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The Journal of Immunology, 1999, 162: 5134-5142.
Copyright © 1999 by The American Association of Immunologists

T Cell Reconstitution of BB/W Rats After the Initiation of Insulitis Precipitates the Onset of Diabetes1

Sheela Ramanathan and Philippe Poussier2

Departments of Immunology and Medicine, University of Toronto, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
One of the diabetes susceptibility genes of the BB/W (Biobreeding/Worcester) rat maps to the lyp locus on chromosome 4. The BB/W lyp allele is responsible for a severe peripheral T lymphopenia. Correction of this lymphopenia by transfer of normal, histocompatible T cells prevents diabetes, providing T cell reconstitution is initiated before insulitis. We have analyzed this time-dependent regulation of the diabetogenic process by normal T cells. We demonstrate that T cell reconstitution after the initiation of insulitis precipitates the onset of diabetes through the recruitment of donor T cells to the autoimmune process. This inability of normal T cells to regulate primed diabetogenic BB/W T cells and their own autoreactive potential were observed when normal T cells outnumbered pathogenic T cells by approximately 1000-fold. Analysis of donor-derived T cells recovered from BB/W rats that were reconstituted before insulitis, and hence protected from diabetes, demonstrates that early T cell reconstitution of BB/W rats does not result in a long term physical or functional depletion of islet cell-specific T cell precursors among donor cells or in the expansion of T cells that can regulate the activation and expansion of diabetogenic T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Biobreeding/Worcester (BB/W)3 rats spontaneously develop a T cell-mediated, insulin-dependent diabetic syndrome that is very similar to that observed in NOD mice and humans (1). Only two diabetes susceptibility loci have been identified in the BB/W rat. Iddm1 maps to the lyp locus on chromosome 4 (2), while Iddm2 maps to the MHC RT1u haplotype (3). Breeding studies have shown that heterozygosity or homozygosity for the RT1u haplotype and homozygosity for the BB/W rat lyp allele are both necessary, although not sufficient, for the spontaneous development of diabetes (3). The lyp mutation is responsible for a peripheral T lymphopenia that is present at birth and results from the combination of decreased thymic output and a short life span of T cells (4, 5). Consequently, there is a 5- to 10-fold reduction in the absolute number of peripheral T cells and, among these cells, a virtual absence of CD4-8+ T cells expressing TCR{alpha}ß (6). The premature death of naive peripheral T cells in the BB/W rat precludes their continued development in the periphery, as evidenced by the absence of peripheral T cells expressing RT6 and CD45RC (7).

The mechanisms through which the BB/W rat lyp allele contributes to the development of antipancreatic autoimmunity remain obscure. A potential pathogenic mechanism is the induction of an excessive production of nitric oxide by macrophages, which are the first mononuclear cells to infiltrate the pancreatic islets of BB/W rats (8, 9). It has been shown that nitric oxide is toxic for ß-cells in vitro. Further, the incidence of diabetes is decreased in BB/W rats that are treated with inhibitors of nitric oxide synthase (10). We have demonstrated that the excessive production of nitric oxide by BB/W rat macrophages is secondary to the T lymphopenic state of these animals (11). Specifically, we have shown that the levels of nitric oxide produced by BB/W macrophages are restored to those produced by macrophages derived from nondiabetes-prone animals when they develop in the context of a normal-sized pool of recirculating T cells.

Two lines of evidence suggest that the T lymphopenic process of the BB/W rat could also compromise the development of T cells that would normally regulate potentially diabetogenic T cells. Adoptive transfer of normal histocompatible T cells to diabetes-prone BB/W rats results in the partial correction of the peripheral T lymphopenia and the prevention of both insulitis and diabetes (12, 13). This protection has been attributed to a subset of donor-derived CD4+, TCR{alpha}ß+ T cells expressing RT6 on their surface (12). Furthermore, administration of depleting anti-RT6 mAb to diabetes-resistant rats, which are genetically related to BB/W rats, results in peripheral T lymphopenia phenotypically similar to that observed in unmanipulated BB/W rats and in the development of type I diabetes when the recipients are simultaneously treated with inducers of IFN-{alpha} (14).

In unmanipulated BB/W rats, infiltration of pancreatic islets by mononuclear cells becomes detectable at 4 wk, and the peak incidence of diabetes is observed around 3 mo (15). The protection of BB/W rats from diabetes afforded by adoptive transfer of histocompatible T cells is time dependent (16). The administration of normal T cells to BB/W recipients at 4–5 wk of age, around the time of initiation of insulitis, prevents the development of diabetes. In marked contrast, the diabetogenic process remains unaltered when normal T cells are adoptively transferred to 2-mo-old diabetes-prone BB/W recipients, in which insulitis is already apparent (16). The analysis of this differential effect of T cell reconstitution on antipancreatic autoimmunity has been hampered by the lack of suitable congenic strains of diabetes-resistant and diabetes-prone rats. In particular, the lack of genetic markers distinguishing donor- and recipient-derived T cells has precluded the recovery of donor-derived T cells that had been used for the reconstitution of 1- and 2-mo-old diabetes-prone recipients. Consequently, it has been impossible to directly analyze the differential capacity of donor-derived T cells to regulate the diabetogenic process in vivo.

