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Partial versus Full Allogeneic Hemopoietic Chimerization Is a Preferential Means to Inhibit Type 1 Diabetes as the Latter Induces Generalized Immunosuppression¹

David V. Serreze^2,*, Melissa A. Osborne^*, Yi-Guang Chen^*, Harold D. Chapman^*, Todd Pearson, Michael A. Brehm and Dale L. Greiner

^* The Jackson Laboratory, Bar Harbor, ME 04609; and Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605

	Abstract

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In both humans and NOD mice, particular combinations of MHC genes provide the primary risk factor for development of the autoreactive T cell responses causing type 1 diabetes (T1D). Conversely, other MHC variants can confer dominant T1D resistance, and previous studies in NOD mice have shown their expression on hemopoietically derived APC is sufficient to induce disease protection. Although allogeneic hemopoietic chimerization can clearly provide a means for blocking T1D development, its clinical use for this purpose has been obviated by a requirement to precondition the host with what would be a lethal irradiation dose if bone marrow engraftment is not successful. There have been reports in which T1D-protective allogeneic hemopoietic chimerization was established in NOD mice that were preconditioned by protocols not including a lethal dose of irradiation. In most of these studies, virtually all the hemopoietic cells in the NOD recipients eventually converted to donor type. We now report that a concern about such full allogeneic chimeras is that they are severely immunocompromised potentially because their T cells are positively selected in the thymus by MHC molecules differing from those expressed by the APC available in the periphery to activate T cell effector functions. However, this undesirable side effect of generalized immunosuppression is obviated by a new protocol that establishes without a lethal preconditioning component, a stable state of mixed allogeneic hemopoietic chimerism sufficient to inhibit T1D development and also induce donor-specific tolerance in NOD recipients.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Studies using the NOD mouse model have indicated type 1 diabetes (T1D)³ results from autoimmune destruction of insulin-producing pancreatic β cells mediated by both CD4 and CD8 T cells (1). Similar to the case in humans, multiple susceptibility (Idd) genes contribute to T1D in NOD mice, but with the primary risk factor being provided by this strain’s unusual H2^g7 MHC (1). Multiple components of the H2^g7 haplotype contribute to T1D susceptibility. Interestingly, these include the H2^g7-encoded K^d and D^b class I molecules, which despite being common variants also found in many nonautoimmune prone strains, aberrantly elicit autoreactive diabetogenic CD8 T cell responses when expressed in NOD mice through interactions with a subset of Idd genes outside the MHC (2). The class II region genes of the H2^g7 haplotype encode the unusual H2-A^g7 variant (histidine and serine at positions 56 and 57 of the β-chain rather than the usual proline and aspartic acid residues), but not an expressible H2-E molecule (3, 4). Both of these class II features contribute to the development of diabetogenic CD4 T cell responses in NOD mice (5, 6, 7, 8, 9).

It is now clear that in addition to serving as restriction elements for β cell-autoreactive CD4 and CD8 T cells, H2^g7 MHC molecules also contribute to T1D in NOD mice through an inability to mediate some of the mechanisms that normally delete or inactivate such pathogenic effectors. This was first demonstrated by findings that T1D is dominantly inhibited in NOD-congenic stocks that express in a heterozygous state a protective MHC haplotype in conjunction with H2^g7 (10, 11, 12). It was subsequently found that even if those encoded by the H2^g7 haplotype remain present, the transgenic or congenic coexpression of certain other MHC molecules corrects the impaired ability of hemopoietically derived APC in NOD mice to trigger mechanisms which delete, anergize, or negatively regulate autoreactive diabetogenic T cells (5, 8, 11, 13, 14, 15, 16, 17). Similarly, certain MHC gene products, such as the DQ6 class II variant, can provide dominant T1D resistance in humans (18). Hence, hemopoietic chimerization, giving rise to APC expressing dominantly protective MHC molecules, holds promise as a means for preventing progression to T1D in susceptible individuals, or allow reversal of disease by cotransplantation of donor-matched pancreatic islets. However, to date, it has not been possible to consider the use of hemopoietic chimerization protocols as a clinical therapy for preventing T1D, or reversing disease by subsequent islet engraftment, because of relatively toxic preconditioning regimens and concerns about the complications of graft-vs-host (GVH) and host-vs-graft (HVG) responses when reconstituting across MHC barriers. Thus, there has been a growing interest in determining whether allogeneic hemopoietic chimerization can be established in NOD mice through the use of relatively benign preconditioning approaches not requiring lethal irradiation of the recipients, and that also prevents the development of GVH or HVG responses.

There have been several reports in which T1D-protective allogeneic hemopoietic chimerization was established in NOD mice that were preconditioned by Ab treatments which transiently depleted host T cells and/or blocked the CD40/CD154 costimulatory pathway just before and after the recipients received a sublethal dose of irradiation followed by donor bone marrow (BM) injection (19, 20). However, while they did so at different rates, in these studies virtually all the hemopoietic cells in the NOD recipients eventually converted to donor type. A potentially important factor to consider about full allogeneic hemopoietic chimeras is that their thymic epithelial cells and peripheral APC are, respectively, of host and donor origin. The respective positive and negative selection of T cells differentiating in the thymus is primarily, but not exclusively, mediated by epithelial cells of nonhemopoietic origin and BM-derived APC (21, 22). Thus, potentially as a consequence of having a sizeable population of T cells that were positively selected by MHC molecules which are not expressed by the only APC available to activate their effector function, full allogeneic hemopoietic chimeras might be immunocompromised. In the present study, we confirm the establishment of full allogeneic hemopoietic chimerism inhibits T1D development in NOD mice, but it does indeed do so at the undesirable cost of also inducing a generalized state of immunosuppression. We also report this undesirable side effect of generalized immunosuppression is obviated by a new protocol that establishes without a lethal preconditioning component, a permanent state of mixed allogeneic hemopoietic chimerism which inhibits T1D and induces donor-specific tolerance in NOD recipients.

