Expression of Antigen on Mature Lymphocytes Is Required to Induce T Cell Tolerance by Gene Therapy

Chaorui Tian; Jessamyn Bagley; John Iacomini

doi:10.4049/jimmunol.169.7.3771

Abstract

Expression of a retrovirally encoded allogeneic MHC class I gene in bone marrow-derived cells can be used to induce tolerance to the product of the retrovirally transduced gene. In this work we examined whether expression of a retrovirally transduced allogeneic MHC class I gene in bone marrow-derived cells from recombinase-activating gene-1 (RAG-1)-deficient mice was sufficient to induce tolerance when transplanted into conditioned hosts together with bone marrow from MHC-matched wild-type mice. Reconstitution of mice with either MHC-matched RAG-1-deficient or wild-type bone marrow transduced with the allogeneic MHC class I gene H-2K^b led to long-term expression of K^b on the surface of bone marrow-derived hematopoietic lineages. T cells from mice reconstituted with H-2K^b-transduced wild-type bone marrow were tolerant to K^b. In contrast, expression of K^b in the periphery of mice reconstituted with a mixture of retrovirally transduced RAG-1-deficient bone marrow and mock-transduced wild-type bone marrow fell below detectable levels by 4 wk after transplantation. T cells that developed in these mice appeared to be hyporesponsive to K^b, demonstrating that expression of K^b on bone marrow-derived APCs was not sufficient to induce tolerance. Our data suggest that induction of tolerance in molecular chimeras requires expression of the retrovirally transduced allogeneic MHC Ag on the surface of mature lymphocytes that populate the host thymus.

Induction of tolerance to transplantation Ags remains a major goal in the field because of its potential to allow for replacement of host organs without the need for lifelong immunosuppression. It has been known for several years that tolerance to allogeneic transplantation Ags can be established through the induction of mixed cellular chimerism after allogeneic bone marrow transplantation (1). Tolerance induced by mixed cellular chimerism is specific and robust, and leaves the host fully immunocompetent (reviewed in Ref. 2). However, bone marrow transplantation across MHC barriers as a means of inducing tolerance to allogeneic organs is complicated by the severity of the preparative regimen required to allow for bone marrow engraftment and the potential for inducing graft-vs-host disease.

Building on the concept of mixed cellular chimerism, we have shown that tolerance can be induced using gene therapy by expressing retrovirally transduced allogeneic MHC genes in host bone marrow-derived cells (3, 4). Reconstitution of lethally irradiated mice with host bone marrow infected with retroviruses carrying the allogeneic MHC class I gene H-2K^b resulted in lifelong expression of K^b on the surface of multiple bone marrow-derived hematopoietic lineages, resulting in a state of molecular chimerism. T cells from molecular chimeras reconstituted with H-2K^b-transduced bone marrow were specifically tolerant to K^b. In addition, mice reconstituted with H-2K^b-transduced bone marrow exhibited long-term acceptance of K^b disparate skin allografts with no additional immunosuppression. Thus, molecular chimerism can induce stable tolerance. In addition, because the induction of molecular chimerism relies on the transfer of genes rather than immunocompetent cells, problems of graft-vs-host disease and engraftment failure associated with allogeneic bone marrow transplants should be avoidable, making this approach clinically attractive.

It has been suggested for a number of years that bone marrow-derived APCs that populate the thymus are perhaps most important for inducing tolerance to MHC Ags (5, 6). However, in mixed cellular chimeras it has been suggested that relatively high levels of donor type T cell chimerism correlates with maintenance of long-term donor-specific tolerance (7). Indeed, thymocyte precursors have been shown to be able to induce tolerance to MHC class I Ags (8), and MHC class I Ags expressed only in CD2⁺ cells induce CD8 T cell tolerance (9). Thus, T cells also have the potential to be tolerogenic. To define the requirements to induce tolerance after the induction of molecular chimerism, we set out to evaluate the ability of distinct hematopoietic cell lineages expressing a retrovirally transduced allogeneic MHC class I Ag to induce tolerance. Expression of H-2K^b in bone marrow-derived cells from recombinase-activating gene (RAG)³-1 mutant mice deficient in mature B and T cells (10) was not sufficient to induce tolerance to K^b when transplanted into conditioned recipients together with wild-type bone marrow. These data suggest that induction of tolerance in molecular chimeras requires expression of the retrovirally transduced allogeneic MHC Ag on the surface of mature lymphocytes.

Materials and Methods

Mice

B10.AKM/SnJ (H-2K^k, I^k, D^q) and B10.MBR (H-2K^b, I^k, D^q) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). RAG-1 mutant mice (10) on the 129/Sv background were crossed to B10.AKM/SnJ mice, and the resulting offspring were backcrossed to the B10.AKM/SnJ strain for six generations. At each generation, mice carrying the mutant RAG-1 allele were selected and used for backcrossing. At the sixth generation, mice were intercrossed to generate homozygous mutants deficient in B and T cells. Resulting RAG-1-deficient mice were intercrossed to generate a colony of RAG-1 mice on the B10.AKM H-2 haplotype. Mice were housed using microisolator conditions in autoclaved cages and maintained on irradiated feed and autoclaved acidified drinking water. All sentinel mice housed in the same colony were viral Ab free. Six- to 12-wk-old mice were used in all experiments.

