that transfer of 16 x 106 could also reduce lesion scores (mean grade of 1.5 ± 0.0), whereas transfer of 12 x 106 or 6 x 106 CD4-enriched cells could not (mean lesion grades of 2.9 ± 1.2 and 3.3 ± 0.3, respectively). In contrast, cotransfer of CD8-enriched cells did not alter lesion severity, but neither did cotransfer of CD8-depleted cells, even though this preparation retained CD4+ cells. The unexpected results from the cotransfer of CD8-depleted normal spleen cells prompted us to further examine whether CD8+ cells had a role in assisting CD4+ cells in their role of reducing lesion scores. We addressed this in two ways. First, we cotransferred 12 x 106 CD4-enriched cells, a dose at which they were unable to moderate lesion development, along with varying numbers of CD8-enriched normal spleen cells. However, the addition of 6, 8, or 12 x 106 CD8-enriched cells did not reduce lesion severity in recipients who had mean lesion grades of 3.4 ± 0.5, 3.8 ± 0.6, and 3.5 ± 0.5, respectively. The second approach was based on reports that dendritic cells are implicated in the regulation of autoimmunity (reviewed in Ref.19) and that there are several dendritic cell populations within mouse spleen that also have surface expression of CD4 or CD8 (20). Although our spleen cell preparations, in general, did not use a method that would have included dendritic cells (21), we nevertheless generated a cotransfer inoculum of normal spleen cells that was depleted only of CD8+ T cells by first removing all T cells with Ab to CD3, then reconstituting the remaining cells with 24 x 106 CD4-enriched cells. But again, we found that this cotransfer did not reduce the lesion grade in recipients (mean grade of 3.2 ± 0.7).


Figure 2
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  		FIGURE 2. Cotransfer of CD4-enriched normal spleen cells results in reduced severity of skin lesions. Mice were shaved, cyclophosphamide pretreated, and given injections of Skn-immune cells alone or with the designated cotransfer inoculum. On day 7 postcell transfer, lesion grades were assessed. Data represent 16 mice for each cotransfer condition 1 and 2, and 5–8 mice for each cotransfer condition 3–6. *, p < 0.005 compared with mice receiving only Skn-immune cells.

 
Together, these data suggested that although CD4+ cells appeared able to moderate the development of skin lesions, the presence of this cell type per se was unlikely to be the only contributing factor.

Elevated IL-7 mRNA levels are associated with reduced lesion severity

Various types of regulatory cells controlling immune pathogenesis are generated in response to and/or produce different cytokines, predominantly, IL-10, TGF-beta (11, 12, 13), and IL-2 (22, 23). To evaluate whether the clinical improvement observed with the cotransfer of normal spleen cells was reflected in the local production of regulatory cytokines, relative mRNA levels were determined from skin biopsies during the prelesional phase (days 0.5–3) or at 7 days postcell transfer. No differences were detected in IL-2, IL-10, or TGF-beta mRNA levels between mice receiving only Skn-immune cells vs those also receiving injections of normal spleen cells in either the prelesional time points (data not shown) or at 7 days posttransfer (Fig. 3).


Figure 3
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  		FIGURE 3. Elevated IL-7 mRNA levels are found with the cotransfer of normal spleen cells. Semiquantitative RT-PCR analysis of cytokine mRNA was determined from skin biopsies taken at day 7 postcell transfer. Animals received standard pretreatments and Skn-immune cells alone (n = 14) or with the cotransfer of unfractionated normal spleen cells (n = 11). Results are expressed as relative units of cytokine mRNA using beta-actin levels for normalization. Data are reported as mean ± SE. *, p ≤ 0.01.

 
Because the recipients in our model were given cyclophosphamide, a compound known to induce leukopenia, IL-7, a cytokine critical for lymphopoiesis, which is constitutively produced by keratinocytes (24), was also evaluated. As expected, injection of cyclophosphamide without transfer of any cells caused elevated levels of IL-7 mRNA in skin biopsies starting at day 1–3 postinjection of cyclophosphamide (IL-7:beta-actin ratio of 0.24 ± 0.19) and continuing throughout the time frame of the experiments (IL-7:beta-actin ratio of 0.47 ± 0.09 on days 5–7). When cells were adoptively transferred, IL-7 mRNA levels declined to constitutive levels (IL-7:beta-actin ratio of 0.12 ± 0.05) and were not different through day 3 between recipients of Skn-immune cells compared with those also receiving injections of normal spleen cells (IL-7:beta-actin ratio of 0.10 ± 0.14 and 0.09 ± 0.06, respectively). However, at day 7 posttransfer (Fig. 3), animals receiving cotransfer of normal spleen cells and whose lesion severity was diminished had significantly elevated IL-7 levels compared with recipients of Skn-immune cells alone. This suggested that IL-7 may be a critical component for providing regulation of lesion formation.