	Materials and Methods

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Mice

NOD/LtDvs mice (H2^g7 = K^d, A^g7, E^null, D^b) are maintained at The Jackson Laboratory by brother-sister matings. Currently, T1D develops in 90% of female and 63% of male NOD mice before a year of age. A T1D-resistant NOD stock congenic for the H2^q (K^q, A^q, E^null, D^q) haplotype is maintained at the N21 backcross generation (23). Similarly, a T1D-resistant NOD stock congenic for the H2^nb1 (K^b, A^nb1, E^k, D^b) haplotype is also maintained at the N21 backcross generation (10). The previously described congenic stock of B lymphocyte-deficient NOD.Igµ^null mice (24) is maintained at the N10 backcross generation. These latter two strains were used as progenitors to generate through an outcross-intercross approach a new NOD.H2^nb1.Igµ^null stock. Previously described T and B lymphocyte-deficient NOD-scid mice (25) are maintained at the N11 backcross generation. Development of a NOD stock transgenically expressing the rearranged TCR (V8)- and β (Vβ2)-chains from the β cell-autoreactive CD8 T cell clone AI4 (designated NOD.AI4) has also been previously described (26). C57BL/6 (B6) mice were obtained from the animal resources unit of The Jackson Laboratory.

Mixed hemopoietic chimerization protocols

Beginning at 4–6 wk of age NOD females were injected i.p. with 0.5 mg of the CD154-specific mAb MR1 on days 0, +3, and +14 relative to their receipt of a low nonlethal irradiation dose (600 cGy) and the subsequent i.v. injection of the indicated number and type of female donor BM cells. Except as indicated, the recipients were also injected i.p. with 0.25 mg of the CD8 T cell-depleting mAb YTS169 on days –3, 0, and +3 relative to BM engraftment. At the indicated time points, PBL or splenic leukocytes from the reconstituted mice were assessed for proportions of donor- and recipient-type cells by the flow cytometric methods described below. In one set of experiments, the recipients were preconditioned with a high dose of irradiation (13 Gy), but no Ab treatments, before being reconstituted with mixtures of the indicated numbers and types of BM cells.

Flow cytometry

The proportions of donor and recipient cells among PBL or splenocytes from the hemopoietic chimeras were assessed by multicolor flow cytometry (FACSCalibur; BD Biosciences) using the CellQuest 3.0 data reduction system. NOD recipient-type cells were detected with the K^d MHC class I-specific mAb SF1-1.1 conjugated to a red fluorescent PE tag. NOD.H2^nb1 donor-type cells were detected with the K^b MHC class I-specific mAb 28-13-3 conjugated to a green fluorescent FITC tag. Deletion and recovery of peripheral CD8 T cells in the chimeras was monitored with the mAb 53-6.72 conjugated to a blue fluorescent allophycocyanin tag. Where indicated, proportions of donor and recipient dendritic cells were determined using these same MHC class I-specific Abs and the CD11c-specific Ab Hl3 conjugated with allophycocyanin. One set of experiments evaluated numbers of AI4-transgenic T cells in the thymi and spleens of mixed hemopoietic chimeras. Splenic AI4 T cells were identified by costaining with the allophycocyanin-labeled CD8-specific Ab and the TCR V8-specific Ab B21.14 conjugated with PE. Three-color flow cytometric analyses were performed to determine the proportions of CD4/CD8 double-positive and CD8 single-positive (SP) thymocytes from the indicated chimeras that expressed the AI4 TCR. In addition to the CD8 and TCR V8-specific Abs respectively conjugated with allophycocyanin and PE, these analyses also used the CD4-specific Ab GK1.5 conjugated with FITC.

Secondary transfer of NOD-derived leukocytes from mixed hemopoietic chimeras

Splenic leukocyte suspensions were prepared from the indicated mixed chimeras with hemopoietic cells of both NOD and NOD.H2^nb1 origin. Leukocytes of NOD.H2^nb1 origin were labeled with the K^b MHC class I-specific mAb 28-13-3 conjugated to biotin. The labeled NOD.H2^nb1-derived cells were then separated from those of NOD origin using streptavidin-conjugated magnetic beads (Miltenyi Biotec) as previously described (27). Subsequent FACS analysis indicated that >99% of the remaining negatively selected splenic leukocytes were of NOD (K^d MHC class I positive) origin. NOD-derived splenic leukocytes from the mixed chimeras were then injected i.v. at a dose of 1 x 10⁷ cells into 4- to 6-wk-old NOD-scid female recipients. Controls consisted of NOD-scid females repopulated with 1 x 10⁷ splenic leukocytes from donors that had received the same preconditioning treatment as the mixed chimeras, but had been reconstituted with syngeneic NOD marrow only. All the secondary NOD-scid recipients were then monitored over a 17-wk period as described below for T1D development. At T1D onset, or at the end of the 17-wk postengraftment observation period, splenic leukocytes from all NOD-scid recipients were assessed by FACS analysis for the presence of contaminating K^b-expressing leukocytes of NOD.H2^nb1 origin. None were found in any of the NOD-scid recipients. Repopulation by T cells of NOD origin from the mixed or syngeneic chimeras was confirmed by FACS analysis using PE-conjugated mAbs specific for the CD4 (GK1.5) and CD8 (53-6.72) markers.

T and B cell responses to soluble Ag

At the indicated times postreconstitution, the designated hemopoietic chimeras were immunized in a rear foot pad or by i.p. injection with 50 µg of keyhole limpet hemocyanin (KLH) emulsified in 50 µl of IFA. Ten days later, T cells were purified from the draining lymph nodes or spleen by the previously described magnetic bead-based negative selection approach (27). Triplicate aliquots of 1–2.5 x 10⁵ T cells (varied between experiments) were seeded into flat-bottom 96-well microtiter plates in a final volume of 200 µl of the previously described culture medium (16) containing varying concentrations of KLH plus 2.5 x 10⁵ of the indicated type of irradiated (20 Gy) splenocytes as a source of APC. Control cultures consisted of T cells incubated with the indicated concentration of Ag, but no APC. Following a 48-h incubation at 37°C in a 95% air/5% CO₂ humidified atmosphere, the cultures were pulsed with 1 µCi/well [³H] thymidine for an additional 24 h. The cultures were then harvested and [³H]thymidine incorporation was determined using a LKB Betaplate 1205 system. Data are presented as mean cpm ± SEM at each concentration of Ag.

Serum samples were also collected from the chimeras and control mice before and 10 days after KLH priming. KLH Ab levels were determined by ELISA as previously described (28). Each serum sample was assayed in duplicate at the indicated dilutions. Data represent the mean OD₄₀₅ ± SD at a given dilution of serum samples from the indicated number of mice in each experimental group.