Retroviruses

Construction and production of vesicular stomatitis virus (VSV)-Kb has been described previously (3). Briefly, the cDNA encoding H-2K^b (K^b) was cloned into the MMP retroviral vector kindly provided by Dr. R. C. Mulligan (Children’s Hospital, Boston, MA) to generate pMMP-K^b. The MMP retroviral vector is a derivative of MFG (11). VSV-G envelope protein pseudotyped viruses were produced by packaging the pMMP-K^b retroviral vector in 293T cells by transient transfection (as described in Refs. 12 and 13) to generate VSV-Kb virus. Functional titers of VSV-Kb retroviral supernatants were determined by analyzing expression of K^b on NIH-3T3 cells by cell surface staining and flow cytometry after infection. All virus preparations were made in affiliation with the Harvard Institute for Human Genetics Gene Therapy Initiative. The viral titer obtained for the preparation of VSV-Kb used in this report was ∼1–2 × 10⁶ infectious particles per milliliter.

Bone marrow harvest, transduction, and transplantation

Bone marrow cells were harvested and transduced as described previously (3, 14). Briefly, bone marrow cells from mice treated 7 days prior with 5-fluorouracil (150 mg/kg) were cultured in Retronectin (TaKaRa Biomedicals, Shiga, Japan)-coated tissue culture plates in transduction medium (DMEM containing 15% lot-tested FCS and cytokines to achieve a final concentration of 100 ng/ml human IL-6 (R&D Systems, Minneapolis, MN), 100 ng/ml recombinant mouse stem cell factor (BioSource International, Camarillo, CA), 50 ng/ml recombinant mouse thrombopoietin (R&D Systems), and 50 ng/ml recombinant mouse Flt-3 ligand (R&D Systems)). Bone marrow cells were cultured at a density of 6 × 10⁶ cells/ml and infected with VSV-Kb virus using a multiplicity of infection of at least 1. Viral supernatants and transduction medium was replaced at 24 and 72 h after the start of the transduction. Twenty-four hours after the last round of infection, bone marrow cells were harvested, washed twice in HBSS, and counted. Mock transductions were conducted in the same manner, except viral supernatants were replaced with transduction medium. Bone marrow cells were used to reconstitute conditioned recipients as described in the text.

Flow cytometry

All cell surface staining and flow cytometry were performed as described previously (3, 15). mAbs specific for CD4 (RM4-5), CD8 (53-6.7), CD3 (2C11), H-2K^b (AF6-88.5), Ly-6G (Gr-1, RB6-8C5), CD19 (1D3), CD11b (Mac-1, M1/70), NK cells (DX5), and CD11c (HL3) were obtained from BD PharMingen (San Diego, CA). Polyclonal rabbit anti-asialo-G_M1 Ab was purchased from Wako BioProducts (Richmond, VA). Expression of K^b on hematopoietic progenitors was performed as described (3). In all cases, mock-infected samples were used to set flow cytometry analysis gates.

PCR assay

DNA was purified from blood using a QIAamp DNA blood mini kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). RNA was prepared from blood cells using a RNeasy mini kit (Qiagen). Complementary DNA was prepared from RNA with oligo(dT) primers with the Superscript first-strand synthesis kit (Invitrogen, Carlsbad, CA). Primer sequences used are as follows: K^b forward primer, 5′-GCTGATCACCAAACACAAGTG-3′; K^b reverse primer, 5′-ATGGCGTTACTTAAGCTAGC-3′; β-actin forward primer, 5′-AACCCCAAGGCCAACCGCGAGAAGATGACC-3′; β-actin reverse primer, 5′-GGTGATGACCTGGCCGTCAGGCAGCTCGTA-3′; Y-chromosome forward primer, 5′-CTCCTGATGGACAAACTTTACG-3′; Y-chromosome reverse primer, 5′-TGAGTGCTGATGGGTGACGG-3′.

CTL assays

CTL assays were performed as described (3).

Results

Retroviral transduction of RAG-1 mutant bone marrow

To examine the ability of bone marrow-derived APCs to induce tolerance, we used RAG-1 mutant mice that were backcrossed to B10.AKM strain for six generations. RAG-1 mutant mice (R⁰) lack mature lymphocytes (10); therefore, by reconstituting conditioned hosts with a mixture of H-2K^b-transduced RAG-1 and mock-transduced wild-type B10.AKM bone marrow it is possible to directly assess the ability of bone marrow-derived APCs to induce tolerance to K^b. R⁰ bone marrow was harvested from mice treated 7 days prior with 150 mg/kg 5-fluorouracil and transduced with VSV-G protein enveloped retroviruses carrying the gene encoding H-2K^b, hereafter referred to as VSV-Kb, as described (3). Immediately following transduction with VSV-Kb, ∼21% of R⁰ bone marrow cells expressed K^b on their cell surface at levels readily detectable by cell surface staining and flow cytometry (Fig. 1⇓). 5-Fluorouracil-treated R⁰ bone marrow was transduced at the same efficiency observed for wild-type B10.AKM bone marrow (24%) (Fig. 1⇓). Analysis of Sca-1⁺, lineage marker negative (Sca-1⁺Lin⁻) hematopoietic progenitors (16) from R⁰ mice and wild-type B10.AKM controls after transduction revealed that hematopoietic progenitors from both strains were transduced at similar efficiencies with VSV-Kb, resulting in expression of K^b on the surface of ∼20–30% of early hematopoietic progenitors (Fig. 2⇓).

FIGURE 1.

Analysis of bone marrow transduction efficiency. Expression of K^b on the surface of VSV-Kb-transduced B10.AKM (A) and R⁰ bone marrow (B) before bone marrow transplantation. The percentage of cells expressing K^b from representative experiments is shown. A total of three independent experiments were performed. In each experiment bone marrow was pooled from either 15 B10.AKM or 20 R⁰ donor mice, transduced, and analyzed by cell surface staining and flow cytometry.

FIGURE 2.

Expression of K^b on the surface of bone marrow hematopoietic progenitor cells. Shown is expression of K^b on lineage marker-negative, Sca-1⁺ R⁰ (A) or B10.AKM (B) bone marrow cells following transduction. Shown is expression of K^b on VSV-Kb (solid line) and mock-transduced (dashed line) lineage marker-negative, Sca-1⁺ cells after transduction. Lineage marker-negative, Sca-1⁺ cells comprise ∼3% of total bone marrow after transduction. The percentage of early progenitors expressing K^b in representative experiments is shown. A total of three independent experiments were performed as described in Fig. 1⇑.

