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The Journal of Immunology, 1998, 161: 2060-2069.
Copyright © 1998 by The American Association of Immunologists

Primary Th1 Cell Immunization Against HIVgp160 in SCID-hu Mice Coengrafted with Peripheral Blood Lymphocytes and Skin1

Nadirah Delhem*, Fabienne Hadida{dagger}, Guy Gorochov{dagger}, Françoise Carpentier{ddagger},¶, Jean-Pierre de Cavel*, Jean-François Andréani§, Brigitte Autran{dagger} and Jean-Yves Cesbron2,*

* Institut National de la Santé et de la Recherche Médicale U167, Institut Pasteur, Lille, France; {dagger} Laboratoire d’Immunologie Cellulaire et Tissulaire, Unité de Recherche Associée 625, Centre d’Etudes et de Recherches: Virologie et Immunologie (CERVI), Hôpital Pitié-Salpétrière, Paris, France; {ddagger} Service d’Anatomie et de Cytologie Pathologiques, Hôpital Victor Provo, Roubaix, France; and § Service de Chirurgie Maxillo Faciale, Hôpital des Armées Scrive, and Université de Lille 2, Lille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
SCID-hu mouse models are of interest in the pathologic investigation of HIV infection, but obtaining a T cell response in SCID-hu-PBL mice is still controversial. We have developed a SCID model by engrafting human skin and autologous PBLs from HIV-seronegative individuals. The study describes the ability of this human-mouse chimera to generate in vivo a primary T lymphocyte response against HIV Ag. The injection of human autologous PBLs was performed 4 to 5 wk after the skin engraftment. Two weeks after injection of PBLs, chimeric mice were immunized with recombinant canary pox virus expressing HIV-1 LAIgp160 (vCP-LAIgp160) and supplemented or not with rIL-2. Intradermal vCP-LAIgp160 injection induced an intradermal perivascular human lymphocytic infiltrate and an epidermic network of CD1a+, CD80+, and CD86+ cells. We derived CD4+ T cell lines (STLs) from the human skin graft of immunized mice, showing that STLs mediated an MHC class II-restricted cytolytic activity directed against HIV-LAIgp160 Ags. Cytokine gene expression in both human skin cells and in STLs showed a predominance of IL-2, IFN-{gamma}, and IL-12 transcripts. Finally, the T cell repertoire analysis using the immunoscope technique showed a very limited CDR3 length polymorphism in the skin infiltrating lymphocytes suggesting an Ag-specific repertoire. The ability to induce a primary Th1 cell response in vivo affords a useful preclinical model for testing vaccine strategies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Severe combined Immunodeficient mice (1), which lack autologous T and B lymphocytes (2, 3), are increasingly used as hosts for adoptive transfer of human PBLs (4) and serve as a model for the study of HIV pathogenesis (5, 6). Using this approach, usually termed the SCID-hu-PBL3 mouse model, human PBLs are inoculated into the mouse peritoneal cavity (7). However, the functionality of such human immunocompetent cells appears to be limited (8). It seems that during the first month after the injection, the human leukocytes might be fully functional. However, after this time, human T cells appear in mouse extralymphoid tissues, such as the lung, liver, and spleen (9, 10, 11). The human T cells are then anergic and unresponsive to stimulation with anti-CD3 or mitogens (11), whereas an Ab response to recall Ags, against which the donor was sensitized, is observed (12, 13, 14) when the mice are challenged with Ag within ~1 wk post-cell transplantation (15). However, the obtention of a primary response in these xenogeneic chimeras is still controversial (8, 9, 15, 16, 17), although this has been reported by several groups (18, 19, 20, 21). Several investigators have tried to improve the engraftment of human immune cells by using immunosuppressive regimens (sublethal irradiation (22), anti-asialo GM1 Ab injection (23)), by increasing the PBL inoculum (22), or by using splenocytes as the source of human mononuclear cells (24). Nevertheless, the obtention of a human MHC-restricted primary T lymphocyte response in a SCID mouse model has not yet been reported. Thus, while these chimeric animal models have been shown to be useful for studying the infection of human T cells with HIV (5, 6), they are less suitable as models for studying a human immune response in vivo. The major limitations for developing a human immune response in this model appear to be linked to the lack of the appropriate human APCs and a suitable microenvironment (8). Thus, we postulated that the engraftment of a human accessory tissue containing APC could contribute to the provision of a more appropriate microenvironment for supporting the survival of naive human T cells, as well as their priming and differentiation following an antigenic stimulation. Skin appeared a good candidate, both because it plays an important role in the induction of the immune response (25, 26) and because it is more easily available for experiments than other human lymphoid organs such as lymph nodes. The demonstration of skin engraftment onto SCID mice was first described using grafts from recessive dystrophic epidermolysis bullosa patients (27). A normal human skin graft onto SCID mice was also reported in a study of the regulation of human endothelial cell-leukocyte adhesion molecules (28, 29), the induction of human papillomavirus V-16 DNA replication (30), the role of UV light in carcinogenesis (31, 32), the induction of delayed hypersensitivity (33), and in human cutaneous mast cell hyperplasia (34).

We therefore developed a SCID model by engrafting both human PBLs and autologous human skin (SCID-hu-PBL/skin) from HIV-seronegative donors and evaluated in this human-mouse chimera the efficacy of raising a primary T lymphocyte response in vivo against HIV-LAIgp160 Ags. Here, we show that intradermal inoculation of a live recombinant canary pox viral vector containing the gene for HIV1-LAIgp160 (vCP-LAIgp160) into a human skin graft induced a perivascular human CD4+ and CD8+ T cell infiltrate with an epidermal recruitment of CD1a+CD80+CD86+ Langerhans cells (LC). In addition, we were able to derive CD4+ T cell lines from such immunized engrafted human skin that mediated in vitro an MHC class II-restricted specificity directed against HIV-LAIgp160 Ags. These HIV-specific CD4+ T cells demonstrated both in vivo and in vitro a Th1 cell differentiation profile.