Viral infection

Pichinde virus, strain AN3739 (PV), stocks was prepared in baby hamster kidney cells (BHK21) as previously described (29). For the generation of acute virus-specific T cell responses, mice were infected i.p. with 1 x 10⁷ PFU of PV.

Intracellular cytokine assay

Cytokine-producing CD8 T cells were detected using the Cytofix/Cytoperm kit Plus (with GolgiPlug; BD Biosciences), as described previously (30). Splenocytes (2 x 10⁶ cells) were incubated with 250 ng/ml anti-mouse CD3 mAb (145-2C11; BD Biosciences) in the presence of 10 U/ml human rIL-2 (BD Biosciences), and 1 µl/ml GolgiPlug at 37°C/5% CO₂ for 5 h. Following the incubation, splenocytes were stained with mAb specific for CD8 (53-6.7) and CD44 (IM7). Samples were then fixed and permeabilized with Cytofix/Cytoperm solution and stained with mAb specific for IFN- (XMG1.2; BD Biosciences) or with an IgG1- isotype control (R3-34; BD Biosciences). The samples were analyzed using a FACSCalibur (BD Biosciences) and FlowJo (Tree Star). When shown, error bars are representative of the SD.

In vitro assessment of allogeneic tolerance

At 22-wk post-BM reconstitution, splenocytes from the indicated NOD mice partially chimerized with NOD.H2^nb1 BM were seeded in tissue culture medium at a concentration of 5 x 10⁵/ml with an equal number of irradiated (20 Gy) splenocytes of either NOD, NOD.H2^nb1, or NOD.H2^q origin. After a 4-day incubation at 37°C, it was determined by multicolor flow cytometry analyses whether any proportion of NOD- or NOD.H2^nb1-derived CD8 cells from the chimeras had been functionally activated as assessed by the acquisition of CD69 expression. CD8 T cells were identified by an allophycocyanin-conjugated Ab (53-6.72), and further determined to be of NOD or NOD.H2^nb1 origin by respective staining with FITC-conjugated Abs specific for the K^d (SF1-1.1) or K^b (28-13-3) MHC class I variants. The proportion of these CD8 T cells that expressed CD69 was assessed using the PE-conjugated Ab H1.2F3.

Skin allograft transplantation

Full-thickness skin grafts, 1–2 cm in diameter, were transplanted onto the dorsal flank of recipient mice as described previously (31). Grafts were examined two to three times weekly and rejection was defined as the first day on which the entire graft surface appeared necrotic. Grafts adherent to the bandage or fully necrotic on day 6 were deemed technical failures and were excluded from analysis (32). Average duration of skin allograft survival is presented as the median. Graft survival between groups was statistically compared by Kaplan-Meier life table analysis (Abacus Concepts). Values of p < 0.05 were considered statistically significant.

Assessment of T1D and insulitis development

T1D development in the indicated hemopoietic chimeras was defined by glycosuric values of 3 as assessed with Ames Diastix (supplied by Miles Diagnostics). Insulitis levels were assessed in any chimera that remained free of overt T1D for a 22-wk period following BM reconstitution. Pancreata from such chimeras were fixed in Bouin’s solution, and sectioned at three nonoverlapping levels. Granulated β cells were stained with aldehyde fuchsin and leukocytes with a H&E counterstain. Islets (at least 20/mouse) were individually scored as follows: 0, no lesions; 1, peri-insular leukocytic aggregates, usually periductal infiltrates; 2, <25% islet destruction; 3, >25% islet destruction; and 4, complete islet destruction. An insulitis score for each mouse was obtained by dividing the total score for each pancreas by the number of islets examined. Data are presented as mean insulitis scores (MIS) ± SEM for the indicated experimental groups.

	Results

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T cell responses to soluble Ag are suppressed in full allogeneic hemopoietic chimeras

Full allogeneic hemopoietic chimerization can protect NOD mice from T1D (19, 20, 33, 34). However, even when established by a preconditioning protocol that does not require lethal irradiation of the recipient, full allogeneic hemopoietic chimerization would not be a desirable way to prevent T1D in susceptible individuals if this approach has the undesirable side effect of eliciting a state of generalized immunosuppression. Studies by other investigators (21, 22) indicated such immunosuppression could occur in full allogeneic hemopoietic chimeras because the MHC molecules expressed by their peripheral APC differ from those of the thymic epithelial cells that primarily mediate T cell-positive selection. It has been previously established that when high doses of donor BM (>2.5 x 10⁷ cells) are used, a preconditioning protocol consisting of a sublethal irradiation dose (600 cGy) and periengraftment treatment with a CD154-specific mAb allows for the development of virtually full allogeneic hemopoietic chimerization in NOD mice (20). Thus, as an initial test of immune competence, we assessed the ability of female NOD mice that had been chimerized using this preconditioning protocol with 1 x 10⁸ NOD.H2^nb1 BM cells, to mount T cell responses to the strong nominal Ag KLH. This strain combination was chosen on the basis that because MHC molecules are the primary determinants of T1D susceptibility or resistance, it seemed to be of potentially important future clinical relevance to determine the extent to which donor cells selected by this criteria alone could be exploited in a BM transplantation system for the prevention of disease. Controls consisted of NOD mice that received the same preconditioning regimen, but were reconstituted with syngeneic marrow.

As expected, over a 22-wk postengraftment period, T1D developed in 87.5% (seven of eight) of the NOD control recipients reconstituted with syngeneic BM (Fig. 1A). In contrast, none (zero of nine) of the recipients chimerized with NOD.H2^nb1 BM developed T1D through 22-wk postengraftment (Fig. 1A). When examined at 22-wk postengraftment, the chimeric NOD females transplanted with NOD.H2^nb1 BM were also found to be insulitis-free (data not shown). The inhibition of insulitis and T1D development was associated with the fact that the level of donor-derived leukocytes in NOD recipients of NOD.H2^nb1 BM increased from 87.5 to 95.6% between 4- and 22-wk postengraftment (Fig. 1B). Following KLH priming in vivo, T cells from both unmanipulated NOD and NOD.H2^nb1 mice responded equivalently upon antigenic restimulation in vitro (Fig. 1C). Thus, both the H2^g7 and H2^nb1 MHC haplotypes competently support strong T cell responses against KLH. However, T cells from the NOD mice that were nearly fully chimeric for H2^nb1-expressing leukocytes failed to demonstrate a KLH Ag-stimulated recall response in vitro in the presence of either donor or recipient type APC (Fig. 1D). In contrast, KLH-primed T cells from control NOD mice reconstituted with syngeneic BM responded readily when restimulated with Ag in vitro (Fig. 1D). Thus, full allogeneic hemopoietic chimerization induces a significantly reduced ability of the NOD recipients to generate a soluble Ag-specific T cell response.