Loss of molecular chimerism in mice reconstituted with a mixture of VSV-Kb-transduced R⁰ and wild-type bone marrow

To examine the ability of APCs to induce tolerance, R⁰ mice were treated with 6-Gy whole-body irradiation, as well as a depleting dose of anti-CD4 (GK1.5; Ref. 17), CD8 (2.43; Ref. 18) and anti-asialo-G_M1 Abs, and reconstituted the following day with 6 × 10⁶ mock-transduced wild-type B10.AKM, 6 × 10⁶ VSV-Kb-transduced R⁰ bone marrow, or a mixture of 6 × 10⁶ mock-transduced B10.AKM and 6 × 10⁶ VSV-Kb-transduced R⁰ bone marrow cells. Hosts were treated with anti-CD4 and CD8 to deplete any residual mature T cells from the B10.AKM donor bone marrow inoculum in vivo. Anti-asialo-G_M1 was used to deplete NK cells from the host. Bone marrow-derived cells expressing K^b on their surface were detectable in the blood of R⁰ mice reconstituted with VSV-Kb-transduced R⁰ bone marrow by cell surface staining and flow cytometry at all time points analyzed over a 22-wk follow-up period (data not shown). As expected, bone marrow-derived cells expressing K^b were not detected in the blood of R⁰ mice reconstituted with mock-transduced bone marrow (data not shown). Bone marrow-derived cells expressing K^b were present in the blood of R⁰ mice reconstituted with a mixture of mock-transduced B10.AKM and VSV-Kb-transduced R⁰ bone marrow cells early after reconstitution; however, the frequency of cells expressing K^b in these mice fell to levels undetectable by cell surface staining and flow cytometry by 4 wk after bone marrow transplantation (Fig. 3⇓).

FIGURE 3.

Expression of K^b on the surface of blood cells from R⁰ mice reconstituted with VSV-Kb-transduced R⁰ bone marrow (A and C) or a mixture of VSV-Kb-transduced R⁰ and wild-type B10.AKM bone marrow (B and D) at 3 (A and B) and 4 (C and D) wk after reconstitution. The percentage of cells expressing K^b is shown. The data are from representative mice in a single experiment. A total of three independent experiments were performed containing five to six mice per group in each experiment.

Mice reconstituted with a mixture of mock-transduced wild-type B10.AKM and VSV-Kb-transduced R⁰ bone marrow contain low levels of cells derived from transduced progenitors

RT-PCR was used to further analyze engraftment of retrovirally transduced cells in reconstituted mice. R⁰ mice reconstituted with VSV-Kb-transduced R⁰ bone marrow as well as R⁰ mice reconstituted with a mixture of mock-transduced wild-type B10.AKM and VSV-Kb-transduced R⁰ bone marrow contained in their blood cells expressing retrovirally encoded K^b for at least 20 wk after transplantation, detectable by RT-PCR using K^b specific primers (Fig. 4⇓). Expression of retrovirally encoded K^b was not detected in control R⁰ mice reconstituted with mock-transduced wild-type B10.AKM bone marrow (Fig. 4⇓). Insofar as expression of K^b on bone marrow-derived cells in mice reconstituted with a mixture of mock-transduced B10.AKM and VSV-Kb-transduced R⁰ bone marrow cells was undetectable by cell surface staining and flow cytometry, these data suggest that a relatively low frequency of cells expressing the retrovirally transduced gene were present in these mice.

FIGURE 4.

A low frequency of cells expressing the retrovirally transduced H-2K^b gene is present in the blood of mice reconstituted with a mixture of VSV-Kb-transduced R⁰ and mock-transduced B10.AKM bone marrow. Shown is analysis of RT-PCR products using K^b- or β-actin-specific primers resolved on a 2% agarose gel stained with ethidium bromide. Shown is amplification of cDNA purified from blood cells of R⁰ mice reconstituted with mock-transduced B10.AKM bone marrow (lanes 1 and 2), VSV-Kb-transduced R⁰ bone marrow (lanes 3–5), or a mixture of VSV-Kb-transduced R⁰ and mock-transduced wild-type bone marrow (lanes 6–8). The data are representative of three independent experiments. In each experiment seven to eight mice were analyzed.

Because R⁰ mice have an abnormal thymic architecture resulting from an absence of T cells, we next examined engraftment of transduced R⁰ bone marrow cells in wild-type B10.AKM recipients. Wild-type B10.AKM female mice were lethally irradiated (10.25 Gy) and treated with a depleting dose of anti-CD4 and CD8 mAbs. The following day, the mice were reconstituted with 6 × 10⁶ mock-transduced B10.AKM female; 6 × 10⁶ VSV-Kb-transduced B10.AKM female; 6 × 10⁶ VSV-Kb-transduced R⁰ female; or a mixture of 6 × 10⁶ mock-transduced B10.AKM female and 6 × 10⁶ VSV-Kb-transduced R⁰ male bone marrow. Bone marrow-derived cells expressing K^b were readily detected in the blood of B10.AKM mice reconstituted with either VSV-Kb-transduced B10.AKM female or VSV-Kb-transduced R⁰ female bone marrow by cell surface staining and flow cytometry (Fig. 5⇓). However, as observed for R⁰ mice reconstituted with a mixture of mock-transduced B10.AKM and VSV-Kb-transduced R⁰ bone marrow, expression of K^b on bone marrow-derived cells fell to below our limits of detection by 4 wk after transplantation of B10.AKM mice with a mixture of mock-transduced B10.AKM female and VSV-Kb-transduced R⁰ male bone marrow (Fig. 5⇓).