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

Homozygous CB-17 scid/scid (SCID) mice derived from breeding stocks provided by Dr M. Liberman (Standford University, Standford, CA), with the permission of Dr M. Bosma (Fow Chase Cancer Center, Philadelphia, PA), were bred and maintained in microisolator cages and used between 4 and 8 wk of age. The mice were kept under pathogen-free conditions without prophylactic administration of antibiotic.

Skin transplantation

Skin transplantation was performed as previously described (28). Briefly, anesthetized mice were prepared for transplantation by shaving the hair from a 5-cm2 area on each side of the lateral dorsal region. A square graft bed, 1.5 x 1.5 cm2, was created on the shaved areas by removing a full thickness of skin down to the fascia. A full-thickness human skin graft of the same size was placed onto the wound beds. The transplant was held in place with nonabsorbed monofilament suture material and covered with an additional layer of micropore cloth tape. Human skin consisted of skin from reconstructive surgery. The protocol was approved by the local Ethical Committee of the "Centre Hospitalier Universitaire et de Recherches de Lille."

Preparation of HIV-seronegative human PBLs and SCID-hu/skin mouse reconstitution

An autologous blood sample from the skin donors was obtained 4 to 5 wk after the surgical intervention. This delay was required to allow the skin to heal. Ficoll-separated PBLs were depleted of NK cells by incubation with anti-CD16+ and anti-CD56+ mAbs (Becton Dickinson, Pont de Clay, France). The negatively selected cells were then harvested and used for SCID reconstitution (20 x 106; i.v.) after immunocytofluorometric analysis controls.

Canary pox virus immunization

Canary pox virus recombinant for the HIV-1 LAIgp160 gene (vCP-LAIgp160; Pasteur Merieux serum vaccine, P.H.S.V., Lyon, France) was a kind gift of Dr. Bernard Meignier. The vaccine products were processed as previously described (35). At 2 wk following PBLs reconstitution, SCID mice were inoculated intradermally with 103 x plaque-forming units (PFU) of vCP-LAIgp160 into the human skin xenograft. A second immunization was conducted under the same conditions 2 wk later. The protocol is schematized in Figure 1Go. Each immunization was performed with 3 or 4 injections for a total volume of 100 µl. The mice received 200 IU rIL-2 (Boehringer, Mannheim, Germany) injected i.p. every 48 h after the first vCP-LAIgp160 injection. A protocol with two injections of vCP-LAIgp160 without rIL-2 complementation was performed as a control. In addition, control mice receiving only wild-type canary pox virus or rIL-2 were also studied. The first three letters of the name of the skin donor became the name of the experimental series. Each mouse was identified individually.



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  		FIGURE 1. Schema for the obtention and study of immunized SCID-hu-PBL/skin mice. Full-thickness human skin was engrafted onto the back of anesthetized SCID mice (1. 5 x 1.5 cm). When the human skin had healed (4–5 wk), autologous NK-depleted PBLs (20 x 106) were injected i.v. Two weeks later, SCID-hu-PBL/skin mice were inoculated with 103 x PFU of vCP-LAIgp160 by three or four intradermal injections into human skin for a total of 100 µl. A second immunization was conducted under the same conditions 2 wk later. The SCID-hu-PBL/skin mice received 200 IU rIL-2 i.p. injected every 48 h after the first vCP-LAIgp160 immunization. At the time of sacrifice, 5 wk post-hu-PBL reconstitution, animals were bled by intracardiac puncture for subsequent cytofluorometric analyses. The skin grafts were carefully dissected from the SCID mice under sterile conditions and divided for further immunohistologic analyses and skin cell isolation.

 
Skin, cell, and plasma collection from human-mouse chimeras

One week after the human PBLs transfer, some human skin grafts were punch biopsied (3-mm diameter punch biopsy; Stiefel, Nanterre, France; see Fig. 1Go). At the time of sacrifice, 5 wk post-human PBL reconstitution, animals were bled by intracardiac puncture. PBLs were isolated using Ficoll-Paque centrifugation and washed with PBS containing 1% BSA for subsequent immunocytofluorometric analysis. The skin graft was carefully dissected from the animals under sterile conditions and divided for further analyses.

Histologic analysis

One piece of each graft was placed in paraformaldehyde, 4% PBS and paraffin embedded. Sections (3 µm) were stained with hematoxylin-eosin saffron (HES) for routine histochemistry analysis. Another piece was placed in OCT compound (Miles-Elkhart, Naperville, OH) and snap-frozen in liquid nitrogen for subsequent immunohistochemical analysis (Dako, Carpinteria, CA). Briefly, the 6-µm frozen sections were fixed in cold acetone. After blocking for 10 min with 0.1% H2O2 and normal horse serum in Tris-buffered saline (TBS) for 10 min, slides were incubated with primary Ab (see below) diluted in TBS 2% FCS for 30 min. After washing, biotinylated rabbit anti-mouse IgG diluted in TBS was added for 15 min. The avidin-biotin complex was added for 10 min followed by 3-amino-9-ethylcarbazole (AEC) for 3 min. Anti-human mAbs CD1a, CD45, CD3, CD8, CD4, CD80, and CD86 and anti-mouse mAbs CD45R/B220 and CD3 were purchased from Becton Dickinson. Anti-human CD16 and CD56 mAbs were from Immunotech (Marseille, France). The third skin fragment was snap frozen in liquid nitrogen for cytokine assays using RT-PCR. The remaining piece of the skin was placed in culture medium to isolate human T cells from the graft.