We have developed a CD45 congenic strain of diabetes-prone BB/W rats, BB/W.7b. At different stages of the diabetogenic process, these animals were reconstituted with T cells isolated from diabetes-resistant BB rats (DR-BB/W) that differed from recipients at the CD45 and lyp loci. As expected, all BB/W.7b animals that were reconstituted with normal T cells at 2 mo of age developed diabetes. Surprisingly, however, the kinetics of onset of diabetes was enhanced compared with those of unreconstituted BB/W.7b animals. We demonstrate that the acceleration of the diabetogenic process resulted from the priming of diabetogenic T cells among donor-derived T cells. Thus, unfractionated T cells from normal donors were unable to regulate primed diabetogenic T cells of recipient origin or to prevent priming and expansion of the diabetogenic T cell precursors present in the donor inoculum. T cell reconstitution of 1-mo-old BB/W.7b rats prevented diabetes. However, the protective donor-derived T cells were unable to regulate primed diabetogenic T cells of BB/W origin in a cotransfer system, even in circumstances where protective T cells outnumbered diabetogenic T cell by >50-fold. Further, the T cells that were protective in a first recipient became diabetogenic when subsequently transferred to a second recipient. Thus, reconstitution of 1-mo-old diabetes-prone recipients with normal T cells was protective, but when donor T cells were rescued 3 mo later and used to reconstitute a 2-mo-old diabetes-prone recipient, the kinetics of onset of diabetes were enhanced. This observation demonstrates that early T cell reconstitution of BB/W rats does not result in a long term physical or functional depletion of islet cell-specific T cell precursors among the protective donor cells. Taken together, the results support the conclusion that normal T cells can prevent the initiation of an antipancreatic autoimmune process in BB/W rats; however, they are unable to regulate an autoimmune response once it has been initiated.


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

Isocongenic diabetes-prone (BB/W) and diabetes-resistant (DR-BB/W) BB rats that differ at the lyp locus were obtained from the University of Massachusetts (Worcester, MA). Wistar Furth (WF) rats were purchased from Charles River (Frederick, MD). BB and WF rats share the same MHC haplotype (RT1u) and are congenic for the two allelic forms of CD45, RT7a for BB rats and RT7b for WF animals. To be able to distinguish mononuclear cells (MNC) of BB/W origin from those of DR-BB/W origin we introduced the RT7b allele of WF rats into the genetic BB/W background. Lymphopenic and diabetic RT7a/b males resulting from the first backcross were used for further backcrosses with BB/W animals. RT7b congenic BB/W rats used in these studies originated from the fourth to sixth backcrosses. Animals were housed in specific pathogen-free conditions, and all the sentinels were negative for circulating Abs specific for the diabetogenic Kilham’s virus. Diabetes-prone rats were tested three times a week for the presence of glycosuria and ketonuria. Once animals became glycosuric, the diagnosis of diabetes was established on the basis of hyperglycemia (blood glucose, >16.7 mM) for 2 consecutive days. Some diabetic rats were treated with s.c. implants of insulin (Linplant, University of Toronto, Toronto, Canada).

mAbs, three-color immunofluorescence, and FACS analysis

The mAbs used in this study were affinity purified from hybridoma culture supernatants on rat anti-mouse Ig-Sepharose or mouse anti-rat Ig-Sepharose, and then conjugated with FITC, biotin, or PE using standard procedures. These mAbs included anti-rat Ig {kappa} (MARK1) (17), anti-CD8 (MRC-OX8) (18), anti-CD4 (W3/25) (18), anti-CD45RC (MRC-OX22) (19), anti-OX41 (a molecule expressed on macrophages and granulocytes in rats; MRC-OX41) (20), anti-CD11b/c (MRC-OX42) (20), and anti-CD5 (MRC-OX19) (21), which were provided by Dr. A. A. Like (Worcester, MA) with the permission of Dr. D. Mason (Oxford, U.K.). R73, a hybridoma secreting an mAb specific for a nonpolymorphic determinant of rat TCR{alpha}ß (22) was a gift from Dr. T. Hünig (Martinsried, Germany). The rat hybridomas DS4.23 (anti-RT6a), 6A5 (anti-RT6b), NDS-58 (anti-RT7a), and 8G6.1 (anti-RT7b) (23) were provided by Dr. D. Greiner (Worcester, MA) and Dr. M. Newton (Oxford, U.K.), respectively. G4.18, a mouse hybridoma secreting an mAb specific for rat CD3{epsilon} (24), was obtained from Dr. G. W. Butcher (Cambridge, U.K.) with the permission of Dr. B. M. Hall (University of New South Wales, Liverpool, Australia). Streptavidin-PE/Texas Red Tandem was purchased from Southern Biotechnology Associates (Birmingham, AL). Affinity-purified normal rat and mouse Ig as well as rat antiserum specific for mouse Ig were purchased from Biocan (Ontario, Canada). Affinity-purified goat anti-rat Ig anti-serum was purchased from Sigma (St. Louis, MO).

Suspensions of MNC were incubated with biotinylated mAb, followed by streptavidin-PE/Texas Red Tandem. PE-labeled and FITC-conjugated mAbs were then added simultaneously. Viable cells were gated using forward and side angle scatter and were analyzed flow cytometrically with a FACScan (Becton Dickinson, San Jose, CA).