FIGURE 5.

Long-term expression of K^b on the surface of blood cells in B10.AKM mice reconstituted with VSV-Kb-transduced R⁰ bone marrow (▪), VSV-Kb-transduced B10.AKM bone marrow (▴), a mixture of VSV-Kb-transduced R⁰ and mock-transduced B10.AKM bone marrow (×), or mock-transduced B10.AKM bone marrow (). Shown are the mean values obtained for between five and six mice per time point. The data are representative of three independent experiments.

Although we were unable to detect K^b-expressing cells in female B10.AKM mice reconstituted with a mixture of mock-transduced B10.AKM female and VSV-Kb-transduced R⁰ male bone marrow 4 wk after transplantation by cell surface staining and flow cytometry, mice in this group contained in their blood male R⁰ bone marrow cells detectable by DNA PCR using Y chromosome-specific primers at this time point (Fig. 6⇓). Similar results were observed at 12 wk after bone marrow transplantation (data not shown). These data suggest that R⁰ bone marrow cells were able to engraft even though the frequency of cells expressing K^b rapidly declined by 4 wk after transplantation. Therefore, the inability to detect K^b-expressing cells is not due to simply a failure of R⁰ bone marrow to engraft.

FIGURE 6.

Engraftment of male R⁰-derived cells in the blood of mice reconstituted with a mixture of VSV-Kb-transduced R⁰ and mock-transduced B10.AKM bone marrow. Shown is analysis of PCR products using Y chromosome- or β-actin-specific primers resolved on a 2% agarose gel stained with ethidium bromide. Shown is amplification of DNA purified from blood cells of female B10.AKM mice reconstituted with mock-transduced female B10.AKM bone marrow (lanes 1–3), VSV-Kb-transduced female R⁰ bone marrow (lanes 4–6), VSV-Kb-transduced female B10.AKM bone marrow (lanes 7 and 8), or a mixture of VSV-Kb-transduced male R⁰ and mock-transduced female B10.AKM bone marrow (lanes 9–11). The data are representative of three independent experiments. In each experiment four to eight mice were analyzed.

T cells from mice reconstituted with a mixture of VSV-Kb-transduced R⁰ and wild-type bone marrow remain responsive to K^b

To examine whether expression of K^b on bone marrow-derived APCs was sufficient to induce tolerance, we assessed the ability of splenocytes from R⁰ mice reconstituted with mock-transduced wild-type B10.AKM; VSV-Kb-transduced R⁰ bone marrow; or a mixture of mock-transduced B10.AKM and VSV-Kb-transduced R⁰ bone marrow cells to kill K^b-expressing target cells 20 wk after bone marrow transplantation. Similarly, we analyzed the ability of splenocytes from B10.AKM mice reconstituted with mock-transduced B10.AKM, VSV-Kb-transduced B10.AKM, VSV-Kb-transduced R⁰, or a mixture of mock-transduced B10.AKM and VSV-Kb-transduced R⁰ bone marrow to lyse K^b-bearing targets 8 wk after bone marrow transplantation. Mice in each group were immunized after bone marrow transplantation with irradiated B10.MBR splenocytes and sacrificed 9 days later, and splenocytes were harvested. Splenocytes were then restimulated in vitro in the presence or absence of 10 U/ml IL-2 for 5 days with irradiated TBA-K^b cells, an Abelson virus-transformed B10.AKM pre-B cell line expressing K^b (3), and CTL assays were performed.

Splenocytes from either R⁰ or B10.AKM mice reconstituted with mock-transduced wild-type B10.AKM bone marrow were able to lyse K^b-expressing targets (Fig. 7⇓). Addition of IL-2 to these cultures led to increased killing of targets expressing K^b (Fig. 7⇓). In contrast, splenocytes from either R⁰ or B10.AKM mice reconstituted with a mixture of mock-transduced wild-type B10.AKM and VSV-Kb-transduced R⁰ bone marrow lysed targets expressing K^b only when IL-2 was provided during in vitro restimulation (Fig. 7⇓). As expected, splenocytes from either control R⁰ or B10.AKM mice reconstituted with VSV-Kb-transduced R⁰ bone marrow were unable to lyse K^b-expressing targets (Fig. 7⇓). In contrast, B10.AKM mice reconstituted with VSV-Kb-transduced B10.AKM bone marrow cells were unable to lyse targets expressing K^b even when IL-2 was added during in vitro restimulation (Fig. 7⇓), consistent with our previous observation that expression of K^b on wild-type bone marrow-derived cells results in tolerance (3). Together, these data suggest that mice reconstituted with a mixture of mock-transduced wild-type B10.AKM and VSV-Kb-transduced R⁰ bone marrow are hyporesponsive rather than tolerant to K^b.

FIGURE 7.

Mice reconstituted with a mixture of VSV-Kb-transduced R⁰ and mock-transduced B10.AKM bone marrow are hyporesponsive rather than tolerant to K^b. Analysis of the ability of splenocytes from R⁰ mice reconstituted with mock-transduced B10.AKM (♦), or a mixture of VSV-Kb-transduced R⁰ and mock-transduced B10.AKM bone marrow (▴) to kill K^b-expressing targets in the absence (A) or presence (B) of exogenous IL-2. Splenocytes from R⁰ mice reconstituted with VSV-Kb-transduced R⁰ bone marrow were unable to kill targets expressing K^b as expected (▪). Shown is an analysis of the ability of splenocytes from B10.AKM mice reconstituted with mock-transduced B10.AKM (♦), VSV-Kb-transduced B10.AKM (×), or a mixture of VSV-Kb-transduced R⁰ and mock-transduced B10.AKM bone marrow (▴) in the absence (C) or presence (D) of exogenous IL-2. Splenocytes from B10.AKM mice reconstituted with VSV-Kb-transduced R⁰ bone marrow were unable to kill targets expressing K^b as expected (▪). In all experiments, the effector cells were normalized based on the total number of splenocytes. Data shown are representative of three independent experiments in which two to three mice per group were analyzed.