View larger version (14K): [in this window] [in a new window]   FIGURE 1. NOD mice fully chimerized with NOD.H2nb1 hemopoietic cells are protected from T1D, but have a suppressed ability to generate T cell responses to soluble Ag. NOD females at 4–6 wk of age received i.p. injections of 0.5 mg of a CD154-specific Ab on days 0 and +3 relative to receipt of a sublethal dose of irradiation (600 cGy), and an i.v. injection of 1 x 108 NOD.H2nb1 BM cells. Controls were NOD mice preconditioned in the same way, but injected with syngeneic marrow. A, Rate of T1D development in the test (•, n = 9) and control chimeras (, n = 8). B, Proportion at various time points postreconstitution of donor cells among PBL (* or spleen at 22-wk postreconstitution) from NOD mice reconstituted with NOD.H2nb1 BM. Donor cells were distinguished from recipient type by respective expression of the Kb vs Kd MHC class I variant. C, Ag-stimulated in vitro recall responses of T cells (2.5 x 105 responders/well) from unmanipulated NOD () and NOD.H2nb1 () mice foot pads primed 10 days earlier with KLH in vivo. Responder T cells were pooled from popliteal lymph nodes of seven mice per experimental group. D, Ag-stimulated in vitro recall responses of T cells (1 x 105 responders/well) from NOD recipients reconstituted with NOD.H2nb1 (• with NOD APC; with NOD.H2nb1 APC) or syngeneic BM () primed 10 days earlier with KLH in vivo. Responder T cells were pooled from seven mice per experimental group. For C and D, data represent the mean cpm ± SEM at each Ag concentration.

T1D can also be prevented in NOD mice by the establishment of mixed, rather than full, allogeneic hemopoietic chimerism (11, 35, 36, 37, 38). A possible advantage of preventing T1D by establishing mixed, rather than full, allogeneic hemopoietic chimerization is this may avoid the possibility suggested by our above studies of the latter approach inducing a generalized immunosuppressed state. Thus, we determined whether a T1D-protective state of stable mixed allogeneic hemopoietic chimerism could be established in NOD mice given sublethal conditioning, and if this also allows the recipients to remain competent in ability to mount a range of immune responses.

In NOD mice preconditioned with anti-CD154 and sublethal irradiation (600 cGy), we observed a threshold effect where either full or no hemopoietic chimerism was established following injection of > or <2.5 x 10⁷ BM cells from B6 or NOD.H2^nb1 donors (data not shown). Another study subsequently found, unlike in other strains, transplantation tolerance to allogeneic skin grafts is not established in NOD mice by CD40/CD154 blockade accompanied by donor-specific transfusion of splenocytes (39). This may be explained in part by the finding that unlike the case in nonautoimmune prone control strains, peripheral CD8 T cells in NOD mice are relatively defective in ability to undergo deletion upon high levels of antigenic stimulation (40, 41). Additionally, donor-reactive CD8 T cells have been shown to be a barrier to establishment of allogeneic bone marrow chimerism (42). Hence, we determined whether transient depletion of host CD8 T cells during the peritransplant period allowed us to establish stable mixed allogeneic hemopoietic chimerism in NOD recipients.

We ultimately settled on a protocol where the NOD recipients were injected i.p. with 0.25 mg of the CD8 T cell-depleting mAb YTS169 on days –3, 0, and +3, and anti-CD154 on days 0, +3, and +14 relative to their receipt of a sublethal irradiation dose (600 cGy) and the subsequent i.v. injection of donor BM cells. When this approach was used with donor NOD.H2^nb1 BM doses of 7.5 x 10⁶ to 2.5 x 10⁷ cells, the NOD recipients developed levels of mixed hemopoietic allogenic chimerization ranging from 50–70% that remained stable through 22-wk postengraftment (Fig. 2A). When analyzed at 22-wk postreconstitution, an equivalent proportion of donor cells was detected in all leukocyte subpopulations. None (0 of 24) of these mixed chimeras developed T1D over the 22-wk postengraftment period (Fig. 2B). In contrast, T1D developed in 88.9% (eight of nine) of NOD recipients that were transplanted with 7.5 x 10⁶ syngeneic BM cells after receiving the same preconditioning treatment as the mixed allogeneic chimeras (Fig. 2B). This latter result demonstrated the preconditioning protocol we used did not have an inherent T1D-protective effect. Furthermore, compared with the control group, insulitis levels were significantly less in the NOD recipients partially chimerized with NOD.H2^nb1 BM (Fig. 2C). It should also be noted that we have not been able to use the above preconditioning approach to establish stable levels of mixed allogeneic hemopoietic chimerism which were <50% by injecting the NOD recipients with <7.5 x 10⁶ donor BM cells. Nonetheless, these results demonstrate the sublethal preconditioning protocol we have developed can be used to establish a stable level of mixed allogeneic hemopoietic chimerization in NOD mice that confers T1D resistance.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. The establishment of stable mixed allogeneic hemopoietic chimerism inhibits the development of T1D and insulitis in NOD mice. NOD females at 4–6 wk of age received i.p. injections of 0.5 mg of a CD154-specific Ab on days –3, 0, and +14 plus 0.25 mg of a CD8-specific Ab on days –3, 0, and +3 relative to receipt of a sublethal dose of irradiation (600 cGy), and an i.v. injection of various numbers of NOD.H2nb1 BM cells. Controls were NOD mice preconditioned in the same way, but injected with syngeneic marrow. A, Proportion of donor cells over time among PBL (* or spleen at 22-wk postreconstitution) of NOD mice chimerized with NOD.H2nb1 BM at doses of 7.5x106 (, n = 6), 1 x 107 (, n = 10), and 2.5 x 107 (•, n = 8) cells. B, Rate of T1D development in NOD mice chimerized with syngeneic (•, n = 9) or NOD.H2nb1 BM at doses of 7.5x106 (, n = 6), 1 x 107 (, n = 10), and 2.5 x 107 (•, n = 8) cells. C, MIS ± SEM at 22-wk postreconstitution of NOD mice chimerized with indicated number of NOD.H2nb1 BM cells. Also depicted is the MIS ± SEM at the time of T1D onset or at 22-wk postreconstitution in NOD mice chimerized with syngeneic BM. The MIS of NOD mice partially chimerized with NOD.H2nb1 BM was significantly less (p < 0.05, Student’s t test) than those chimerized with syngeneic BM.