We then asked whether similar IL-7 levels could be found in animals with reduced lesions severity after cotransfer of fractionated normal spleen cells. In these experiments, IL-7 mRNA was observed to be significantly increased only in the skin of animals receiving cotransfer of CD4-enriched normal spleen cells when compared with recipients of Skn-immune cells alone (Fig. 4). No difference in IL-7 mRNA was observed in animals receiving cotransfer of CD4-depleted, CD8-depleted, or CD8-enriched normal spleen cells (Fig. 4).


Figure 4
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  		FIGURE 4. Elevated IL-7 mRNA levels are found specifically with the cotransfer of CD4-enriched normal spleen cells. Semiquantitative RT-PCR analysis of cytokine mRNA was determined from skin biopsies taken at day 7 postcell transfer. Animals received standard pretreatments and were given injections of Skn-immune cells alone or with the designated cotransfer inoculum. Results are expressed as relative units of cytokine mRNA using beta-actin levels for normalization. Data are reported as mean ± SE and represent 14–16 mice for each cotransfer condition 1 and 2, and 5–8 mice for each cotransfer condition 3–6. *, p ≤ 0.01 compared with mice receiving only Skn-immune cells.

 
When IL-7 message levels were evaluated in the context of both lesion grade and presence of subpopulations of normal spleen cells, the data suggested that lesion severity was diminished only with the combination of increased expression of IL-7 mRNA in the skin and cotransfer of CD4+ normal spleen cells. This is clearly seen in Fig. 5 when lesion grade is plotted against IL-7 mRNA levels. The conclusion is further supported in that, while CD8-enriched cell cotransfer resulted in a variable IL-7 mRNA level in the skin with some mice having elevated IL-7 expression, diminished lesion severity was not observed in the absence of CD4+ cells. Likewise, cotransfer of CD8-depleted cells, which retained CD4+ cells, could not modulate lesions without adequate expression of IL-7 mRNA.


Figure 5
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  		FIGURE 5. A composite graph of IL-7 mRNA levels vs mean lesion grade shows that both the presence of CD4+ T cells in the cotransfer inoculum and elevated IL-7 mRNA are required for reduced lesion severity. Data were determined from mice described in Figs. 2 and 4.

 
In vivo anti-IL-7 treatment and reduction of IL-7R expression on cotransferred CD4+ cells abrogate reduced lesion serenity

To further examine the significance of IL-7 and its association with CD4+ cells in contributing to the reduction in lesion severity, we performed two additional experiments.

In the first experiment, we tested whether in vivo administration of anti-IL-7 mAb attenuated the ability to support decreased lesion grades. A mouse IgG anti-human IL-7 mAb that cross-reacts and neutralizes mouse IL-7 (25) and that has previously been shown to inhibit T lymphopoiesis (25, 26) was used. For these studies, mice received either Skn-immune cells alone, Skn-immune cells with cotransfer of normal spleen cells, or Skn-immune cells with normal spleen cells plus in vivo treatment with anti-IL-7 mAb. As shown previously, mice receiving injections of Skn-immune cells alone developed lesions at grade 3.3, whereas those administered Skn-immune cells plus normal spleen cells developed lesions of reduced severity at 1.6 (Table I, Expt. 1). However, animals receiving cotransfer of Skn-immune cells plus normal spleen cells and that also were treated with anti-IL-7 mAb developed lesions that did not differ in severity from those animals given Skn-immune cells alone (Table I, Expt. 1). The reversal of reduced lesion severity by anti-IL-7 further supports a role for IL-7 in limiting lesion progression. In the second experiment, we hypothesized that down-regulation of IL-7R expression on cotransferred cells would also interfere with the ability to reduce lesion severity. In this study, we took advantage of the fact that culture of freshly isolated T cells in the presence of IL-7 results in a substantial decrease in IL-7R expression when measured by flow cytometry (27, 28) (our unpublished results). We then performed another set of transfer experiments in which mice were given injections either of Skn-immune cells alone, Skn-immune cells with cotransfer of CD4-enriched normal spleen cells, or Skn-immune cells plus CD4-enriched normal spleen cells that had been cultured in the presence of rIL-7. As previously shown, mice cotransferred with CD4-enriched normal spleen cells exhibited reduced lesion severity when compared with recipients of Skn-immune cells alone. In contrast, recipients cotransferred with cultured CD4-enriched spleen cells developed lesions comparable to mice given injections of Skn-immune cells alone (Table I, Expt. 2). These results strengthen the association between IL-7 and CD4+ normal spleen cells in promoting reduction in lesion development.