Analysis of hematopoietic lineages expressing K^b in the thymus of reconstituted mice

To examine why R⁰ bone marrow failed to induce tolerance, we analyzed expression of K^b in the thymus of wild-type B10.AKM recipients reconstituted with VSV-Kb-transduced R⁰ bone marrow, VSV-Kb-transduced B10.AKM bone marrow, or a mixture of VSV-Kb-transduced R⁰ and mock-transduced B10.AKM bone marrow. B10.AKM mice reconstituted with VSV-Kb-transduced B10.AKM bone marrow contained a significant number of cells expressing K^b in their thymuses (Fig. 8⇓). While macrophages and NK cells expressing K^b were detected in the thymus of these mice, the majority of cells expressing K^b were CD3⁺ thymocytes. The frequency of K^b-expressing cells in the thymus of B10.AKM mice reconstituted with VSV-Kb-transduced B10.AKM bone marrow was similar to the overall frequency of cells expressing K^b in the blood (Fig. 5⇑). In contrast, B10.AKM mice reconstituted with VSV-Kb-transduced R⁰ bone marrow contained relatively fewer K^b-expressing cells in their thymus (Fig. 8⇓). Expression of K^b was detected on NK cells and macrophages in the thymuses of these mice. The frequency of K^b-expressing cells in the thymus of B10.AKM mice reconstituted with VSV-Kb-transduced R⁰ bone marrow was significantly lower than the frequency observed in the blood (Fig. 5⇑). We were unable to detect cells expressing K^b in the thymus of B10.AKM mice reconstituted with a mixture of VSV-Kb-transduced R⁰ and mock-transduced B10.AKM bone marrow (Fig. 8⇓).

FIGURE 8.

Expression of K^b in the thymus of B10.AKM mice reconstituted with VSV-Kb-transduced R⁰ (upper panels), VSV-Kb-transduced B10.AKM (middle panels), or a mixture of VSV-Kb-transduced R⁰ and mock-transduced B10.AKM bone marrow (lower panels). Thymuses were analyzed ∼3 wk after bone marrow transplantation. Similar data were observed at 8 wk. Shown is expression of K^b on CD3⁺ cells (left panels), DX5⁺ NK cells (middle panels), and Mac-1-positive cells (right panels). Data shown are from representative mice from two independent experiments. Two to three mice per group were analyzed in each experiment.

Discussion

In this study we evaluated the ability of distinct hematopoietic cell lineages to induce tolerance after the induction of molecular chimerism. While expression of the retrovirally transduced H-2K^b gene in B10.AKM bone marrow-derived cells was sufficient to induce tolerance to K^b, expression of K^b on R⁰ bone marrow-derived cells induced hyporesponsiveness rather than tolerance to K^b. Analysis of reconstituted mice revealed that macrophages and NK cells expressing K^b were detected in the thymus of B10.AKM mice reconstituted with either VSV-Kb-transduced R⁰ or B10.AKM bone marrow. However, the ability to induce tolerance to K^b correlated with the presence of T cells expressing K^b in the thymus of B10.AKM mice reconstituted with VSV-Kb-transduced B10.AKM bone marrow. While we cannot rule out the possibility that B cells contributed to the induction of tolerance in these mice, we favor the possibility that T cells rather than B cells were required because we could not reliably detect B cells expressing K^b in the thymus of tolerant mice (data not shown). Furthermore, we do not think that T or B cells expressing K^b in peripheral tissues were required to induce tolerance, because we have recently shown that induction of CD8 T cell tolerance induced by gene therapy occurs by a central deletional mechanism in the thymus (19).

Our data suggest that expression of the retrovirally transduced MHC class I gene in all bone marrow-derived lineages except mature B and T cells is not sufficient to induce robust tolerance. Therefore, either expression of K^b on APCs alone is insufficient to induce tolerance or an insufficient number of APCs expressing K^b may have led to hyporesponsiveness rather than tolerance. Mice reconstituted with a mixture of VSV-Kb-transduced R⁰ and wild-type B10.AKM bone marrow contained only low levels of hematopoietic cells derived from transduced progenitors by 4 wk after transplantation. It is unlikely that inefficient transduction of early hematopoietic progenitors in R⁰ bone marrow led to a decline in K^b expression in mice reconstituted with a mixture of VSV-Kb-transduced R⁰ and mock-transduced B10.AKM bone marrow because hematopoietic progenitors from R⁰ mice were transduced at a frequency similar to that observed for wild-type bone marrow. In addition, R⁰ and wild-type mice reconstituted with VSV-Kb-transduced R⁰ bone marrow exhibited expression of K^b on bone marrow-derived cells long term, suggesting that early progenitors were transduced. We suggest that cells expressing K^b in mice reconstituted with a mixture of VSV-Kb-transduced R⁰ and wild-type B10.AKM bone marrow may have undergone rejection as they developed. The persistent low level of K^b expression detected by RT-PCR may reflect engraftment of H-2K^b-transduced hematopoietic stem cells, which have been suggested to be immunoprivileged (20). Therefore, it is possible that the transduced stem cells persist while progeny expressing K^b are actively rejected. Consistent with this idea, cells expressing K^b were detectable in mice receiving a mixture of VSV-Kb-transduced R⁰ and wild-type B10.AKM bone marrow only at relatively early time points after bone marrow transplantation, before T cell recovery took place (data not shown). Hyporesponsiveness may result from activation-induced cell death of K^b-reactive T cells. It is also possible that low levels of K^b expressed on resting APCs induced anergy rather than deletional tolerance, which could be overcome by providing exogenous IL-2.