We next assessed whether the establishment of mixed, rather than full, allogeneic hemopoietic chimerization allowed the NOD recipients to more competently mount a range of immunological responses. New groups of NOD-recipient mice were produced that were either fully or partially chimerized with NOD.H2^nb1 BM (99.3 ± 1.1% vs 61.8 ± 2.4% donor-type leukocytes) using the two approaches described earlier. A subset of mice in each group was assessed for ability to generate T cell responses to soluble Ag by in vivo priming and in vitro recall with KLH. As expected, T cells from NOD or NOD.H2^nb1 control mice reconstituted with syngeneic BM both responded strongly upon KLH restimulation (Fig. 3A). However, as observed previously, compared with those from the above controls, KLH-restimulated T cells from full NOD allogeneic hemopoietic chimeras exhibited significantly lower responses in the presence of either donor or host origin APC (Fig. 3B). Conversely, T cells from the mixed chimeras responded robustly to Ag restimulation in vitro in the presence of NOD APC (Fig. 3B). A somewhat lower response was observed in the presence of NOD.H2^nb1 APC (Fig. 3B). These results indicate the ability to generate T cell responses to soluble Ag is impaired in full, but not mixed, NOD allogeneic hemopoietic chimeras.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. T and B cell responses to soluble Ag are more robust in mixed, than full, NOD allogeneic hemopoietic chimeras. Groups of NOD female mice were either fully or partially chimerized with NOD.H2nb1 BM cells by the protocols respectively described for Figs. 1 and 2. At 6-wk postreconstitution, donor cell levels in the full vs mixed chimeras were, respectively, 99.3 ± 1.1% (n = 8) and 61.8 ± 2.4% (n = 8). As controls, other NOD and NOD.H2nb1 mice were reconstituted with syngeneic BM. Subsets of mice in each group were then compared for ability to generate T and B cell responses to soluble Ag. A, At 6-wk postreconstitution, NOD and NOD.H2nb1 mice reconstituted with syngeneic BM were primed by i.p. injection of KLH in IFA. Ten days later, two pools of splenic T cells per group were prepared from two mice each, and then cultured in triplicate (2.5 x 105/well) with an equal number of irradiated NOD or NOD.H2nb1 splenocytes as a source of APC plus the indicated concentration of KLH. The cultures were labeled with [3H]thymidine over the final 24 h of a 72-h incubation period. B, NOD mice partially or fully chimerized with NOD.H2nb1 BM were primed with KLH. Ten days later, two pools of splenic T cells per group were prepared from two mice each, and assessed in the presence of NOD and NOD.H2nb1 APC for KLH-induced recall responses in vitro as described for A. Data in A and B represent the mean cpm ± SD at each Ag concentration of the two T cell pools per group cultured in triplicate with each type of APC. C, ELISA analyses of KLH-specific Abs in the same chimeras assessed for T cell responses to this soluble Ag in A and B. *, Response of the full allogeneic hemopoietic chimeras significantly less (p < 0.05, ANOVA) than that of NOD mice reconstituted with syngeneic BM.

Other mice within these same sets of full or mixed NOD allogeneic hemopoietic chimeras were compared for ability to generate virus-specific memory CD8 T cell responses in vivo. Previous studies have demonstrated that memory, but not naive, CD8 T cells are capable of producing IFN- within 5 h of TCR signaling induced by anti-CD3 treatment (43, 44). Thus, this phenotype was used to determine whether infection with PV 8 days earlier resulted in differential induction of memory CD8 T cell responses in the full vs mixed NOD allogeneic hemopoietic chimeras. NOD and NOD.H2^nb1 mice fully chimerized with syngeneic BM and subsequently infected with PV served as controls. After a 5-h incubation with anti-CD3, CD8 T cells from each group of mice previously infected with virus were examined for the expression of CD44 to assess the overall level of activation (Fig. 4A), and for IFN- production by intracellular cytokine assay (Fig. 4B). CD8 T cells from NOD mice that were either full syngeneic or partial allogeneic chimeras and subsequently infected with virus, responded equivalently in terms of CD44 expression and IFN- production following the 5-h anti-CD3 stimulation. In contrast, relative to those from all other infected groups, the virus-induced response of CD8 T cells from full NOD allogeneic chimeric mice was significantly impaired as demonstrated by lower levels of CD44 expression (p < 0.001 relative to all other groups) and diminished IFN- production (p < 0.01 relative to all other groups). Moreover, two of four full allogeneic chimeras were unable to completely clear virus from their fat pads (180 and 200 PFU/fat pad, respectively) after 8 days of infection, while the viral levels in all other mice were below the limit of detection (100 PFU/fat pad). Together, these results demonstrate that full allogeneic chimerization will induce a generalized state of immunosuppression and prevent the optimal generation of virus-specific T cells.

View larger version (17K): [in this window] [in a new window]   FIGURE 4. Viral-induced in vivo CD8 T cell responses are more robust in mixed, than full, NOD allogeneic hemopoietic chimeras. NOD female mice were either fully or partially chimerized with NOD.H2nb1 BM cells by the protocols, respectively, described for Figs. 1 and 2. Controls consisted of NOD and NOD.H2nb1 mice reconstituted with syngeneic BM. At 6-wk postreconstitution, all mice were infected with PV. At 8 days postinfection, splenic leukocytes from each mouse were stimulated in vitro for 5 h with anti-CD3 and the CD8 T cells subsequently assessed by flow cytometry for CD44 expression (A) and intracellular levels of IFN- (B). Data represent the mean proportion ± SEM of CD8 T cells from each group of virally preinfected mice that were positive for CD44 expression or intracellular IFN- following the 5-h anti-CD3 stimulation.