Purification of T cells

T cells were enriched by negative selection using a rosetting procedure as previously described (25, 26). Briefly, donor T cells (DR-BB/W and WF) used in reconstitution experiments were purified from pooled splenic and lymph node MNC through the depletion of macrophages, B lymphocytes, and NK cells. The cell suspension was incubated with a mixture of mouse mAbs consisting of MARK-1, OX41, OX42, and 3.2.3. mAb-coated cells were mixed with SRBC (Cederlane, Hornby, Canada) coated with rat anti-mouse Ig. The cell suspension was rotated for 30 min at 4°C to allow rosette formation. To maximize aggregate formation and hence the degree of purification, the cell suspension was then mixed with normal mouse Ig-coated SRBC for 30 min at 4°C. Rosettes were separated by centrifugation at 200 x g for 1 min. The supernatant containing the T cells was recovered and analyzed. The degree of enrichment was assessed by FACS analysis. The purity of T cells from nonlymphopenic animals was routinely >98%.

Following reconstitution of diabetes-prone BB/W.RT7b and BB/W rats with T cells from DR-BB/W and WF donors, respectively, cells of donor origin were purified from splenic MNC as described above with some modifications due to the fact that the anti-RT7 mAbs used for the depletion of host T cells are of rat origin. The depleting Ab mixture included anti-RT7 mAb in addition to the mouse mAbs. This was followed by an incubation of the MNC with rat anti-mouse Ig {kappa} mAb 187 (27) to coat all the cells that had to be depleted with rat Abs. mAb-coated splenocytes were mixed with SRBC coated with goat anti-rat Ig. The cell suspension was rotated for 30 min at 4°C to allow rosette formation. The rosettes were separated by centrifugation at 200 x g for 1 min. The supernatant was collected and centrifuged at 600 x g for 10 min. The pellet was resuspended, mixed again with goat anti-rat Ig-coated SRBC, and rotated for 30 min at 4°C. The cell suspension was then mixed with normal rat Ig-coated SRBC for 30 min at 4°C. Rosettes were separated by centrifugation at 200 x g for 1 min. The supernatant containing donor-derived T cells was recovered and analyzed. More than 98% of the cells in the resulting cell suspension were donor derived.

RT6+ T cells of DR-BB/W origin were sorted from splenic and lymph node MNC using a FACStar (Becton Dickinson). Splenic T cells from acutely diabetic rats were isolated by three-step rosetting as described above.

T cell reconstitution of diabetes-prone BB rats and adoptive transfer of diabetes

T cell reconstitution of 1- and 2-mo-old diabetes-prone BB/W rats was achieved through one i.v. injection of T cells derived from 2-mo-old normal donors.

Thymectomized and sublethally irradiated (TX) rats were used as T cell recipients in adoptive transfer experiments. Three-week-old rats were thymectomized (28), rested for a week, and then exposed to gamma radiation immediately before T cell transfer. The sublethal doses of gamma radiation for 3- to 4-wk-old BB/W and WF rats were 5 and 7 Gy, respectively. Donor T cells were transferred immediately after purification or after in vitro activation. T cell activation was performed in bacteriological petri dishes at a concentration of 106 cells/ml in Iscove’s modified Dulbecco’s medium supplemented with 10% FCS. Cells were incubated with PMA (20 ng/ml; Sigma), ionomycin (25 µM; Calbiochem, San Diego, CA), and 20 U/ml of rIL-2 at 37°C for 72 h. At the end of the culture, an aliquot of the cells was kept for phenotypic analysis, and the remaining cells were injected i.v. into the recipients. In cotransfer experiments, cells were mixed following activation in the indicated ratios. Thereafter, recipients were checked three times per week for the development of diabetes. Diabetic animals were killed after 3 consecutive days of stable hyperglycemia. Nondiabetic recipients were sacrificed at various intervals after transfer. The pancreas was fixed in 1% formalin and paraffin embedded, and sections were stained with hematoxylin and eosin. MNC from lymph nodes and spleen were analyzed to determine the degree of engraftment of donor T cells.

Statistics

Statistical analysis of significance was performed using unpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
T cell reconstitution at 2 mo precipitates the onset of diabetes in BB/W rats

It has been previously demonstrated that T cell reconstitution of BB/W rats early in life (~1 mo old) prevents the development of diabetes, whereas reconstitution later in life (~2 mo old) is not protective (16). To gain insight into the mechanisms underlying this protection, or lack thereof, we developed a CD45 congenic strain of diabetes-prone BB/W rats, named BB/W.7b, which provided the opportunity to distinguish T cells of the isocongenic, diabetes-resistant strain DR-BB/W from those of BB/W origin. As illustrated in Table IGo, the prevalence and peak incidence of diabetes in BB/W.7b animals resulting from the fourth to sixth backcrosses, were similar to those observed in inbred, RT7a homozygous BB/W rats. The genetic heterogeneity of BB/W.7b used in this study, although limited, could have influenced their diabetogenic phenotype and resulted in minor histocompatibility differences with the DR-BB/W strain. These concerns can be ruled out for the following reasons. BB/W.7b animals resulting from the ninth backcross (BC) have now been generated in our laboratory. These animals exhibit a peak incidence of diabetes and a cumulative incidence of the disease similar to those in animals resulting from the fourth to sixth BC. Further, the kinetics and magnitude of T cell reconstitution of BB/W.7b rats from the ninth BC following injection of DR-BB/W T cells are superimposable on those observed in animals resulting from the fourth to sixth BC (data not shown). We therefore analyzed the differential effects of i.v. injection of 25–30 x 106 DR-BB/W T cells into BB/W.7b rats either early or late in life on the development of diabetes.