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  		Table I. Effect of IL-7 modifications on lesion severity

 
IL-7 gene therapy controls Skn-targeted autoimmune lesions

Because elevated levels of IL-7 were clearly associated with decreased lesion grades, we examined whether exogenous IL-7 expression within the skin could induce lesion control without cotransfer of normal CD4+ spleen cells. It has been shown that topical application of naked plasmid DNA results in gene expression in murine skin (29). Using this delivery method, a vector encoding mouse IL-7 cDNA under the control of a CMV promoter and containing a FLAG sequence for N terminus tagging (pCMV-Tag1-IL-7) was selected. To determine whether administration of pCMV-Tag1-IL-7 could reduce lesion severity, we applied the IL-7 plasmid expression vector to mice that had been pretreated by shaving and cyclophosphamide and given injections of Skn-immune cells.

Preliminary experiments were conducted to establish an optimal dose, timing, and site of application of plasmid DNA. Daily deliveries of a total of 15 µg of pCMV-Tag1-IL-7 in areas adjacent to, but not directly on, the shaved skin sites were chosen. The hair in the delivery sites was clipped short, but not shaved. Control applications consisted of either pCMV-Tag1-null vector or vehicle alone; results were comparable and these control applications were used interchangeably. Mice receiving daily delivery of 15 µg of pCMV-Tag1-IL-7 for 7 days demonstrated significantly reduced lesion severity (mean grade of 1.1 ± 1.3; p = 0.002) when compared with animals receiving control applications (mean lesion grade of 3.3 ± 1.1). Using the tag (FLAG) associated with the plasmid vector, endogenous vs plasmid IL-7 transcripts could be distinguished. From the shaved skin site of pCMV-Tag1-IL-7 recipients, FLAG-tagged IL-7 mRNA was readily detected by RT-PCR (FLAG-IL-7:beta-actin ratio of 0.23 ± 0.12), while endogenous IL-7 messages were not observed. These data strongly imply that exogenous plasmid-derived IL-7, independent of endogenous IL-7, was contributing to reduced lesion severity.

To determine whether pCMV-Tag1-IL-7 gene therapy altered other potential regulatory cytokine levels, day 7 skin biopsies were evaluated for IL-2, IL-4, IL-10, IFN-{gamma}, and TGF-beta mRNA. Whereas IL-7 levels were significantly elevated, no differences were found for any of the other cytokines in pCMV-Tag1-IL-7 recipients vs nonplasmid recipients (Fig. 6). Although these data do not preclude altered gene expression in cytokines not tested, the results suggest that IL-7-associated lesion modulation was most likely acting independently of other reported regulatory cytokines.


Figure 6
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  		FIGURE 6. pCMV-Tag1-IL-7 topical delivery does not alter other skin-derived cytokine mRNA levels. Semiquantitative RT-PCR analysis of cytokine mRNA was determined from skin biopsies taken at day 7 postcell transfer. Animals received standard pretreatments, were given injections of Skn-immune cells, and received daily topical treatment with pCMV-Tag1-IL-7 or with vehicle (nonplasmid recipients). Results are expressed as relative units of cytokine mRNA using beta-actin levels for normalization. Data are reported as mean ± SE as combined data from three experiments. *, p < 0.001 vs nonplasmid recipients.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the Skn model of autoimmunity, lesion formation relies on short-term immunosuppression for introduction of autoreactive cells into recipients. The development of clinical disease is based on the hypothesis that immunosuppression interferes with peripherally located regulatory cells that would otherwise control the adoptively transferred self-reactive lymphocytes. In this study, we have investigated the ability to restore suppression of autoreactivity in these animals and have found that the transfer of adequate numbers of normal CD4+ spleen cells in conjunction with sufficient expression of endogenous IL-7 was associated with reduced lesion severity. In addition, topical gene therapy with an IL-7-expressing plasmid could replace cotransfer of CD4+ normal spleen cells and elicit a similar reduction in lesion grade.