Previous studies found that β cell-autoreactive T cells still develop, but are functionally suppressed in NOD stocks where transgenic H2-A or H2-E class II molecules were the only T1D-protective MHC gene product expressed on the complete spectrum of APC (5, 8). However, specific NOD-derived β cell-autoreactive T cell clones were shown to be deleted or rendered permanently anergic when forced to mature in the presence of a complete spectrum of APC expressing the multiple genes of the T1D-protective H2^nb1 MHC haplotype (K^b, A^nb1, E^k, D^b) (14, 15, 17). Thus, we determined the extent to which H2^nb1-expressing APC in the mixed chimeras mediated T1D resistance by inducing an active immunoregulatory mechanism(s) vs deleting and/or anergizing β cell-autoreactive T cells.

We reasoned that if protection resulted from the induction of an active immunoregulatory process, then NOD-derived T cells from the mixed chimeras would demonstrate a restored ability to induce T1D when liberated from the influence of H2^nb1-expressing APC. Conversely, if diabetogenic effectors had been deleted or permanently inactivated when maturing in the presence of H2^nb1-expressing APC, then purified NOD-derived T cells from the mixed chimeras should not demonstrate a restored ability to induce disease. The mixed chimeras we used as donors were those depicted in Fig. 2 in which 50% of the leukocytes were of NOD.H2^nb1 origin (generated with 7.5 x 10⁶ donor BM cells). We assessed whether splenocytes from these mixed chimeras that had been depleted of H2^nb1-expressing cells could adoptively transfer T1D to NOD-scid recipients. CD4 and CD8 T cells in the transfer inoculum repopulated NOD-scid recipients (Fig. 5A), and exhibited normal proliferative responses to anti-CD3 stimulation (data not shown), but did not induce T1D (Fig. 5B). Importantly, no H2^nb1-expressing cells could be detected in the NOD-scid recipients. As a control, we repopulated NOD-scid recipients with splenocytes from NOD mice that had been reconstituted 8 wk previously with syngeneic BM only, but had not yet developed overt T1D. Splenocytes from these controls induced T1D in 87.5% of NOD-scid recipients by 17-wk posttransfer (Fig. 5B). These collective results indicated the H2^nb1-expressing APC in the mixed chimeras mediated T1D protection by inducing the deletion and/or permanent inactivation of pathogenic T cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. H2nb1-expressing APC in mixed hemopoietic chimeras mediate the deletion and/or functional inactivation of NOD-derived diabetogenic T cells. At 22-wk postreconstitution, splenocytes from the NOD mice depicted in Fig. 2 that were chimerized with 7.5 x 106 NOD.H2nb1 BM cells were depleted of H2nb1-expressing cells. Aliquots of 1 x 107 of the remaining NOD-derived leukocytes from these chimeras were then injected i.v. into 4- to 6-wk-old female NOD-scid recipients. Controls consisted of NOD-scid recipients of 1 x 107 splenic leukocytes from NOD mice chimerized with syngeneic BM 8 wk previously. A, Engraftment levels in NOD-scid recipients at T1D onset or at 17-wk posttransfer of NOD-derived CD4 and CD8 T cells from the mixed (, n = 5) or syngeneic (, n = 8) chimeras. B, Rate of T1D development in NOD-scid recipients of NOD-derived splenic leukocytes from the mixed (•, n = 5) or syngeneic (, n = 8) chimeras.

We previously found that AI4 CD8 T cells, which are normally an important population of autoreactive diabetogenic effectors in NOD mice (45), undergo intrathymic negative selection in a system where all APC congenically expressed the H2^nb1 haplotype in a heterozygous fashion (17). However, it was unknown what proportion of H2^nb1-expressing APC must be present to efficiently mediate the negative selection of diabetogenic T cells. Although the level of H2^nb1-expressing APC needed to mediate the negative selection of various diabetogenic T cell clonotypes may differ, we could at least address this question for the AI4 population. Provided they do inhibit T1D in NOD recipients by enhancing the negative selection of β cell-autoreactive T cells, the data in Fig. 2 indicates a 50% frequency of H2^nb1-expressing leukocytes is sufficient to mediate this disease protective process. Thus, we determined whether the efficient elimination of diabetogenic AI4 T cells could still occur when H2^nb1-expressing leukocytes were present at a frequency <50%. As described earlier, we have been unable to use our CD154 blockade and transient CD8 T cell depletion preconditioning protocol in a way that allows leukocytes derived from NOD.H2^nb1 BM to stably engraft NOD recipients at levels <50%. Hence, for this portion of our studies we used recipients preconditioned by a high dose of irradiation (13 Gy) before being reconstituted with various admixtures of BM. Furthermore, we had found that even after receiving a 13-Gy irradiation dose, standard NOD mice retain sufficient residual T activity to eliminate any H2^nb1-expressing BM cells. To overcome this difficulty, we used (NOD x NOD.H2^nb1)F₁ hybrids as recipients. Such F₁ hybrids develop T1D when reconstituted with NOD BM alone (11). However, when repopulated with a 1:1 mixture of NOD and NOD.H2^nb1 BM, stable mixed (50/50) chimerism results, and the F₁ recipients are now protected from T1D (11).

We were also interested in determining what subtypes of chimeric APC (B lymphocytes, macrophages, dendritic cells) must express H2^nb1 molecules to induce T1D-protective effects. The types of APC present in the thymus that can mediate negative selection include dendritic cells and macrophages, but very few B lymphocytes (21, 46). For this reason, we hypothesized the T1D-protective effect of H2^nb1 expression is primarily mediated by dendritic cells and/or macrophages. A B lymphocyte-deficient NOD.H2^nb1.Igµ^null stock was generated to test this hypothesis.