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  		Table I. Incidence of insulitis and diabetes in unmanipulated and T cell-reconstituted BB/W strains

 
In accordance with previous results (16), none of the BB/W.7b recipients that were reconstituted at 1 mo of age developed diabetes (Table IGo). Further, when these animals were sacrificed at 4 mo of age, their pancreatic islets were free of insulitis (not shown). In contrast, all animals that were reconstituted at 2 mo of age became diabetic, but importantly, diabetes occurred 2 wk earlier (p < 0.01) than in unmanipulated BB/W.7b animals (Table IGo). This precipitation of diabetes onset following T cell reconstitution at 2 mo of age was not peculiar to BB/W.7b rats, as it was also observed in BB/W recipients of DR-BB/W T cells (Table IGo). These results demonstrate that early and late T cell reconstitutions have an opposite effect on the antipancreatic autoimmune response of BB/W rats. A possible explanation for this observation is that the former reconstitution results in the expansion of T cells that can regulate priming and/or expansion of autoreactive T cells, while the latter T cell reconstitution leads to the recruitment of ß-cell-specific T cells of donor origin to the diabetogenic process. These possibilities were assessed using an adoptive transfer system.

Late T cell reconstitution of diabetes-prone BB/W rats results in the activation of donor-derived diabetogenic T cells

It has been demonstrated that T cells from acutely diabetic BB/W rats can adoptively transfer diabetes to young syngeneic recipients as long as donor T cells are preactivated in vitro (29). T cells of DR-BB/W origin that were used for reconstitution of BB/W.7b rats were purified from recipient splenocytes and subsequently tested for their capacity to adoptively transfer diabetes. Four-week-old thymectomized and sublethally irradiated (TX) BB/W rats were used as recipients of DR-BB/W T cells to rule out a possible contribution of recipient-derived T cells in the adoptive transfer system. In the absence of T cell transfer, TCR{alpha}ß+ T cells accounted for about 3% of the lymph node MNC, and <1% of splenic MNC in TX BB/W rats 1 mo after irradiation. One month after T cell transfer, secondary lymphoid organs of TX recipients contained 5- to 10-fold more T cells than those of nontransferred recipients (Fig. 1Go, A and B). Importantly, the nontransferred TX animals uniformly fail to develop diabetes or insulitis (Tables II and III).



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  		FIGURE 1. Phenotypic analysis of MNC present in the spleen (left) and lymph nodes (right) of TX BB/W rats that did not receive (A) or received (B) nonactivated DR-BB/W T cells. TX animals were 1 mo old at the time of transfer and 2 mo old at the time of analysis. DR-BB/W T cells that were adoptively transferred to TX BB/W recipients were isolated from diabetic BB/W.7b rats that had been T cell reconstituted at 2 mo. Expression of CD4 and TCR{alpha}ß on the surface of MNC from two representative animals is shown. Numbers indicate the percentages of cells in the corresponding quadrants.

 
DR-BB/W T cells were isolated from 3-mo-old diabetic BB/W.7b animals that had been reconstituted at 2 mo of age. At the time of sacrifice, donor-derived cells accounted for 49.8 ± 8.8% of splenic T cells (n = 8; Fig. 2Go). After purification, recipient-derived T cells accounted for <0.5% of the resulting cell suspension (Fig. 2Go). Following in vitro activation, T cells accounted for >90% of the total MNC population, and the proportion of T cells that expressed RT7b was <0.5% (Fig. 2Go).



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  		FIGURE 2. Phenotypic analysis of BB-DR/W T cells isolated from the spleens of 4-mo-old BB/W.7b rats that had been T cell reconstituted at 1 mo (left) or 2 mo (right). Expression of CD5 and RT7b on the surface of splenic MNC from two representative animals is shown before (upper panel) and after (middle panel) depletion of recipient-derived MNC as well as after activation (lower panel) of purified donor-derived T cells. Numbers indicate the percentages of cells in the corresponding quadrants.

 
Three weeks after adoptive transfer of 106 activated DR-BB/W T cells derived from BB/W.7b rats that had been reconstituted at 2 mo, all the TX recipients developed type 1 diabetes (Table IIGo). It could be argued that the few BB/W.7b T cells contaminating DR-BB/W T cells were responsible for the adoptive transfer of diabetes to TX recipients. Two observations minimize this concern. First, it has been demonstrated that T cells of BB/W origin need be preactivated to adoptively transfer diabetes ((29) (Table IIIGo). Therefore, we assessed the ability of DR-BB/W T cells used for the reconstitution of 2-mo-old BB/W.7b to adoptively transfer diabetes to TX rats in the absence of preactivation. As described in Table IIGo, all the TX recipients of nonactivated DR-BB/W T cells became diabetic. Further, the minimal number of preactivated BB/W T cells required for adoptive transfer of diabetes to TX recipients exceeds the number of BB/W T cells contaminating the inoculum of DR-BB/W T cells by >2 orders of magnitude (Table IIIGo). Specifically, while all TX recipients of 1.2 x 106 activated BB/W T cells developed diabetes, TX recipients of as many as 0.24 x 106 activated BB/W T cells remained diabetes and insulitis free (Table IIIGo). This number of BB/W T cells (0.24 x 106) is 100-fold larger than the number of BB/W.7b T cells contaminating the 6 x 104 DR-BB T cells that were still able to adoptively transfer diabetes to TX rats (Table IIGo).