Skin cell isolation and generation of skin infiltrating T cell lines (STL)

Briefly, the skin graft was gently minced in RPMI medium supplemented with 10% human serum AB. Effector skin cells were set up in culture with 5 x 104/ml 5 k rad irradiated allogeneic PBLs as feeder cells in the presence of purified PHA 1 µg/ml (Murex Diagnostics, Dartford, U.K.), 20 IU/ml rIL-2 (Boehringer) at day 0 in RPMI supplemented with 10% human AB serum, 0.3 mg/ml glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Cergy Pontoise, France). Recombinant IL-2 alone (20 IU/ml) was subsequently added every 3 days during the 3 wk of culture growth, before testing for cytolytic activity. All the T cell culture assays were conducted blind according the same procedure without any in vitro Ag-specific restimulation.

Immunofluorescence staining

Human lymphocytes, either from the SCID-hu-PBL/skin mice blood or from infiltrating human skin explants were analyzed for their phenotype. Lymphocytes (100 x 103 cells/sample) were incubated for 30 min on ice with a mixture of appropriate fluorescence-labeled mAb. The labeled Abs used recognize specific surface molecules: anti-CD45 FITC, anti-CD4-phycoerythrin (PE), and anti-CD8-peridinin chlorophyll protein (PerCP). Control Abs consisted of nonspecific IgG conjugated with FITC, PE, or PerCP. Nonreconstituted SCID mice were also used as controls. After washing, three-color fluorescent analysis of human Ags was performed on a FACScan analyser (Becton Dickinson, San Jose, CA).

Cytotoxicity assays

CTL assays were performed 3 wk after generation of CTL line cultures from skin. HLA-matched or mismatched allogeneic EBV-transformed B-lymphoblastoid cell lines were used as target cells after infection with recombinant vaccinia virus constructs expressing the Env HIV-1 protein (Vac-LAIgp160) and the wild-type Copenhagen strain (wt-Vac; Transgène, Strasbourg, France) at a multiplicity of infection of 5 PFU per cells, as previously described (36). Target cells were labeled with Na251Cr04 (Amersham, Les Ulis, France) and added in triplicate microtiter plates (Dutscher, Brumath, France) to CTL at various E:T ratios, as previously described (36). After a 4-h incubation period at 37°C, the supernatants were harvested and chromium release was measured in a gamma counter (Clinigamma, ECG Instruments, Evry, France). Spontaneous 51Cr values were 15 to 20% of total incorporated radioactivity. The percentage of 51Cr release was calculated as follows: 100 x (experimental release) - (spontaneous release)/(total release) - (spontaneous release).

HLA typing

HLA typing of human PBLs was performed using standard serologic and molecular methods for HLA class I and HLA class II molecules at the "Laboratoire d’Immunologie Cellulaire et Tissulaire" at Pitié Salpétrière Hospital, Paris, France.

Cytokine determination

The whole procedure was performed as described previously (37) with minor modifications and adaptation for human cytokines. Total RNA from ex vivo frozen skin and CTL lines were isolated with RNAzol (2 ml/100 mg of tissues and 0.2 ml/106 cells; Tel-Test, Friendswood, TX). Purification of mRNA was performed as previously reported (38). cDNA was prepared using Stratascript (Stratagene, La Jolla, CA). Each sample (10 OD/ml) was subsequently amplified by PCR using primers for hypoxanthine-guanine phosphoribosyltransferase (HPRT, a housekeeping enzyme) and cytokines as published (38) (Table IGo). Reaction buffer was used as a control and was always negative. Ten-microliter samples were used for each reaction. PCR amplification using the primers specific for different gene transcripts was performed in 50-µl volumes with 1 U of Taq polymerase (Perkin-Elmer, Emeryville, CA) and a 96-well thermocycler (Perkin-Elmer) under the following conditions: 30 cycles of 30 s at 94°C; 30 s at 55°C; and 1 min at 72°C. The amplified product (10 µl) was separated by electrophoresis on a 1% agarose gel at 75 V/cm for 30 min.


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  		Table I. Oligonucleotide sequences (5' to 3')

 
TCR BV repertoire analysis

Total RNA was extracted from skin biopsies and PBLs, according to Chomezynski (39), and reverse transcribed using the Stratagene single-strand synthesis kit according to the manufacturer’s instructions. Amplification reactions were performed using Cß1- and Cß2-specific primers (CßC) and a Vß-specific primer, along with a C{alpha} primer set as an internal control of amplification. Families BV1 to BV24 were studied, using appropriate oligonucleotide probes (Table IGo) as previously described (40). Briefly, 2 µl of the RT product (corresponding to 2.104-2.105 CD4+ or CD8+ T cells) were brought to a final reaction volume of 50 µl containing 20 pmol of each oligonucleotide, 0.2 mM of each dNTP, and 2.5 U of Taq DNA polymerase (Boehringer) blocked for hot start with 1.1 µg of TaqStart Ab (Clontech, Palo Alto, CA). After an initial denaturation step of 3 min at 95°C, the reactions were subjected to 30 cycles of PCR (94°C, 30 s; 60°C, 1 min; 74°C, 1 min), followed by a final extension step of 5 min at 74°C. One nested specific dye-labeled oligonucleotide (Table IGo) (Joefluorophore, Applied Biosystems, Foster city, CA) was used in run-off reactions as previously described (41). The extension reaction consisted in a 3-min denaturation step at 95°C followed by 12 cycles of 30 s at 94°C, 30 s at 60°C, and 2 min at 72°C. A final 10-min incubation at 72°C was performed.