It has previously been observed that relatively inefficient transduction and expression of H-2K^b in bone marrow-derived cells results in hyporesponsiveness to K^b, which can be overcome by providing T cell help (21, 22). Interestingly, Fraser et al. (21) were able to observe expression of K^b on the surface of macrophages and B220⁺ cells; however, K^b expression was not detected on CD4 or CD8 T cells in the majority of mice reconstituted with H-2K^b-transduced bone marrow. We suggest that the inability to achieve tolerance in that study may have been related to inefficient expression of K^b on T cells, or restricted expression in hematopoietic cell lineages such as myeloid cells, which we show here are unable to induce tolerance.

It has been suggested that bone marrow-derived APCs are critical mediators of negative selection in the thymus (5, 6). Yet is has been shown, in mixed cellular chimeras, that a relatively high level of donor type T cell chimerism correlates with maintenance of long-term donor-specific tolerance (7), and it has been shown that thymocyte precursors can induce tolerance to MHC class I Ags (8). In mice reconstituted with VSV-Kb-transduced R⁰ bone marrow, relatively few cells expressing K^b were present in the thymus of reconstituted mice. In contrast, the frequency of cells expressing K^b was significantly higher in the thymus of mice reconstituted with VSV-Kb-transduced wild-type bone marrow. We suggest that the requirement for T cells to induce tolerance may be related to the ability of T lineage cells to deliver Ag to the thymus. Because T lineage cells comprise ∼85% of the hematopoietic cells in the thymus, expression of K^b on T cell precursors or mature single positive T cells may be required to achieve a threshold level of K^b expression in the thymus to induce negative selection of K^b-reactive T cells.

It has recently been described that reconstitution of non-myeloablated recipients with CD4 and CD8 double knockout bone marrow can result in state of mixed host and donor hematopoietic chimerism without inducing tolerance (23). In contrast, we did not observe a dissociation of chimerism and tolerance. K^b was not expressed at significant levels in the periphery of nontolerant mice. Although the results of Umemura et al. (23) are consistent with the notion that mature T cells are required to induce tolerance, in that study it is possible that transplant rejection was related to minor Ag disparities between the knockouts used as bone marrow donors and the skin graft donors. Nevertheless, insofar as tolerance was not achieved in mixed chimeras generated using bone marrow from CD4 and CD8 double knockout mice, we suggest that the failure to induce tolerance may have been related to low levels of alloantigen expressed in the thymus resulting from a lack of bone marrow-derived T cells.

We speculate that mature lymphocytes expressing K^b are required to bring the retrovirally transduced alloantigen to the thymus, establishing a critical threshold of expression that must be achieved to establish negative selection of alloreactive T cells. T lineage cells may be well suited for this purpose because they comprise the vast majority of cells in the thymus, and therefore may have the greatest potential for delivering Ag. We are currently examining this possibility. Defining the minimum expression level required to induce and maintain tolerance after the induction of molecular chimerism could be used to predict whether stable tolerance will be established. Insofar as our results demonstrate that efficient trafficking of cells expressing K^b to the thymus is required to induce tolerance, we suggest that strategies designed to enhance trafficking of Ag to the thymus may facilitate tolerance induction. In addition, we suggest that, in addition to genetic engineering of bone marrow, it may be possible to induce tolerance to transplantation Ags by treating transiently immunosuppressed hosts with autologous peripheral lymphoid cells expressing allogeneic MHC genes introduced using viral delivery vehicles.

Acknowledgments

We thank Drs. Richard C. Mulligan and Jeng-Shin Lee for providing the MMP retroviral vector, packaging system, and technical advice as well as the Harvard Gene Therapy Initiative Vector Core, in part supported by the Association Français contre les Myopathies, for production of viral vectors. In addition, we thank Drs. David H. Sachs and Joren Madsen for critical review of the manuscript and members of the Iacomini laboratory for helpful discussions. We also thank Lan Wang for technical assistance and Jessica Lynch for expert secretarial assistance.

Footnotes

↵1 This work was supported by Grant RO1 AI43619 from the National Institutes of Health (to J.I.). J.B. was supported in part by National Institutes of Health Training Grant T32 AI07529 and a grant from the Children’s A-T Project.
↵2 Address correspondence and reprint requests to Dr. John Iacomini, Transplantation Biology Research Center, Massachusetts General Hospital East, Building 149, 13th Street, Boston, MA 02129. E-mail address: iacomini{at}helix.mgh.harvard.edu
↵3 Abbreviations used in this paper: RAG, recombinase-activating gene; VSV, vesicular stomatitis virus.

Received May 14, 2002.
Accepted August 7, 2002.