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  		Table II. Adoptive transfer of diabetes by unfractionated DR-BB T cells used for the reconstitution of BB/W.7b rats

 

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  		Table III. Adoptive transfer of diabetes by T cells from diabetic BB/W donors to 4-wk-old TX recipients

 
Strains of rats genetically unrelated to BB/W animals have a potentially diabetogenic T cell repertoire

DR-BB/W and BB/W rats are isocongenic animals differing at the lyp locus (30), and the former strain can develop type 1 diabetes after various manipulations. For example, diabetes can be induced in DR-BB/W rats by cyclophosphamide treatment (31), sublethal irradiation (32), or the combined administration of anti-RT6 mAb and poly(I:C) (33). In the same circumstances, other strains of animals, including those sharing the MHC haplotype of BB/W rats, fail to develop diabetes (31). It could therefore be argued that the presence and activation of islet cell-specific cells among DR-BB/W T cells used to reconstitute 2-mo-old BB/W.7b rats are related to the isocongenic nature of donor and recipient strains. To determine whether this is the case, we substituted DR-BB/W T cells with WF T cells for the reconstitution of diabetes-prone BB/W animals.

We and others have previously demonstrated that the life span of naive T cells in BB/W rats does not exceed 6 days as a consequence of the lyp mutation (4, 5). Rescue of BB/W recent thymic emigrants from premature apoptotic death can occur through activation by their specific Ag (5). Therefore, the capacity of BB/W rat peripheral T cells to respond to a given Ag requires the persistence of a continuous thymic output of T cells and/or previous exposure to this Ag. When BB/W rats are thymectomized, their pool of recirculating T cells is only comprised of T cells that have been exposed to their specific Ag before thymectomy. We have previously illustrated this point using the minor histocompatibility differences that exist between the BB/W and WF strains. Thus, injection of WF T cells in euthymic BB/W rats is followed by the rapid rejection of donor cells. In contrast, injection of WF T cells in thymectomized BB/W rats is followed by an expansion of donor cells (5). However, if BB/W rats are thymectomized after the injection and subsequent rejection of WF T cells, the recipients can mount a secondary immune response against another injection of WF T cells (5).

Similarly, thymectomy of BB/W rats before the initiation of insulitis prevents diabetes, possibly because naive precursors of diabetogenic T cells disappear rapidly from the pool of recirculating T cells after thymectomy (Table IVGo). In contrast, thymectomy at 2 mo has little effect on the diabetogenic process (Table IVGo), possibly because activation of diabetogenic T cells occurred before thymectomy.


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  		Table IV. Development of insulitis and diabetes in thymectomized BB/W rats reconstituted with WF T cells

 
We used these observations on the differential effects of early and late thymectomy of BB/W rats on allogenic and autoimmune responses to assess the effects of early and late T cell reconstitution of BB/W rats with WF T cells. BB/W rats were thymectomized at 1 or 2 mo and 5 days later received an injection of 25 x 106 T cells purified from 2-mo-old WF donors. None of the BB/W rats that were thymectomized at 1 mo developed diabetes or insulitis independent of T cell reconstitution (Table IVGo). Thymectomy at 2 mo, without subsequent T cell reconstitution, had no effect on the incidence of diabetes in BB/W rats, although it slowed the progression of the disease. Thus, 88% of thymectomized BB/W rats became diabetic, with a peak incidence at 110 ± 9 days (Table IVGo). Two of the three BB/W rats that were thymectomized at 2 mo and received WF T cells developed diabetes 15–21 days after reconstitution (Table IVGo). At sacrifice >90% of splenic T cells were of WF origin in reconstituted BB/W rats. This high proportion of donor-derived T cells found as early as 3 wk after reconstitution is most likely the consequence of the rapid disappearance of approximately 80% of BB/W peripheral T cells after thymectomy (5, 34).

T cells of WF origin were isolated 1 mo after reconstitution from the spleen of BB/W recipients that had developed diabetes and/or insulitis and were adoptively transferred with or without preactivation to syngeneic or BB/W TX recipients (Table VGo). All TX recipients developed type 1 diabetes rapidly, independent of the activation state of WF T cells (Table VGo). In contrast, none of the TX recipients of WF T cells isolated from BB/W rats thymectomized and reconstituted at 1 mo developed diabetes or insulitis (Table VGo).


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  		Table V. Adoptive transfer of diabetes to thymectomized and irradiated recipients by WF T cells used for the reconstitution of thymectomized BB/W rats

 
The lack of development of diabetes in BB/W rats that were thymectomized and reconstituted with WF T cells at 1 mo (Table IVGo) and the inability of the reconstituting WF T cells to subsequently transfer diabetes to TX recipients (Table VGo) strongly suggest that the diabetogenicity of WF T cells used in the reconstitution of 2-mo-old BB/W rats is not the consequence of a GVH reaction. Importantly, histological analysis of the skin, liver, gut, and exocrine pancreas of TX BB/W recipients showed no evidence of GVH reaction (not shown).