The size and area of the peaks corresponding to the DNA products of TCR CDR3 were determined using the Immunoscope software (C. Pannetier, Paris, France). The percentage of representation of each peak size among all Cß-Vß segments was subsequently calculated. The observed peaks are usually separated by three bases, corresponding to in-frame transcripts of TCRs. Windows of analysis were centered on expected sizes corresponding to TCR transcripts encoding a 10-residue-long CDR3 region. The CDR3 region was defined according to Kabat.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Presence of human LCs in the human skin engrafted on SCID-hu-PBL/skin mice

Six series of skin transplantation from six HIV-seronegative donors were performed with a total of 40 SCID mice. The skin graft appeared normal 5 to 6 wk after skin engraftment, as defined by the absence of inflammation, skin breakdown, skin contraction, and the supple aspect of the epidermis, which persisted during the whole time course of experiments (>1 yr for some mice). However, we noted a 5% graft failure due to pericicatricial necroses.

A first histologic analysis of the human skin grafted onto SCID mice was performed 1 wk post-human PBL injection (Fig. 1Go). The cutaneous grafts were well preserved in every case, without inflammation or necrosis of the different cutaneous components; epidermis, dermis, annexa, and blood vessels were still recognizable and normally arranged without cellular alteration (Fig. 2GoA). To determine the number of murine cells infiltrating the human skin, we biopsied a junctional human/mouse skin region and performed an immunohistologic analysis using an anti-H-2k{alpha} MHCI mouse Ag mAb. This mAb labeled cells within a few human dermal vessels, indicating the presence of rare murine cells in human capillaries (not shown). Conversely, using an anti-human MHC class I Ag, no reactivity was observed in the mouse dermal tissues (not shown). No positivity for anti-human CD3+ (Fig. 2GoB), CD45+, CD4+, or CD8+ mAb was found on the human skin sections, indicating the absence of human T cells in the human engrafted skin 1 wk post-human PBL transfer.



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  		FIGURE 2. Histologic and immunohistologic analyses of human skin engrafted onto SCID-hu-PBL/skin mice. Photomicrographs A–C correspond to samples from nonimmunized SCID-hu-PBL/skin mice biopsied 1 wk after the hu-PBL reconstitution, while D–G illustrate results from human engrafted skin 1 wk after the vCP-LAIgp160 intradermal immunization. Data are from a representative mouse, DAR II-2. The experimental protocol is summarized in the legend to Figure 1Go. A, Junctional human/mice skin biopsy HES showed a preservation of epidermal and dermal structures. Mouse skin is on the left, and human skin is on the right. Grains of pigments from a tattoo are seen in the dermis. B, Immunoperoxidase stains of frozen skin biopsy showed no positivity for anti-CD3+ mAb. C, The anti-CD1a+ cells represented a low level of epidermal compartment cells. D, Biopsy of vCP-LAIgp160-immunized skin HES showed perivascular lymphocyte infiltration (arrow). Some CD45+ cells passed through the basement membrane. E, Immunoperoxidase studies revealed a perivascular infiltration of CD4+ cells (arrow); and F, a massive dermal and epidermal infiltration of CD45+. Intradermal vCP-LAIgp160 injection induced a massive CD1a+ cell recruitment into the epidermis (G). The control mice that received rIL-2 alone or wt-vCP presented no inflammation, lymphocyte recruitment, or positivity for the anti-human leukocytes markers, used and the histologic analyses were similar to those in A–C. Magnification, x 200.

 
In contrast, CD1a+ dendritic cells were observed, exclusively confined to the human epidermal compartments. These CD1a+ cells were present in the same proportions as in normal human skin (Fig. 2GoC) and progressively disappeared during the 3 mo after human skin engraftment. SCID mice engrafted with human skin but without human PBLs were also used for long term studies (>1 yr for some mice) as controls. No significant differences in the immunohistologic data were observed in human skin samples, except in one case (1/40) in which a minor basophil-mediated degeneration of collagen and a minor elastosis were observed in the dermis.

Injection of autologous HIV-seronegative PBLs was performed 4 wk after the human skin engraftment (Fig. 1Go). At the time of sacrifice, 5 wk later, circulating human PBLs were evaluated in the peripheral blood. Of 24 SCID-hu-PBL/skin mice, 22 showed approximately 5 to 8% CD45+, 2% CD4+, and 3% CD8+ in total leukocytes (Fig. 3).

Intradermal vCP-LAIgp160 injection induces human leukocyte and CD1a+ cells recruitment into the human skin graft

Two vCP-LAIgp160 injections were performed intradermally 2 and 4 wk after the human PBL transfer. One hour after each injection of recombinant canary pox virus, an inflammatory erythematous reaction of the human skin was clearly observable, with a maximum reaction 4 h later. The skin recovered a normal aspect 48 h postinjection. One week after the first and the second vCP-LAIgp160 injection, histologic analyses of skin sections were performed. The cutaneous grafts immunized by vCP-LAIgp160 alone presented a discrete perivascular lymphocytic infiltration. The cutaneous grafts immunized by vCP-LAIgp160 with added rIL-2 showed severe intradermal perivascular or periadnexal infiltrates, or diffuse lymphocytic infiltrates, with some neutrophils, eosinophils, and a few plasma cells (Fig. 2GoD). The epidermal basement cell layer was sometimes infiltrated by these inflammatory cells. Numerous histiocytes and fibroblasts and a variable number of intravascular fibrinous microthrombi were detectable in some cases. Immunohistochemistry analyses confirmed the infiltration of human CD4+ (Fig. 2GoE), CD45+ (Fig. 2GoF), CD8+, and CD3+ (not shown) cells in the dermis with a perivascular predominance. Some CD45+ cells passed through the basement membrane, as shown in Figure 2GoF. However, we observed different levels of leukocyte infiltration, according to the conditioning of SCID-hu-PBL/skin mice. The inflammation and the lymphocyte infiltrate were more marked when vCP-LAIgp160-injected mice received rIL-2 than in those inoculated with vCP-LAIgp160 alone. To check the human origin of the lymphocytic infiltrate, we performed an immunohistologic analysis on skin biopsies of the human-mouse junction using an anti-mouse CD45R/B220 mAb and anti-CD3 mAb. No positivity was found, indicating the absence of mouse T cells in the human engrafted skin. The control mice that received wt-vCP presented a transient inflammatory reaction, but not the rIL-2-treated mice. Very few labeled cells for human leukocyte markers (<10 cells/section) could be observed in the engrafted skin (Table IIGo).