References

↵
Ildstad, S. T., D. H. Sachs. 1984. Reconstitution with syngeneic plus allogeneic or xenogeneic bone marrow leads to specific acceptance of allografts or xenografts. Nature 307: 168
OpenUrl CrossRef PubMed
↵
Sykes, M.. 2001. Mixed chimerism and transplant tolerance. Immunity 14: 417
OpenUrl CrossRef PubMed
↵
Bagley, J., C. Tian, D. H. Sachs, J. Iacomini. 2002. Induction of T cell tolerance to a MHC class I alloantigen by gene therapy. Blood 99: 4394
OpenUrl Abstract/FREE Full Text
↵
Bagley, J., J. Bracy, C. Tian, E.-S. Kang, J. Iacomini. 2002. Establishing immunological tolerance through the induction of molecular chimerism. Front. Biosci. 7: 1331
OpenUrl CrossRef
↵
Jenkinson, E. J., P. Jhittay, R. Kingston, J. J. Owen. 1985. Studies of the role of the thymic environment in the induction of tolerance to MHC antigens. Transplantation 39: 331
OpenUrl PubMed
↵
Charlton, B., H. Auchincloss, Jr, C. G. Fathman. 1994. Mechanisms of transplantation tolerance. Annu. Rev. Immunol. 12: 707
OpenUrl CrossRef PubMed
↵
Tomita, Y., D. H. Sachs, A. Khan, M. Sykes. 1996. Additional monoclonal antibody (mAB) injections can replace thymic irradiation to allow induction of mixed chimerism and tolerance in mice receiving bone marrow transplantation after conditioning with anti-T cell mABs and 3-Gy whole body irradiation. Transplantation 61: 469
OpenUrl CrossRef PubMed
↵
Shimonkevitz, R. P., M. J. Bevan. 1988. Split tolerance induced by the intrathymic adoptive transfer of thymocyte stem cells. J. Exp. Med. 168: 143
OpenUrl Abstract/FREE Full Text
↵
Schulz, R., A. L. Mellor. 1996. Self major histocompatibility complex class I antigens expressed solely in lymphoid cells do not induce tolerance in the CD4⁺ T cell compartment. J. Exp. Med. 184: 1573
OpenUrl Abstract/FREE Full Text
↵
Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa, V. E. Papaioannou. 1992. RAG-1 deficient mice have no mature B and T lymphocytes. Cell 68: 869
OpenUrl CrossRef PubMed
↵
Riviere, I., K. Brose, R. C. Mulligan. 1998. Effects of retroviral vector design on expression of human adenosine deaminase in murine marrow transplant recipients engrafted with genetically modified cells. Proc. Natl. Acad. Sci. USA 92: 6733
OpenUrl Abstract/FREE Full Text
↵
Finer, M. H., T. J. Dull, L. Qin, D. Farson, M. R. Roberts. 1994. kat: a high-efficiency retroviral transduction system for primary human T lymphocytes. Blood 83: 43
OpenUrl Abstract/FREE Full Text
↵
Ory, D. S., B. A. Neugeboren, R. C. Mulligan. 1996. A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc. Natl. Acad. Sci. USA 93: 11400
OpenUrl Abstract/FREE Full Text
↵
Bracy, J. L., J. Iacomini. 2000. Induction of B cell tolerance by retroviral gene therapy. Blood 96: 3008
OpenUrl Abstract/FREE Full Text
↵
Sawada, T., Y. Wu, D. H. Sachs, J. Iacomini. 1997. CD4⁺ T cells are able to reject class I disparate allografts. Transplantation 64: 335
OpenUrl CrossRef PubMed
↵
Aguila, H. L., K. Akashi, J. Domen, K. L. Gandy, E. Lagasse, R. E. Mebius, S. J. Morrison, J. Shizuru, S. Strober, N. Uchida, et al 1997. From stem cells to lymphocytes: biology and transplantation. Immunol. Rev. 157: 13
OpenUrl CrossRef PubMed
↵
Dialynas, D. P., Z. S. Quan, K. A. Wall, A. Pierres, J. Quintans, M. R. Loken, M. Pierres, F. W. Fitch. 1984. Characterization of the murine T cell surface molecule designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu 3/T4 molecule. J. Immunol. 131: 2445
OpenUrl Abstract/FREE Full Text
↵
Sarmiento, M., A. L. Glasebrook, F. W. Fitch. 1980. IgG or IgM monoclonal antibodies reactive with different determinants on the molecular complex bearing Lyt2 antigen block T cell-mediated cytolysis in the absence of complement. J. Immunol. 125: 2665
OpenUrl Abstract
↵
Kang, E.-S., J. Iacomini. 2002. Induction of central deletional T cell tolerance by gene therapy. J. Immunol. 169: 1930
OpenUrl Abstract/FREE Full Text
↵
Fandrich, F., X. Lin, G. X. Chai, M. Schulze, D. Ganten, M. Bader, J. Holle, D. S. Huang, R. Parwaresch, N. Zavazava, B. Binas. 2002. Preimplantation-stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning. Nat. Med. 8: 171
OpenUrl CrossRef PubMed
↵
Fraser, C. C., M. Sykes, R. Stanton-Lee, D. H. Sachs, C. LeGuern. 1995. Specific unresponsiveness to a retrovirally transferred class I antigen is controlled through the helper pathway. J. Immunol. 154: 1587
OpenUrl Abstract
↵
Stanton-Mayfield, R., H. Hayashi, T. Sawada, K. Bergen, C. LeGuern, M. Sykes, D. H. Sachs, J. Iacomini. 1997. The mechanism of specific prolongation of class I mismatched skin grafts induced by retroviral gene therapy. Eur. J. Immunol. 27: 1177
OpenUrl CrossRef PubMed
↵
Umemura, A., H. Morita, X. C. Li, S. Tahan, A. P. Monaco, T. Maki. 2001. Dissociation of hemopoietic chimerism and allograft tolerance after allogeneic bone marrow transplantation. J. Immunol. 167: 3043
OpenUrl Abstract/FREE Full Text

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Show more CELLULAR IMMUNOLOGY AND IMMUNE REGULATION

[1] ↵
Ildstad, S. T., D. H. Sachs. 1984. Reconstitution with syngeneic plus allogeneic or xenogeneic bone marrow leads to specific acceptance of allografts or xenografts. Nature 307: 168
OpenUrl CrossRef PubMed

[2] ↵
Sykes, M.. 2001. Mixed chimerism and transplant tolerance. Immunity 14: 417
OpenUrl CrossRef PubMed

[3] ↵
Bagley, J., C. Tian, D. H. Sachs, J. Iacomini. 2002. Induction of T cell tolerance to a MHC class I alloantigen by gene therapy. Blood 99: 4394
OpenUrl Abstract/FREE Full Text