(NOD.Igµ^null x NOD.H2^nb1.Igµ^null)F₁ hybrids received a 13-Gy irradiation dose and then reconstituted with BM from F₁ plus NOD.AI4 TCR-transgenic donors at ratios of either 1:2 or 1:4. We reasoned this approach would allow engraftment of each BM type and limit the types of APC-expressing H2^nb1 molecules to variable numbers of dendritic cells/macrophages. Controls consisted of the same type of irradiated F₁ hybrids reconstituted with NOD plus NOD.AI4 BM at ratios of 1:2 and 1:4. At 7-wk postreconstitution, the proportions of splenic dendritic cells derived from each donor BM type were assessed, as well as the numbers of splenocytes and CD8 SP thymocytes that expressed the AI4 TCR. As expected, in the control chimeras no H2^nb1-expressing APC were detected, and large numbers of AI4 T cells were present in both the splenic and CD8 SP thymocyte compartments (Fig. 6). Between the two groups of test chimeras, the proportions of H2^nb1-expressing dendritic cells ranged from 15 to 40% (Fig. 6). Compared with levels in the controls, the number of AI4 CD8 SP thymocytes in both groups of test chimeras was greatly reduced (Fig. 6). There was a strong positive correlation between the proportion of H2^nb1-expressing dendritic cells and the extent to which AI4 CD8 SP thymocytes were reduced in the test chimeras. The number of AI4 T cells in the spleens of both test chimera groups were also much less than in the corresponding controls (Fig. 6). However, in contrast to what was observed intrathymically, at all levels of engraftment, the H2^nb1-expressing dendritic cells elicited an essentially equivalent reduction in splenic AI4 T numbers compared with the controls (Fig. 6). Indeed, the level of H2^nb1-expressing dendritic cell engraftment required to elicit a 50% reduction in AI4 CD8 SP thymocytes (25–30%) was approximately twice that needed to induce a similar decrease in the spleen.

View larger version (28K): [in this window] [in a new window]   FIGURE 6. Chimeric allogeneic dendritic cells/macrophages induce a greater extent of diabetogenic T cell deletion in the periphery than in the thymus. After receiving a 13-Gy irradiation dose, (NOD.Igµnull x NOD.H2nb1.Igµnull)F1 hybrids were reconstituted with BM from syngeneic and NOD.AI4 TCR-transgenic donors at ratios of either 1:2 or 1:4. Controls were the same type of F1 hybrids reconstituted with NOD and NOD.AI4 TCR-transgenic BM at ratios of 1:2 or 1:4. At 7-wk postreconstitution, the proportions of splenic dendritic cells derived from each donor BM type were assessed, as well as the numbers of splenocytes and CD8 SP thymocytes that expressed the AI4 TCR. The left panels depict the numbers of AI4 CD8 SP thymocytes (upper) and splenocytes (lower) in chimeras, respectively, reconstituted with the indicated BM mixtures at 1:2 or 1:4 ratios. Right panels, The extent to which AI4 CD8 SP thymocyte (upper) or splenocyte (lower) numbers are decreased as a function of the proportion of H2nb1-expressing dendritic cells in the test chimeras.

The establishment of mixed allogeneic hemopoietic chimerism in NOD mice induces donor-specific tolerance

The studies described to this point demonstrate the establishment of mixed allogeneic hemopoietic chimerism inhibits the initial development of T1D in NOD mice by preventing the generation or activation of β cell-autoreactive T cells. However, if any approach of establishing mixed allogeneic hemopoietic chimerization is ultimately to be of potential clinical use, it would be important that the protocol also allows for the reversal of already established T1D by rendering the recipient tolerant to allogeneic islet grafts obtained from the same individual providing the reconstituting BM. Thus, we initially examined in vitro whether T cells from the NOD chimeras depicted in Fig. 2, in which 50% of the leukocytes were of NOD.H2^nb1 origin (generated with 7.5 x 10⁶ donor BM cells), were truly tolerant of donor alloantigens. This was done by determining whether donor- and recipient-type CD8 T cells in these chimeras, respectively, identified by the presence of the K^b and K^d MHC class I variant, became activated as assessed by the induction of CD69 expression when cultured in the presence of irradiated NOD or NOD.H2^nb1 splenocytes. As a control, CD8 T cells from the chimeras were also assessed for their ability to respond to third-party NOD.H2^q splenocytes. Both donor- and recipient-type CD8 T cells from the chimeras failed to become activated by either NOD or NOD.H2^nb1 stimulators (Fig. 7, A and B). However, third-party NOD.H2^q stimulators elicited a response by both donor- and recipient-type CD8 T cells from the chimeras, indicating neither population was characterized by nonspecific functional incompetence. To further test the establishment of donor-specific tolerance, we transplanted another group of mixed chimeric NOD mice in which 50–60% of the leukocytes were of NOD.H2^nb1 origin with donor-type or third-party (NOD.H2^q) skin allografts. As expected, third-party skin grafts were rapidly rejected (Fig. 7C, median survival time (MST) = 11 days), while H2^nb1 skin grafts uniformly survived throughout the period of observation (Fig. 7C, median survival time >30 days). Thus, in addition to inhibiting the development of autoimmune responses against β cells, the establishment of stable mixed allogeneic hemopoietic chimerism in NOD mice also induces a state of donor-specific immunological tolerance.

View larger version (20K): [in this window] [in a new window]   FIGURE 7. The establishment of mixed allogeneic hemopoietic chimerism in NOD mice induces donor-specific tolerance. Splenocytes from four of the NOD mice depicted in Fig. 2 that had been partially chimerized with 7.5 x 106 NOD.H2nb1 BM cells were individually cultured for the indicated period of time with irradiated splenocytes of NOD (•), NOD.H2nb1 (), or NOD.H2q () origin. At the indicated time points, flow cytometry was used to determine whether chimera-derived CD8 T cells of NOD (A) or NOD.H2nb1 (B) origin (identified by Kd vs Kb MHC class I expression) had been functionally activated in any of the cultures as assessed by expression of the CD69 marker. Data represent the mean proportion ± SEM of CD8 T cells that expressed CD69. *, Significantly higher CD69 expression (p < 0.05, Student’s t test) than on chimeric CD8 T cells cultured with irradiated NOD stimulators. C, Skin allograft survival in mixed allogeneic chimeric NOD mice. NOD mice chimerized 8 wk earlier with NOD.H2nb1 BM received skin allografts of NOD.H2nb1 (donor-type) or NOD.H2q (third-party) origin. Levels of H2nb1-expressing leukocytes ranged from 50 to 60% at the time of skin grafting. Skin allograft survival of NOD.SWR-H2q (third-party) grafts was significantly shorter than NOD.NON-H2nb1 (donor-type) grafts (p < 0.01).