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  		Table II. Semiquantitative immunohistochemical analysis of human engrafted skin and flow cytoflorometric analysis of cell lines isolated from the skin grafts

 
The injections of vCP-LAIgp160 increased the numbers of human CD1a+ cells that were detectable in human engrafted skins (Fig. 2GoG). This led to the establishment of a dense network of dendritic CD1a+ cells adjacent to the epidermis and near the basement membrane (Fig. 4GoA). These dendritic cells displayed similar positivity for both CD80 and CD86 molecules, which were activation markers of dendritic cells (Fig. 4Go, B and C). However, the increase in the number of CD1a+ cells was ~50% less prominent when mice received vCP-LAIgp160 alone, without any rIL-2 complementation. In control mice injected with only wt-vCP or only rIL-2, no or only very few CD1a+ cells were observed. No CD1a+ cells were observed in adjacent mouse skin after vCP-LAIgp160 inoculation. Visual semiquantitative immunohistochemistry analysis data from 2 representative series are summarized in Table IIGo.



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  		FIGURE 4. CD1a+, CD80+, and CD86+ cells can be observed in human skin engrafted onto SCID-hu-PBL/skin mice 1 wk after VCP-LAIgp160 immunization. A, VCP-LAIgp160 immunization led to the constitution of a dense network of epidermis adjacent CD1a+ cells near the basement. Magnification, x400. B and C, Dendritic cell activation was evaluated using anti-D80 and anti-CD86 mAb. Magnification, x200. Data are from a representative mouse, DAR II-2. CD1a+ cells were less numerous when mice received vCP-LAIgp160 alone without any rIL-2 complementation. In control mice injected with wt-vCP or rIL-2, few CD1a+ cells were observed (not shown).

 
Generation of skin-derived HIV-1 gp160-specific T cells lines from human skin engrafted onto SCID-hu-PBL/skin mice

From two of the six series we derived STLs from the skin of immunized mice by coculturing skin cells with allogeneic feeder cells in the absence of in vitro HIV Ag restimulation. At 3 wk of culture, we obtained six cell lines from the SCID-hu-PBL/skin mice immunized with vCP-LAIgp160 without rIL-2 (VER I; DAR I) or with rIL-2 (VER II; DAR II-2; DAR II-7). In contrast, we failed to expand any STL from control mice that were not immunized (VER/wt-vCP and DAR/wt-vCP) except for DAR/IL-2. The STL phenotypes analyzed by cytometric analyses are summarized in Table IIGo. A predominance of CD4+ T cells was observed in five of six STLs independently of the conditioning regimen. Isolated T cells did not proliferate in the presence of recombinant gp160 (data not shown).

From these six STLs, four could be expanded sufficiently for further characterization (VER I, VER II, DAR II-2, and DAR II-7), and we analyzed their reactivity against HIV-1 gp160. The two STL cell lines derived from mice immunized by vCP-LAIgp160 in the presence of rIL-2 (VER II and DAR II-2) mediated an HIV-1 gpl60-specific cytotoxic activity as assessed by lysis of an HLA-matched EBV-transformed B cell line expressing the HIV-1 LAI Env gene product (Fig. 5). In contrast, we failed to detect any HIV-1 gp160-specific CTL activity in vitro either with DAR II-7, which was composed mostly of CD8+ T cells, or from the CD4+ VER I, which derived from mice immunized with vCP-LAIgp160 alone. Using different HLA-matched allogeneic Vac-LAIgp160-infected target cells, we tested the HLA class II-restricted cytotoxic activity. The restricting elements were HLA-DR14 for the DAR-CD4+ STL and HLA-DR4 for the VER-CD4+STL. We purified the CD4+ cells from both the VER II and the DAR II-2 lines and confirmed the DAR-MHC class II-restricted cytolytic activity of CD4+ T cell lines specific for HIV-gp160 (Fig. 6). This MHC class II restriction of HIV1-gp160 recognition agreed with the CD4+ phenotypes of the T cell lines obtained. In the case of the CD8+ T cell line, no reactivity against the HIV1-gp160 Ag could be detected.

In vivo and ex vivo cytokine gene expression in human skin cells

To determine whether vCP-LAIgp160 injection could induce in vivo production of human cytokines, we performed a qualitative RT-PCR analysis on mRNA extracted from the human engrafted skin of the five DAR SCID-hu-PBL/skin mice, 5 wk after the human PBL transfer and 1 wk after the second vCP-LAIgp160 immunization (Fig. 1Go). The expression of the following human cytokine genes was tested: IL-2, IL-4, IL-5, IL-6, IL-7, IFN-{gamma}, IL-10, and IL-12. IL-2 mRNA transcript (458 bp) was detectable in skin samples from DAR II-2, DAR II-7, and DAR I mice. IFN-{gamma} transcripts (346 bp) were observed in skin samples from DAR II-2 and DAR II-7, while the IL-12 transcript (354 bp) was detectable in only one case (DAR II-2). In summary, we could detect mRNA transcripts of IL-2, IFN-{gamma}, and IL-12 in skin samples from mice that were both immunized and treated by rIL-2 (DAR II-2 and DAR II-7). The IL-2 mRNA transcript could also be detected in mice immunized with vCP-LAIgp160 alone. No cytokine mRNA was detectable in the skin explants of control mice that received either rIL-2 or wt-vCP alone. A similar amount of PCR product was obtained in all cases using housekeeping gene-specific oligonucleotides (not shown).