[4] ↵
Bagley, J., J. Bracy, C. Tian, E.-S. Kang, J. Iacomini. 2002. Establishing immunological tolerance through the induction of molecular chimerism. Front. Biosci. 7: 1331
OpenUrl CrossRef

[5] ↵
Jenkinson, E. J., P. Jhittay, R. Kingston, J. J. Owen. 1985. Studies of the role of the thymic environment in the induction of tolerance to MHC antigens. Transplantation 39: 331
OpenUrl PubMed

[6] ↵
Charlton, B., H. Auchincloss, Jr, C. G. Fathman. 1994. Mechanisms of transplantation tolerance. Annu. Rev. Immunol. 12: 707
OpenUrl CrossRef PubMed

[7] ↵
Tomita, Y., D. H. Sachs, A. Khan, M. Sykes. 1996. Additional monoclonal antibody (mAB) injections can replace thymic irradiation to allow induction of mixed chimerism and tolerance in mice receiving bone marrow transplantation after conditioning with anti-T cell mABs and 3-Gy whole body irradiation. Transplantation 61: 469
OpenUrl CrossRef PubMed

[8] ↵
Shimonkevitz, R. P., M. J. Bevan. 1988. Split tolerance induced by the intrathymic adoptive transfer of thymocyte stem cells. J. Exp. Med. 168: 143
OpenUrl Abstract/FREE Full Text

[9] ↵
Schulz, R., A. L. Mellor. 1996. Self major histocompatibility complex class I antigens expressed solely in lymphoid cells do not induce tolerance in the CD4⁺ T cell compartment. J. Exp. Med. 184: 1573
OpenUrl Abstract/FREE Full Text

[10] ↵
Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa, V. E. Papaioannou. 1992. RAG-1 deficient mice have no mature B and T lymphocytes. Cell 68: 869
OpenUrl CrossRef PubMed

[11] ↵
Riviere, I., K. Brose, R. C. Mulligan. 1998. Effects of retroviral vector design on expression of human adenosine deaminase in murine marrow transplant recipients engrafted with genetically modified cells. Proc. Natl. Acad. Sci. USA 92: 6733
OpenUrl Abstract/FREE Full Text

[12] ↵
Finer, M. H., T. J. Dull, L. Qin, D. Farson, M. R. Roberts. 1994. kat: a high-efficiency retroviral transduction system for primary human T lymphocytes. Blood 83: 43
OpenUrl Abstract/FREE Full Text

[13] ↵
Ory, D. S., B. A. Neugeboren, R. C. Mulligan. 1996. A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc. Natl. Acad. Sci. USA 93: 11400
OpenUrl Abstract/FREE Full Text

[14] ↵
Bracy, J. L., J. Iacomini. 2000. Induction of B cell tolerance by retroviral gene therapy. Blood 96: 3008
OpenUrl Abstract/FREE Full Text

[15] ↵
Sawada, T., Y. Wu, D. H. Sachs, J. Iacomini. 1997. CD4⁺ T cells are able to reject class I disparate allografts. Transplantation 64: 335
OpenUrl CrossRef PubMed

[16] ↵
Aguila, H. L., K. Akashi, J. Domen, K. L. Gandy, E. Lagasse, R. E. Mebius, S. J. Morrison, J. Shizuru, S. Strober, N. Uchida, et al 1997. From stem cells to lymphocytes: biology and transplantation. Immunol. Rev. 157: 13
OpenUrl CrossRef PubMed

[17] ↵
Dialynas, D. P., Z. S. Quan, K. A. Wall, A. Pierres, J. Quintans, M. R. Loken, M. Pierres, F. W. Fitch. 1984. Characterization of the murine T cell surface molecule designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu 3/T4 molecule. J. Immunol. 131: 2445
OpenUrl Abstract/FREE Full Text

[18] ↵
Sarmiento, M., A. L. Glasebrook, F. W. Fitch. 1980. IgG or IgM monoclonal antibodies reactive with different determinants on the molecular complex bearing Lyt2 antigen block T cell-mediated cytolysis in the absence of complement. J. Immunol. 125: 2665
OpenUrl Abstract

[19] ↵
Kang, E.-S., J. Iacomini. 2002. Induction of central deletional T cell tolerance by gene therapy. J. Immunol. 169: 1930
OpenUrl Abstract/FREE Full Text

[20] ↵
Fandrich, F., X. Lin, G. X. Chai, M. Schulze, D. Ganten, M. Bader, J. Holle, D. S. Huang, R. Parwaresch, N. Zavazava, B. Binas. 2002. Preimplantation-stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning. Nat. Med. 8: 171
OpenUrl CrossRef PubMed

[21] ↵
Fraser, C. C., M. Sykes, R. Stanton-Lee, D. H. Sachs, C. LeGuern. 1995. Specific unresponsiveness to a retrovirally transferred class I antigen is controlled through the helper pathway. J. Immunol. 154: 1587
OpenUrl Abstract

[22] ↵
Stanton-Mayfield, R., H. Hayashi, T. Sawada, K. Bergen, C. LeGuern, M. Sykes, D. H. Sachs, J. Iacomini. 1997. The mechanism of specific prolongation of class I mismatched skin grafts induced by retroviral gene therapy. Eur. J. Immunol. 27: 1177
OpenUrl CrossRef PubMed

[23] ↵
Umemura, A., H. Morita, X. C. Li, S. Tahan, A. P. Monaco, T. Maki. 2001. Dissociation of hemopoietic chimerism and allograft tolerance after allogeneic bone marrow transplantation. J. Immunol. 167: 3043
OpenUrl Abstract/FREE Full Text

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Expression of Antigen on Mature Lymphocytes Is Required to Induce T Cell Tolerance by Gene Therapy

Abstract

Materials and Methods