	Discussion

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There have been several reports of preconditioning protocols using transient depletion of host T cells and blockade of the CD40/CD154 costimulatory pathway that allowed for the establishment of a T1D-protective state of allogeneic hemopoietic chimerism in NOD mice without lethal cytoreductive conditioning (19, 20, 47). However, in these studies at some time point postreconstitution, virtually all the hemopoietic cells (>90%) in the NOD recipients had converted to donor type. In the current study, we show that the establishment of full allogeneic hemopoietic chimerization is not a desirable way to prevent T1D because this approach also results in a state of severe, generalized immunosuppression. Both a previous (48) and our current study found such immunosuppression does not occur in mixed allogeneic hemopoietic chimeras. We also report development of a relatively benign protocol to establish, without a lethal preconditioning dose of irradiation, a stable state of mixed rather than full allogeneic hemopoietic chimerism in NOD mice that results in T1D resistance, but not generalized immunosuppression. Liang et al. (38) recently reported another protocol stated to establish stable mixed allogeneic hemopoietic chimerization in NOD recipients that received no irradiation, but were instead preconditioned with two 500-µg injections of a CD3-specific Ab. However, this protocol required the use of quite high doses of donor BM cells (one to two injections of 1–2 x 10⁸ cells) raising significant concerns about the extent it could be translated for clinical use.

The generalized immunosuppression observed in the full allogeneic hemopoietic chimeras is likely due to the fact that their T cells were positively selected by host MHC molecules expressed on thymic epithelial cells, but then these effectors cannot be activated in the periphery because all APC express donor MHC molecules. This does not occur in mixed chimeras because there are APC available that express the same MHC molecules which mediated T cell-positive selection. It should be noted that while much weaker than those mediated by host MHC molecules, we did observe donor MHC-restricted T cell responses in the mixed chimeras. This has been previously observed in mixed chimeras and found to be due to the fact that hemopoietically derived APC can mediate some degree of positive selection, albeit much less efficiently than thymic epithelial cells (48). However, it should be noted that we cannot completely discard the possibility that the immunocompromised state of the full allogeneic hemopoietic chimeras results from a low-level undetected GVH response.

Previous studies have demonstrated it is possible to establish stable mixed allogeneic hemopoietic chimerism in nonautoimmune-prone strains through preconditioning protocols in which the recipients are treated with agents that block the CD40 and/or CD28 costimulatory pathways plus a low nonlethal dose of irradiation (49, 50). However, in our current studies, we found such an approach was unsuccessful when using NOD recipients, perhaps owing to this strain’s generalized resistance to transplantation tolerance induction (51). We previously demonstrated transplantation tolerance could not be established to allogeneic skin grafts in NOD mice by treatment with donor-specific transfusion and anti-CD154, owing in part to a failure to delete or inactivate alloreactive CD8 T cells (39). Thus, we determined whether a preconditioning protocol that also included the transient elimination of host CD8 T cells at the time of donor BM transfer now allowed the establishment of stable mixed allogeneic hemopoietic chimerism in NOD recipients. This proved to be true, but we have yet to find a way that our protocol can be further modified that allows donor hemopoietic cells to engraft at levels <50%.

The establishment of an 50% level of allogeneic hemopoietic chimerism successfully inhibited T1D development in NOD recipients. Two-tier adoptive transfer studies indicated the engrafted allogeneic APC mediated T1D resistance in the NOD recipients through the elimination or permanent inactivation of diabetogenic T cells. Also of importance was the finding that in addition to having attenuated β cell autoimmunity, the mixed chimeras were also rendered tolerant to donor alloantigens. This suggests our mixed chimera protocol, or one similar to it, could be used to reverse already established T1D by engrafting islets from the same donor that supplied the BM. However, it should be noted that our studies to date have only assessed tolerance of mixed allogeneic hemopoietic chimeras to skin, but not islet allografts.

Because to date our mixed chimera protocol has not allowed NOD recipients to become engrafted with donor hemopoietic cells at levels <50%, we were unable to determine the minimum extent to which they must be present to block T1D development. However, we feel it is likely that T1D resistance could be induced in NOD recipients by allogeneic APC engraftment levels much <50%. This supposition is based on studies that used NOD recipients preconditioned with a 13 Gy dose of irradiation, and found that compared with controls in which they were absent, an allogeneic APC engraftment level of 20% resulted in an 80% decrease in the numbers of a known diabetogenic T cell clonotype (AI4). Interestingly, an equivalent level of allogeneic leukocyte engraftment resulted in a greater decrease in AI4 T cell numbers in the periphery than in the thymus. There are several possible explanations for this finding. One is H2^nb1-expressing APC induce the deletion of AI4 T cells more efficiently in the periphery than during their development in the thymus. Alternatively, AI4 T cells may undergo deletion when first encountering an H2^nb1-expressing APC regardless of whether this occurred during their development in the thymus or at a later time point in the periphery, resulting in an accumulative loss of these diabetogenic effectors.

In conclusion, our results indicate it is preferable to inhibit T1D development by the establishment of partial, rather than full, allogeneic hemopoietic chimerization because the latter results in a generalized state of immunosuppression. We also report development of a protocol that overrides the difficulty of establishing stable mixed allogeneic hemopoietic chimerization in autoimmune prone NOD mice, without lethal cytoreductive conditioning. It would seem likely that the same type of barriers may limit the establishment of mixed hemopoietic chimerism in humans at risk for T1D, and they too could possibly be overcome by a protocol similar to the one we have developed for use in NOD mice. Furthermore, our results suggest only a relatively low level of allogeneic APC may be needed to block T1D development in susceptible humans or reverse already established disease with donor-matched islet grafts. For all these reasons, we believe that an allogeneic hemopoietic chimerization approach should not be completely discarded as a future clinical means to present or reverse autoimmune T1D.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

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

¹ This work was supported by National Institutes of Health Grants DK51090, DK46266, AI46629, and DK53006; Cancer Center Support Grant CA34196; Diabetes Endocrinology Research Center Grant DK32520; as well as by grants from the Juvenile Diabetes Research Foundation; and a fellowship to M.A.B from the Charles A. King Trust, Bank of America, Co-Trustee.

² Address correspondence and reprint requests to Dr. David V. Serreze, The Jackson Laboratory, Bar Harbor, ME 04609. E-mail address: dave.serreze{at}jax.org

³ Abbreviations used in this paper: T1D, type 1 diabetes; BM, bone marrow; GVH, graft vs host; HVG, host vs graft; PV, Pichinde virus; SP, single positive; KLH, keyhole limpet hemocyanin; MIS, mean insulitis score.

Received for publication August 16, 2005. Accepted for publication August 25, 2006.

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