Previous studies had indicated that T cell reconstitution of 2-mo-old BB/W rats had no effect on the ongoing diabetogenic process of recipients (16). In contrast, we demonstrate the pathogenicity of late T cell reconstitution in BB/W rats that results from the priming and expansion of islet cell-specific T cells of donor origin. Further, our results show that within the limits of our experimental conditions, the recruitment of ß-cell-specific T cells, present among unfractionated, donor-derived T cells, to the diabetogenic process cannot be regulated.

Lack of evidence for the existence of T cells that can regulate primed diabetogenic T cells

DR-BB/W T cells that were used for the reconstitution of 1-mo-old BB/W.7b rats were isolated from recipient splenocytes 2 mo after reconstitution. At that time, donor-derived cells accounted for 55–60% of splenic T cells (not shown). Adoptive transfer of 2 x 106 of these protective DR-BB/W T cells to TX BB/W recipients failed to induce either diabetes or insulitis among recipients (Table IIGo) in the following 3 mo. This innocuity of DR-BB/W T cells was independent of their state of activation at the time of transfer (Table IIGo). Similar results were obtained when DR-BB/W T cells were substituted with WF T cells (Table VGo). BB/W rats that were thymectomized at 1 mo did not develop diabetes regardless of whether they were reconstituted with WF T cells or left unmanipulated. When WF T cells were recovered 3 mo after reconstitution of 1-mo-old thymectomized BB/W rats and adoptively transferred to syngeneic or BB/W TX rats, none of the recipients developed diabetes or insulitis, independent of the activation state of WF T cells (Table VGo).

Increased levels of MHC class I expression and transcription of IFN-{alpha} are the first abnormalities that can be detected in the islet cells of unmanipulated 1-mo-old BB/W rats (35, 36). It is unclear how T cell reconstitution at this initial stage of the inflammatory process of pancreatic islets results in complete protection of the recipients from both diabetes and insulitis. T cell reconstitution could prevent activation of diabetogenic T cells and/or regulate the effector function of a limited number of primed, self-specific T cells. Using an adoptive cotransfer system, we sought to determine whether DR-BB/W T cells lost their diabetogenic potential and/or acquired the capacity to modulate diabetogenic T cells during their expansion in 1-mo-old BB/W.7b rats.

DR-BB/W T cells used for the reconstitution of 1- and 2-mo-old BB/W.7b rats were recovered from 4-mo-old recipients, mixed in a ratio of four protective T cells to one diabetogenic T cell, and transferred, without preactivation, to TX BB/W rats. As illustrated in Table IIGo, DR-BB/W T cells derived from BB/W.7b recipients that had been reconstituted at 1 mo and protected from diabetes were unable to prevent the adoptive transfer of diabetes induced by DR-BB/W T cells recovered from diabetic BB/W.7b rats that had been reconstituted at 2 mo. This lack of protection was reproduced in two other experiments, one of which involved the transfer of T cell populations that were mixed in a similar 4:1 ratio after in vitro activation (data not shown). It is therefore unlikely that the DR-BB/W T cells that expanded in 1-mo-old BB/W.7b rats protected these animals from pancreatic inflammation through regulation of the effector functions of diabetogenic T cells. However, the limited numbers of DR-BB/W T cells recovered from reconstituted BB/W.7b rats did not allow us to test higher ratios of protective to diabetogenic T cells in our cotransfer system. It could be argued, therefore, that the clonal expansion of diabetogenic T cells among DR-BB/W T cells recovered from diabetic BB/W.7b donors far exceeded the regulatory capacity of DR-BB/W T cells recovered from protected BB/W.7b donors. We reasoned that the optimal ratio of regulatory to potentially diabetogenic T cells would be found in the protective DR-BB/W T cell population that was used for the reconstitution of 1-mo-old BB/W.7b rats. We determined whether this T cell population would remain nondiabetogenic after transfer into animals whose pancreatic islets are the target of an ongoing autoimmune response mediated by endogenous T cells.

Protective DR-BB/W T cells can become diabetogenic

We reconstituted 2-mo-old BB/W.7b rats with 5 x 106 DR-BB/W T cells that had been previously used for the reconstitution of 1-mo-old BB/W.7b rats and had persisted in these initial recipients for 3 mo. The subsequent 2-mo-old BB/W.7b recipients of these DR-BB/W T cells became diabetic 3 wk after reconstitution (Table IGo). The relatively low level of T cell reconstitution observed in these animals was probably due to the low number of DR-BB/W T cells injected at 2 mo (Table IGo). DR-BB/W T cells used in two successive reconstitutions were purified from the second BB/W.7b recipients, activated, and then injected into TX BB/W recipients. All the TX BB/W recipients became diabetic 21 days after transfer (Table IIGo). These results demonstrate that T cell reconstitution of 1-mo-old BB/W.7b rats does not result in a long term physical or functional depletion of islet cell-specific T cell precursors among donor cells or in the expansion of T cells that can regulate the activation and expansion of diabetogenic T cells.