The cytokine profile expressed in vivo in the skin transplant of the DAR II-2 mouse (IFN-{gamma}, IL-2, IL-12) was then compared with the cytokine transcripts detected in vitro in the CD4+ STL derived from the same skin graft after in vitro stimulation with the recombinant Vac-LAIgp160. As a control, this CD4+ STL was stimulated using an anti-CD3 or anti-CD28 mAb, or using wt-Vac. IFN-{gamma} and IL-12 transcripts were amplified under in vitro Vac-LAIgp160 stimulation, while in the presence of wt-Vac, we observed only the 346-bp IFN-{gamma} amplimer (Fig. 7B). IL-2 mRNA was also detectable after CD3+/CD28+ stimulation.

TCR BV repertoire analysis

A semiquantitative RT-PCR analysis of the expressed TCRß repertoire was undertaken using 24 different BV-specific probes. Mice DAR II-2 and DAR II-7 were analyzed. Total RNA was extracted both from human PBLs before engraftment (Fig. 1Go) and from the human skin graft biopsies at the day of sacrifice. STLs from mice DAR II-2 and DAR II-7 were also analyzed. TCR transcripts corresponding to all VB families could be detected among the PBL samples using the set of oligonucleotide probes. Human TCR transcripts were detected in the human skin grafts of mice DAR II-2 et DAR II-7, but only with 8 VB probes (not shown). Human TCR transcripts were barely detectable in the skin grafts harvested from the nonstimulated mouse, DAR IV. The presence of particular T cells therefore seems to be specific to the immunized animals. To further investigate the nature of the T cells infiltrating the human skin grafts following Vac-LAIgp160 immunization, the distribution of CDR3 sizes of TCR ß-chains expressed by the human PBLs and the skin-infiltrating lymphocytes (SIL) from DAR II-2 mice was compared using the immunoscope technique. SIL presented a very limited CDR3 length polymorphism, since only a few major peaks were observed for each BV family analyzed. Thus, the cutaneous T cell infiltrates appeared to be highly oligoclonal, while distributions of CDR3 lengths expressed by total PBLs were found to present gaussian, polyclonal patterns in all of the corresponding subsets (Fig. 8). Using the same approach, we confirmed that the T cell lines assayed in vitro actually corresponded to single clonal expansions of T cells initially present in the skin samples (not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Xenogeneic transplantation of human PBLs into SCID mice, first described by Mosier in 1988 (4), has permitted a new approach to study human lymphoid differentiation (42), tumor biology (43), and autoimmune (44, 45) and infectious diseases (5, 46, 47). However, long term studies of the SCID-hu-PBL model showed that human T cells from these mice become anergic and unresponsive to TCR stimulation when tested 4 to 8 wk after reconstitution. Such T cell anergy might either result from a graft-vs-host disease or reflect the lack of an appropriate microenvironment in mouse tissues that would support human lymphocyte trafficking and differentiation for human immune responses to be induced (8, 11, 48). Thus, a human primary immune response has proven to be difficult to obtain in this model, although it has been reported on some occasions (18, 19, 20, 21). To overcome this major limitation of the original SCID-hu mouse model, we coengrafted human skin and autologous PBLs from normal HIV-seronegative donors and demonstrated the ability of this model to allow primary T cell immunization against the HIV Ag and the development of a type 1 CD4 T cell response against HIV-1 gp160.

We first confirmed that xenografted human skin preserves a fully differentiated human epidermis and dermis up to 12 mo posttransplantation, with a success rate of >90% (27, 28, 29, 30, 31). No mouse cell infiltration was detectable in the human skin, as noted in previous observations (24, 33, 49). This finding contrasted with observations made after intradermal inoculations of TNF, which induced a murine polymorphonuclear leukocyte infiltrate (29). The human engrafted skin harbored dendritic cells that had characteristics of LCs, while the number of LCs slowly decreased with time and were no longer detectable at 3 to 4 mo postengraftment. Such kinetics are in agreement with the observations performed after allogeneic bone marrow transplantation in humans in which the skin APC of recipient origin disappear 3 mo post-bone marrow eradication and are replaced by LCs of donor origin (50).

Intradermal inoculation of a live vector recombinant for the HIV-LAIgp160 protein was followed by a typical inflammatory response combining a perivascular T cell infiltrate and activated CD1a+ LCs that displayed surface molecules involved in Ag presentation (CD80 and CD86). The source of such LC recruitment might be human PBL detectable in the murine peripheral blood of SCID-hu-PBL/skin mice. PBLs are known to contain dendritic cell progenitors and might therefore replenish the skin’s dendritic cell compartment. We can hypothesize, therefore, that the coengraftment of human skin and PBLs, together with exogenous rIL-2 addition, allowed appropriate conditions for differentiation and migration in engrafted skin of both human dendritic CD1a+ cells and human Ag-specific lymphocytes. However, the induction of immune responses is supposed to take place in draining lymph nodes rather than within the skin itself. In skin, T cells are found to be activated memory lymphocytes and are usually found in the dermal perivascular areas (25, 26). The capacity of human PBLs to migrate into murine lymphoid organs has been reported previously (51). Therefore, human skin CD1a+ dendritic cells might have migrated from the site of immunization to the draining lymph nodes where human T cells could be primed in the presence of exogenous rIL-2. Once primed, gp160-specific T cells would have migrated back to the skin at the site of Ag inoculation, mediating a classical inflammatory sequence of events. However, this sequence of migration would require a functional migration capacity of human lymphocytes in SCID chimeric mice. Alternatively, the data presented here could also suggest that a primary Ag-specific T cell activation might occur in the skin, as suggested by Fichtelius and coworkers in a classic but unconfirmed experiment (52). Similarly, Chen et al. have suggested that the costimulatory activation of human B and T cell is not dependent of the microenvironment of the germinal centers (53).