CD4+ T cells have consistently been identified as regulatory cells in various animal models of autoimmunity (9, 10, 30). Therefore, it was not unusual to find that the CD4-enriched fraction of normal spleen cells could reduce lesion severity when cotransferred with Skn-immune lymphocytes. However, it was surprising and seemed inconsistent to observe that cotransfer of spleen cells depleted of CD8+ cells, which retained normal numbers of CD4+ cells, lost the ability to control lesion development. One potential explanation is that CD8+ cells were necessary for CD4+ cells to perform their regulatory activity. However, we found that with the cotransfer of reduced numbers of CD4-enriched normal spleen cells, supplementation with CD8-enriched cells did not result in reduced lesion scores, suggesting that CD8+ cells were unlikely to have a direct role in the regulatory function observed with CD4+ cells. An alternative possibility is that CD4+ and CD8+ cells are part of a regulatory cascade. As our CD8 depletion procedure removed only CD8+ cells from a complex mixture of spleen cells, candidate cells in a regulatory cascade could come from any of a number of major cell types or subsets. Indeed, several recent reports show that in the suppression of autoimmune diabetes and experimental allergic encephalomyelitis, NKT cells enhance the development of regulatory dendritic cells (31, 32, 33), which in turn promote the induction of regulatory CD4+ T cells (33). At this time, we have no additional evidence that would lend support to such a mechanism for controlling lesions in our model based on cotransfer alone, in that our CD4-enriched cells were most likely depleted of NKT cells by means of Ab to CD49b and our spleen cell preparations did not use a method that would have included dendritic cells (21). Yet we cannot rule out the possibility that participating cells could come from endogenous reconstitution of recipients or even from the Skn-immune cell inoculum.

However, the most likely explanation is that the cytokine milieu influenced or directly determined the function of CD4+ cells in recipients. Skin, the target organ for Skn autoimmunity, is known to constitutively express many cytokines (reviewed in Ref.34) and can actively respond to various stimuli through altered cytokine gene expression (35). Although cytokines linked with regulation of autoreactivity in other disease models did not correlate with reduced lesion severity, we did observe that IL-7 mRNA levels were elevated in mice, demonstrating lesion reduction, but only when there were concurrent CD4+ cells in the cotransfer inoculum. The relationship between CD4+ cells and IL-7 is clear, such that if one of these conditions was not met, lesions scores remained high. This is evident in mice receiving cotransfer of CD8-depleted normal spleen cells in which IL-7 mRNA levels were extremely low and lesions scores were high. Likewise, when the number of cotransferred CD4-enriched cells fell below a critical number (12 x 106), lesion grades remained high despite the fact that IL-7 mRNA levels were elevated (data not shown). What still remains unclear is the mechanism directing the increase of IL-7 in recipients. Although elevated IL-7 mRNA appears to be driven by cotransfer of normal spleen cells, particularly cotransfer of CD4+ cells, it is not an absolute consequence with cotransfer. At this time, we have only the observable relationship, but no mechanism to explain these data. Because various cell types express IL-7Rs (reviewed in Ref.36) and IL-7 has recently been shown to have both dose (37) and temporal (38) effects in its regulatory capacity, further studies will be needed to establish which additional factors could affect changes in IL-7 expression to resolve the conflicting results noted above.

Our finding that skin-derived increases in IL-7 mRNA are a critical component in animals with reduced lesion severity is intriguing in light of two prevailing concepts in understanding the significance of the peripheral T cell repertoire. First, there is substantial evidence that IL-7 is essential for peripheral T cell homeostasis (reviewed in Refs.39 and 40) and contributes to the survival and proliferation of naive CD4+ and CD8+ (41, 42), and memory CD4+ (43, 44, 45) and CD8+ (42, 46) T cells in lymphopenic conditions. Second, an underlying cause of autoimmunity in lymphopenic individuals can be due to abnormal T cell homeostasis (47, 48, 49, 50).

In our model, a lymphopenic condition, which we provided by pretreatment with cyclophosphamide, is required for subsequent development of autoimmune skin lesions (2). And while normal murine and human keratinocytes can secrete IL-7 protein in biologically relevant amounts (24), pretreated mice receiving Skn-immune cells alone appear to have negligible IL-7 expression in their skin throughout the time frame of our experiments (this study). Therefore, our adoptive transfer conditions for autoimmunity produced an animal that not only was lymphopenic, but, most likely, also one without the capacity to restore peripheral T cell homeostasis, and likewise control of autoimmunity, of its own accord. However, as noted above, when adequate numbers of normal CD4+ T cells together with elevated levels of endogenous IL-7 were present, or when an exogenous supply of IL-7 was introduced, lesion severity was diminished. Furthermore, mice cotransferred with a source of normal CD4+ spleen cells, but also treated with anti-IL-7 Ab, no longer demonstrated reduced lesion severity. Based on these data, we speculate that IL-7 via CD4+ T cells plays the critical role in controlling the development of lesions most likely through the re-establishment of peripheral T cell homeostasis. However, it remains to be determined how IL-7 might perform this function, in that in our model, both autoreactive Skn-immune lymphocytes (51) and the cells that regulate lesion severity (this study) predominantly reside in the CD4+ subpopulation.