T cell subsets, including RT6+ T cells, from unmanipulated DR-BB/W donors become diabetogenic when cotransferred with low numbers of T cells from diabetic BB/W donors

The recruitment of adoptively transferred DR-BB/W T cells to the diabetogenic process of BB/W recipients occurs when T cell reconstitution is performed after the initiation of insulitis. This observation suggests that an established inflammatory process in the recipient islets is a prerequisite for the priming and expansion of diabetogenic cells among donor-derived T cells. We determined whether T cells from DR-BB/W rats would become diabetogenic when adoptively transferred to insulitis-free TX recipients containing primed diabetogenic T cells in numbers too low to induce insulitis by themselves.

Decreasing numbers of activated T cells isolated from acutely diabetic and unmanipulated BB/W donors were injected alone or together with a fixed number of activated DR-BB/W T cells into TX recipients (Table IIIGo and V). Importantly, the DR-BB/W T cells used in the initial series of cotransfers were isolated from the spleens of 3-mo-old BB/W.7b animals that had been T cell reconstituted at 1 mo. Therefore, induction of diabetes or insulitis could only originate from BB/W T cells. A minimum of 106 T cells from acutely diabetic BB/W donors were required to transfer diabetes or insulitis when injected alone, and these cells had to be activated in vitro before transfer (Table IIIGo). However, diabetes could be induced by adoptive transfer of as few as 103 activated BB/W T cells when these cells were coinjected with DR-BB/W T cells (Table VIGo). This induction of diabetes by the cotransfer of BB/W and DR-BB/W T cells was observed even in the absence of activation in vitro (not shown). Neither diabetes nor insulitis could be induced when T cells from 1-mo-old prediabetic BB/W rats were substituted for T cells from diabetic BB/W donors in our cotransfer system (not shown), suggesting the need for in vivo priming and expansion of diabetogenic T cells in our cotransfer model. Three conclusions can be drawn from these results. Advanced insulitis does not seem necessary for the priming and clonal expansion of diabetogenic cells among DR-BB/W T cells. The frequency of diabetogenic cells among splenic T cells from diabetic BB/W rats is at least 10-3. DR-BB/W T cells recovered from BB/W.7b rats that had been T cell reconstituted at 1 mo failed to regulate diabetogenic BB/W T cells even when DR-BB/W T cells exceeded BB/W T cells by a factor of 700.


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  		Table VI. Transfer of diabetes and insulitis to 4-wk-old TX BB/W rats by various combinations of T cell subsets derived from DR-BB/W donors and T cells from diabetic BB/W rats

 
The results of the above cotransfer experiments led us to determine whether T cell subsets isolated from unmanipulated and diabetes-resistant DR-BB/W rats could mediate diabetes in TX BB/W recipients when these cells were coinjected with subdiabetogenic numbers of BB/W T cells. The lack of peripheral T cells expressing RT6 on their surface in diabetes-prone BB/W rats, the induction of diabetes in normal rats through chronic, Ab-mediated depletion of RT6+ T cells, and the protection from diabetes afforded by reconstitution of BB/W rats with RT6+ T cells have all suggested that the RT6+ T cell subset contains cells with the capacity to regulate potentially diabetogenic T cells (12, 13, 14). However, when we cotransferred 7 x 105 purified RT6+ T cells of DR-BB/W origin with 104 T cell from diabetic BB/W donors to TX BB/W recipients, diabetes ensued within 3 wk (Table VIGo). Thus, a large excess of RT6+ T cells is unable to regulate a low number of primed, self-specific T cells, and in fact, RT6+ T cells become diabetogenic.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The lyp allele of the diabetes-prone BB/W strain is responsible for the premature apoptotic death of recent thymic emigrants and the lack of long-lived naive T cells (4, 5, 37). Consequently, peripheral T cells with a given Ag specificity are either T cells that emigrated from the thymus in the previous 24–48 h or T cells that were rescued from premature apoptosis through Ag activation (5). The differential effect of early and late thymectomy on the development of diabetes in BB/W rats results from the short life span of naive T cells in these animals (4, 5, 37). Thymectomy before the initiation of islet inflammation protects animals from diabetes as a consequence of the rapid apoptotic death of potentially diabetogenic recent thymic emigrants (38). In contrast, when thymectomy is performed after the initiation of insulitis, the course of the diabetogenic process is unaltered (S. Ramanathan, unpublished observation). A likely basis for this observation is that at the time of surgery, islet cell-specific T cells have already been rescued from apoptosis through activation by immunogenic islets. In this context, it is puzzling that T cell reconstitution of diabetes-prone BB/W rats after the initiation of insulitis did not modify the course of the diabetogenic process (16). It has been demonstrated that adoptive transfer of T cells to animals that are T cell deficient results in an Ag-driven expansion of donor-derived T cells (39). The unaltered time course of antipancreatic autoimmunity after late T cell reconstitution of BB/W rats suggested that donor-derived T cells were unable to regulate recipient-derived, autoreactive T cells and yet were capable of prohibiting their own enrollment into the ongoing autoimmune process. This apparent paradox led us to develop CD45 congenic strains of diabetes-prone and diabetes-resistant rats to analyze the regulatory or, alternatively, diabetogenic potential of T cells derived from diabetes-resistant donors that had been used for the reconstitution of diabetes-prone animals.