Whatever the precise mechanism involved, T cell infiltration and CD1a+ cell recruitment occurred within the human skin graft after intradermal injection of vCP-LAIgp160. One has to be surprised that no skin leukocyte infiltration was observed after the inoculation with wt-vCP. As previously observed, immunization with canary pox virus does not elicit canary pox CTLs in mice (54). The difference observed between the SCID-hu mice immunized with wt-vCP and those inoculated with vCP-LAIgp160 may be explained by the lack of exogenous rIL-2 in the mice inoculated with wt-vCP alone. Since SCID mice were grafted with skin from immunized with HIV-seronegative healthy individuals, development of HIV-1 gp160 CD4+ T cell lines reflects a primary immunization. The SIL have an MHC class II-restricted antigenic specificity against the HIV-1-LAI env protein, as shown by the reactivity of STL against HLA-matched EBV-transformed B cells. Both CD4+ and CD8+ cells were detectable in vCP-gp160-immunized skin grafts and in STL 3 wk after the initiation of the culture, but long term cell culture resulted in a predominance of human CD4+ T cell clones. This may be the result of a bias of long term culture, rather than a property of the Ag used. The cytolytic potential of the skin-derived CD4+ T cell clones observed in vitro is compatible with a Th1-type function suggested by cytokine mRNA profiles showing the predominance of IL-2, IFN-{gamma}, and IL-12 transcripts both in the skin and in STL. Previous studies reported the capacity to elicit in vitro HIV-1 gp160-specific cytolytic CD4+T cell clones from immunized donors (55). Although the in vivo relevance of such virus-specific CD4+ CTLs remains questionable, the Th1 differentiation reported herein might be of interest with regard to the association of HIV-specific CD4+ Th1 cells with control of HIV replication recently in long term nonprogressors (56).

Previous studies of SCID mice combining the engraftment of human skin and the injection of PBLs have shown the ability of human T cells to induce first-set rejection of allogeneic human skin (24, 49). Delayed hypersensitivity reactions were also obtained after intradermal injection of tetanus toxin following i.p. inoculation of human PBLs from preimmunized donors (33). In the latter report, no immune response was obtained in the SCID-hu-PBL/skin model when PBLs from nonimmunized donors were used, indicating the absence of a primary immune response. The difference between the two models previously reported and ours might consist in the route of reconstitution (i.v. vs i.p.), the Ag (a live vector vs a toxin), and the use of rIL-2. We clearly observed that the response was absent or weak without IL-2 supplementation, suggesting that the addition of IL-2 might have bypassed the requirement for an optimal Th cell functional status during the Ag-priming events. In other SCID mouse models, administration of human rIL-2 following human T cell engraftment (57) improved the homing and engraftment of PBLs from rheumatoid arthritis patients in SCID mice (58).

The SCID mouse model has already been used to evaluate vaccine strategies. SCID mice grafted with PBLs from gp160-vaccinated donors were shown to resist HIV infection (59). Adoptive transfers of a Nef-specific human CTL clone into SCID mice protected them from an HIV challenge, although nonspecifically (60). The induction of an antiviral CTL response is believed to be an important component of a protective anti-HIV vaccine (35, 61). The ability of a human-mouse chimeric model to generate CTLs in vivo would represent a further advancement toward establishing a model for evaluation of vaccine strategies in naive donors. This approach has been explored by Segall et al. (22), who succeeded in obtaining an anti-nef CTL response in SCID mice by immunization with a recombinant vaccinia-nef virus, within the first few weeks posttransplantation through conditioning of recipient mice by sublethal irradiation and using a large inoculum of human PBLs (80 x 106 per mouse). However, human T cell lines were not derived from the SCID-hu-PBL mice, rendering the demonstration of the MHC-restricted and nef-specific response incomplete. More recently, the obtainment of a secondary CTL response against influenza virus after injection into the SCID mice of human PBLs stimulated in vitro has been reported (62). However, in both reports, a T cell response was detected only during the first week after engraftment, before the antigenic repertoire of the human T cells deviated toward the xenoreactive clones (48).

The T cell line developed in vitro in the presence of IL-2 and feeder cells in the absence of gp160 proliferated poorly. Therefore, gp160 proliferation assays could be performed only once and appeared to be negative, while the specificity for gp160 was checked twice in standard chromium assays. These T cells could be expanded in vitro after IL-2 addition without Ag-specific restimulation, thus reflecting their in vivo activated status, and could not be considered to result from an in vitro priming.