Re-establishment of peripheral homeostatic regulation of T cell subpopulations is reported to be accomplished simply by the presence of large numbers of bystander T cells, most likely ones responsive to IL-7 (52). With respect to cotransfer of unfractionated normal spleen cells, our data are consistent with these findings. Indeed, we found that injection of normal spleen cells in numbers equal to Skn-immune cells could not diminish lesion severity, whereas increasing the number of normal spleen cells to twice that of Skn-immune cells could moderate lesion development in recipient animals. However, total number of cotransferred cells alone was not responsible for lesion reduction, as titering of CD4-enriched normal spleen cells for cotransfer suggested, at least in part, that the number of CD4+ cells was what was important in regulating the Skn-immune cells.

Others suggest that peripheral T cell homeostasis comes through competition for IL-7 and/or TCR signals (53, 54) or between memory and naive status (55). Our data point to a difference in responsiveness to IL-7, in that treatment with IL-7 gene therapy did not exacerbate, but rather diminished, autoreactivity, indicating that Skn-immune CD4+ T cells were unlikely to be sensitive to IL-7. In contrast, when CD4+ T cells from normal animals were pretreated so as to down-regulate surface expression of the IL-7R, their ability to moderate lesion severity was lost, demonstrating that responsiveness to IL-7 was needed to moderate autoimmunity. Important too in driving this responsiveness to IL-7 is the dose of IL-7 available to the lymphopenic host, wherein as IL-7 levels increase, the proliferation of naive T cells with low affinity TCR interactions can occur (37, 56). During the time frame of our experiments, we found that a significantly increased dose of IL-7 mRNA was a critical component in reducing lesion severity vs the dose observed in recipients with high lesion scores. With respect to competition via TCR signaling or through memory/naive status, the constraints in the setup of our model have hampered our ability to make meaningful determinations at this time, in that we do not yet have the capability to determine the frequency or phenotypic state of Skn-specific autoimmune vs Skn-specific and/or nonspecific regulatory CD4+ T cells. Additional experiments are needed and may require additional knowledge of Skn as an Ag.

Another possibility is that regulatory CD25+CD4+ T cells affect the homeostatic expansion of CD4+ T cells and prevent autoimmunity (57). For a limited number of animals, we examined the expression of CD4 and CD25 or CD8 on PBL at 13 days posttransfer and found no differences in pretreated mice given injections of anti-Skn immune cells whether or not they were also treated with IL-7 gene therapy (our unpublished observations), implying that there was no correlation between decreased lesion grade and CD25+CD4+ T cell number. This is similar to findings reported by Barthlott et al. (58), who suggest that T cell regulation in lymphopenia can be the result of competition from expansion of normal T cells, without the need to have properties dedicated to suppressor function.

The clinical significance of our data may best be demonstrated in the finding that IL-7 gene therapy augmented the ability to reduce lesion severity without the overt cotransfer of CD4+ T cells. Our results appear to be consistent with previous reports that show a relationship between exogenous IL-7 delivery and improved clinical outcome. For example, IL-7 immunotherapy in some tumor models augments tumor regression (Ref.59 and references therein; Ref.60). More apropos to Skn-directed autoimmunity, there are several murine models of graft-vs-host disease, whereby administration of IL-7 to lymphopenic recipients enhances lymphocyte recovery after bone marrow transplant, while not aggravating graft-vs-host disease (61, 62, 63, 64). In the graft-vs-host disease models, the effect of IL-7 can be dependent upon host conditioning treatments and parameters of IL-7 delivery (64).

In summary, the data presented in this study demonstrate the effect of reducing autoimmune lesions by the introduction of normal CD4+ T cells concomitant with increased endogenous IL-7 gene expression in mice prepared for Skn-targeted autoimmune skin disease. Moreover, the similar results obtained by topical gene delivery with IL-7 alone suggest that this cytokine may have potential therapeutic value for other autoimmune diseases or chronic disorders.


   Acknowledgments
 
We thank St. Mary’s Hospital Cancer Center for irradiating the mice, and Shivaleela Keerthy and Kevin Saunders for expert technical assistance. We also thank Dr. Benjamin Rich for the gift of the IL-7 cDNA, and Dr. Todd Green for the gift of the C2 mouse muscle cells.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by National Institutes of Health Grant AI 34421. Back

2 Address correspondence and reprint requests to Dr. Susan H. Jackman, Department of Microbiology, Immunology, and Molecular Genetics, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25704. E-mail address: jackman{at}marshall.edu Back

Received for publication March 3, 2005. Accepted for publication January 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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