Our results demonstrate that T cell reconstitution of BB/W rats after the initiation of insulitis is not neutral vis-a-vis the ongoing autoimmune process of the recipients. Rather, it is deleterious due to the recruitment of donor-derived T cells to the diabetogenic process. This precipitation of diabetes after late T cell reconstitution demonstrates that unfractionated T cells originating from diabetes-resistant donors are unable to regulate primed diabetogenic BB/W T cells even in circumstances where normal T cells outnumber pathogenic T cells by almost 1000-fold. Furthermore, the reconstituting T cells fail to regulate their own autoreactive potential.

This is the first demonstration of the exacerbation of an ongoing autoimmune process through adoptive transfer of unfractionated T cells derived from normal donors. This observation probably results from the effects of the BB/W lyp allele on the life span of T cells. In most cases where regulation of an ongoing autoimmune process by adoptively transferred T cells was attempted, the recipients were not lymphopenic, and/or the life spans of donor and recipient T cells were similar. If activation of donor-derived, autoreactive T cells occurred in these circumstances, their clonal expansion was probably limited compared with that of recipient-derived T cells and hence unlikely to profoundly alter the course of the ongoing autoimmune process (40, 41). This is not the case in the model of BB/W rats reconstituted with T cells, in which both resting and activated T cells of recipient origin are short lived (5), while donor T cells have a normal life span. Consequently, the clonal expansion of donor-derived, islet-specific T cells following their adoptive transfer in lymphopenic BB/W rats and the subsequent contribution of these donor T cells to the ongoing antipancreatic autoimmune process are likely to overcome those of recipient T cells. Our results also show that the lyp allele of the BB/W rat, which is known to contribute to the development of diabetes, is at least partly responsible for the slow progression of T cell-mediated ß-cell destruction in unmanipulated animals.

It has been suggested that one potential mechanism through which the BB/W lyp allele contributes to the development of antipancreatic autoimmunity is the impaired development of T cells that express RT6 and can regulate self-specific T cells (16, 33, 42, 43, 44, 45). This hypothesis is based on three observations. BB/W rats lack peripheral T cells expressing RT6 on their surface (43). Diabetes can be induced in nonlymphopenic rats that are normally diabetes resistant, through Ab-mediated depletion of RT6+ T cells (33, 44, 45). Early T cell reconstitution of diabetes-prone BB/W rats with cells expressing RT6 prevents diabetes (42). Our cotransfer experiments demonstrate that RT6+ T cells derived from diabetes-resistant donors are potentially diabetogenic and fail to regulate pathogenic T cells even in circumstances where RT6+ T cells outnumber diabetogenic T cells by almost 100-fold. These results argue against a regulatory role for a subset of RT6+ T cells against antipancreatic autoimmunity, at least when this subset is part of unfractionated T cells, or a T cell population enriched in RT6+ cells.

While our results confirm that early T cell reconstitution of BB/W rats protects these animals from diabetes (16), they provide indirect evidence that this protection does not result from the development of an islet cell-specific regulatory T cell subset. As previously reported, we could not detect an infiltration of pancreatic islets by mononuclear cells in protected animals (16). The protective T cells that were allowed to expand in BB/W recipients for 3 mo were unable to regulate very low numbers of diabetogenic T cells in our cotransfer experiments. More importantly, the diabetogenic potential of these protective T cells, as shown by our "double-parking" experiments and our cotransfer experiments, is indistinguishable from that of T cells derived from unmanipulated, diabetes-resistant donors.

How, then, does T cell reconstitution of 1-mo-old recipients mediate protection? BB/W rat T cells can mount an immune response against alloantigens and nominal Ags, including self Ags (5). Although these T cell responses are relatively slow compared with those observed in animals carrying a different lyp allele, they are vigorous enough to reject an allograft and destroy pancreatic islets (1, 5). It is therefore tempting to propose that the protective effect of normal T cells early in life does not result from the response of these protective T cells to a specific Ag, but, rather, from their capacity to restore a pattern of recirculation and/or cytokine secretion critical to preventing pancreatic ß-cells from becoming immunogenic. This protection from diabetes afforded by T cells would require a functional lyp gene.


   Acknowledgments
 
We thank Michael Julius for his critical reading of this manuscript and C. Cantin (Cytometrics, Inc.) for expert assistance with flow cytometry.


   Footnotes
 
1 This work was supported by a grant from the Medical Research Council of Canada and a postdoctoral fellowship from the Juvenile Diabetes Foundation International (to S.R.). Back

2 Address correspondence and reprint requests to Dr. Philippe Poussier, Arthritis and Immune Disorder Research Center, 620 University Avenue, Suite 700, c/o Ontario Cancer Institute, Toronto, Ontario, Canada M5G 2M9. E-mail address: Back

3 Abbreviations used in this paper: BB/W, diabetes-prone Biobreeding/Worcester rats; DR-BB/W, diabetes-resistant BB/W rats; WF, Wistar Furth; MNC, mononuclear cells; PE, phycoerythrin; TX, thymectomized and sublethally irradiated; BC, backcross; IDDM, insulin-dependent diabetes mellitus. Back

Received for publication December 3, 1998. Accepted for publication February 8, 1999.


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