Finally, immunoscope analysis showed that SIL present a very limited CDR3-length polymorphism, since only a few major peaks were observed for each BV family analyzed, while distributions of CDR3 lengths expressed by total PBLs before injection were found to present gaussian polyclonal patterns in all of the corresponding subsets. We could probe in vivo T cells, which were proved to be HIV specific in CTL assays according to their particular CDR3 size and BV family usage. The highly oligoclonal cutaneous T cell infiltrates suggest an Ag-specific repertoire rather than a nonspecific, inflammatory infiltrate. Indeed, these data are consistent with a CDR3-specific, and therefore Ag-specific, process driving the expansion of the T cells at the site of immunization, unless they reflect a low engraftment efficiency of CTL precursors, illustrating the limitations of the SCID model (8, 63). On the other hand, it seems unreasonable to expect that enough CTL would be generated in the skin to provide a protective response against a systemic HIV inoculation. Finally, anti-gp160 Abs were not detectable, suggesting the absence of a B primary immune response. But in fact, human T cells constituted the majority of the cells detectable in the SCID-hu mice (>95%) soon after the reconstitution (8), and B cells are frequently not detectable or oligoclonal (9).

Our report is the first to show the generation and further isolation and characterization of specific human T cell lines in the SCID-hu model. This study constitutes the first step in an expanding strategy. Efforts to validate this model with various Ags and by comparisons with humans have to be conducted. The results presented here underline the interest of this approach for testing vaccine strategies. Since many tumors or leukemias can also be transplanted into SCID mice, these could provide a system for generating antitumor T cells and for evaluating the efficacy of tumor immunizations.



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  		FIGURE 3. Determination of the presence of human T cells in the SCID-hu-PBL/skin mice. PBLs were isolated at the time of the sacrifice of the mice and were analyzed by three-color flow cytometry for the expression of human CD45, CD4, and CD8. The percentages of cells in each quarter are indicated. The data are representative of 16 mice.

 


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  		FIGURE 5. Identification of the HIV-1-LAI Env specificity of the different STL isolated from the DAR and VER mice. CTL reactivity of CD4+ or CD8+ STL from the mice against HLA-matched EBV-transformed B cell lines sharing HLA-DR14 for DAR and HLA-DR4 for VER preinfected with recombinant vaccinia virus expressing HIV-1-LAIgp160 protein ({blacksquare}) or the wt-vCP ({square}) as control (E:T ratio, 13:1).

 


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  		FIGURE 6. Recognition and presentation of the target cells expressing HIV-1-LAI Env gene product to HLA-DR14-restricted CTL from DAR-STL (HLA-A3-A11/B35-B18/C4-C5/BW6/DR14). CTLs derived from mice DAR immunized by vCP-LAIgp160+ IL-2 (DAR II-2) were tested against different HLA-matched EBV-transformed B cell lines compatible for HLA-A3, A11, B18, B35, or DR14. The different symbols indicate the HLA molecule shared by effector and target cells.

 


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  		FIGURE 7. RT-PCR determination of human cytokine expression in human engrafted skin of the DAR series and in the subsequently derived STLs. A, RT-PCR was performed on total RNA extracted from the human skin grafts of the five DAR SCID-hu-PBL/skin mice for each mouse with specific primers for the human cytokines IL-2, IL-4, IL-5, IL-6, IL-7, IFN-{gamma}, IL-10, and IL-12. Positive detection and corresponding controls are presented. IL-4 mRNA, lanes 1 and 8; IFN-{gamma}, lanes 2, 9, 13, and 14; IL-12 mRNA, lanes 3, 6, 12, 16, and 18; IL-2 mRNA, lanes 4, 10, 11, 15, and 17; IL-7 mRNA, lanes 5 and 7. B, Total RNA extracted from Vac-LAIgp160-stimulated CD4+ STL derived from the DAR II-2 mouse was analyzed. As controls, cell homogenates for RT-PCR were prepared from CD4+ STL stimulated using wt-Vac or anti-CD3 and anti-CD28 mAb. IL-2 mRNA, lanes 1, 4, and 10; IFN-{gamma} mRNA, lanes 2, 6, and 9; IL-12 mRNA, lanes 5 and 8; IL-4 mRNA, lanes 3 and 7.

 


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  		FIGURE 8. Distribution of CDR3 sizes of TCR ß-chains expressed by human PBLs and SIL. Immunoscope profiles corresponding to BV families overexpressed in the skin are presented in comparison with those obtained from autologous PBLs. Data are from mice DAR II-2. The corresponding BV-specific primer used is indicated in the window of analysis. The graphs represent the intensity of fluorescence in arbitrary units as a function of the CDR3 length, given in amino acids, of Vß-Cß ssDNA run-off products.

 

   Acknowledgments
 
We thank Dr. Bernard Meignier for providing us the recombinant canary pox virus, Dr. Ioannis Theodorou for HLA typing, and Christophe Parizot for technical assistance. We also thank Dr. Ray Pierce, Prof. Monique Capron, and Dr. Stanley Plotkin for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by the "Agence Nationale de Recherches sur le SIDA" and SIDACTION. Back

2 Address correspondence and reprint requests to Jean-Yves Cesbron, Unité INSERM U167, Institut Pasteur, Rue du Professeur A. Calmette, B.P. 245-59019 Lille, Cédex France. E-mail address: Back

3 Abbreviations used in this paper: SCID-hu-PBL, SCID mouse inoculated with human PBL; HES, hematoxylin-eosin saffron stain; LCs, Langerhans cells; PFU, plaque-forming unit; SIL, skin-infiltrating lymphocytes; STL, skin-infiltrating T cell lines; wt-Vac, vaccinia virus expressing the wild-type Copenhagen strain; Vac-LAIgp160, recombinant vaccinia virus expressing HIV-1 LAIgp160; vCP-LAIgp160, recombinant canary pox virus expressing HIV-1 LAIgp160; wt-vCP, wild-type canary pox virus; CDR, complementarity-determining region. Back

Received for publication December 19, 1997. Accepted for publication April 17, 1998.


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 Introduction
 Materials and Methods
 Results
 Discussion
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