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The Journal of Immunology, 2004, 172: 4624-4629.
Copyright © 2004 by The American Association of Immunologists

Terminal Deoxynucleotidyltransferase Deficiency Decreases Autoimmune Disease in Diabetes-Prone Nonobese Diabetic Mice and Lupus-Prone MRL-Faslpr Mice1

Ian F. Robey, Melissa Peterson, Marc S. Horwitz, Dwight H. Kono, Thomas Stratmann, Argyrios N. Theofilopoulos, Nora Sarvetnick, Luc Teyton and Ann J. Feeney2

Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The wide diversity of the T and B Ag receptor repertoires becomes even more extensive postneonatally due to the activity of TdT, which adds nontemplated N nucleotides to Ig and TCR coding ends during V(D)J recombination. In addition, complementarity-determining region 3 sequences formed in the absence of TdT are more uniform due to the use of short sequence homologies between the V, D, and J genes. Thus, the action of TdT produces an adult repertoire that is both different from, and much larger than, the repertoire of the neonate. We have generated TdT-deficient nonobese diabetic (NOD) and MRL-Faslpr mice, and observed a decrease in the incidence of autoimmune disease, including absence of diabetes and decreased pancreatic infiltration in NOD TdT-/- mice, and reduced glomerulonephritis and increased life span in MRL-Faslpr TdT-/- mice. Using tetramer staining, TdT-/- and TdT+/+ NOD mice showed similar frequencies of the diabetogenic BDC 2.5 CD4+ T cells. We found no increase in CD4+CD25+ regulatory T cells in NOD TdT-/- mice. Thus, TdT deficiency ameliorates the severity of disease in both lupus and diabetes, two very disparate autoimmune diseases that affect different organs, with damage conducted by different effector cell types. The neonatal repertoire appears to be deficient in autoreactive T and/or B cells with high enough affinities to induce end-stage disease. We suggest that the paucity of autoreactive specificities created in the N region-lacking repertoire, and the resultant protection afforded to the newborn, may be the reason that TdT expression is delayed in ontogeny.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The nuclear enzyme TdT plays a critical role in generating immune receptor diversity. During rearrangement of Ig and TCR V region genes, TdT catalyzes the addition of N nucleotides without a DNA template to V-D and D-J junctions in both Ig and TCR as well as the V-J junctions in the TCR (1, 2, 3). During the fetal and newborn period, TdT activity is absent, resulting in an N region-deficient Ag receptor repertoire significantly lacking in the diversity observed in the junctional regions of Igs and TCRs of adult animals (4, 5, 6, 7). The absence of TdT also limits the diversity of the junctions that are created in the neonate, because junctions are far more likely to be formed at the site of short sequence homologies, a common feature between the 3' end of DH and 5' end of JH segments (4, 8, 9, 10). The coding ends of the TCR V region genes have few of these short sequence homologies; therefore, neonatal TCR junctions are primarily restricted only by the lack of N additions (11). The resulting fetal/neonatal Ag receptor repertoire is thus much smaller than, and different from, the adult repertoire.

The reason for having a smaller and different neonatal immune repertoire is unknown, but it has been suggested that the early repertoire is purposefully restricted to ensure the reproducible production of particular VDJ combinations with the appropriate junctional sequences, which may produce certain key specificities (9, 12). One such example is anti-phosphorylcholine Abs, which are important for neutralizing Streptococcus pneumonia infection. These Abs do not contain N regions and possess uniform VDJ junctions created by homology-directed recombination (9, 13). Mice with an inactivated TdT gene become healthy adults and live normal life spans (2, 3, 14). These animals mount immune responses comparable to their wild-type littermates, including CTL responses to viruses and proliferative T cell responses to the dominant peptide epitopes of protein Ags. Comparable serum Ab responses to a variety of Ags were also observed (14). Thus, these mice have not shed light on the reason for having a TdT-deficient repertoire early in life, followed by a TdT-containing repertoire later on.

The TdT-deficient B cell repertoire was shown to demonstrate a lower degree of polyreactivity compared with wild-type B cells (15). In contrast, the T cell repertoire in TdT-/- mice showed a higher level of epitope promiscuity in a study of CTL clones (16), and furthermore, T cells from TdT-/- mice display increased thymic positive selection (17). It has therefore been proposed that the germline repertoire may be biased toward self-reactivity, and that the absence of the diverse N region diverse repertoire may increase susceptibility to development of autoimmune disease. Contrary to this hypothesis, however, two strains of TdT-deficient lupus-prone mice, (NZB x NZW)F1 and MRL-Faslpr, showed that the TdT deficiency resulted in increased survival and less severe autoimmune disease (18, 19, 20), and the nonlupus TdT-deficient B6-Faslpr mice also show lower titers of anti-dsDNA.

In this study, we extend these observations to a very different autoimmune disease, type I diabetes. We bred the TdT deficiency onto nonobese diabetic (NOD)3 mice, and these NOD TdT-/- mice are far less prone to development of diabetes than NOD TdT+/+ littermates. Histological examination of age-matched prediabetic pancreata from TdT+/+ and TdT-/- NOD mice reveals decreased severity of islet infiltration in TdT-/- NOD animals. Thus, in two very distinct autoimmune diseases, TdT deficiency decreases the onset of end-stage disease, and diminishes many of the earlier symptoms of disease. Our hypothesis is that the early T or B repertoire, or both, may have a lower frequency of autoreactive lymphocytes with high enough affinity to cause autoimmune disease. Therefore, the neonate is protected against autoimmune disease by the early immune repertoire that shows a paucity of cells with autoreactive potential, and this may be the reason for the delayed onset of TdT expression.


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

NOD and MRL-Faslpr mice were obtained from the breeding colony at The Scripps Research Institute. TdT-/- mice were kindly given to us by D. Mathis and C. Benoist (Harvard University, Cambridge, MA) several years ago via M. Bevan (University of Washington, Seattle, WA), and are maintained in our breeding colony under specific pathogen-free conditions. TdT-deficient mice were backcrossed onto the NOD background and were screened for TdT on tail DNA (16). Heterozygotes were then intercrossed to obtain fourth through ninth generation NOD TdT-/- and TdT+/+ littermates for the analyses shown in this study. Selected N4 backcross mice were screened for some Idd loci (Idd1, 3, 10, 16, 17, 18) by Genescan for microsatellite markers on chromosomes 3, 15, and 17 (21), and any that were heterozygous at any of those loci were not used for breeding. Four N5 backcross mice were screened for 62 microsatellite markers covering all the chromosomes, and only two markers, in addition to the one near TdT on chromosome 19, showed heterozygosity. Some analyses were done on NOD TdT+/+ and TdT-/- homozygous lines that were generated from NOD TdT+/+ and TdT-/- N7 or N9 generation parents. MRL-Faslpr TdT-/- mice had been backcrossed for 13 generations, and lines of MRL-Faslpr TdT-/- and MRL-Faslpr TdT+/+ mice were established from N13 intercross littermates.

Assessment of diabetes

NOD TdT-/- and NOD TdT+/+ mice were analyzed for blood glucose levels using a one-step Bayer Glucometer Elite (Bayer, Elkhart, IN) after 20 wk of age and monitored every 2 wk for 1 year. Mice with blood glucose levels higher than 300 mg/dl after two consecutive readings were scored as diabetic. Animals surviving for 1 year without developing hyperglycemia were sacrificed.

Pathology

Lymphocytic infiltration of the pancreas was evaluated on H&E-stained paraffin sections of pancreas from NOD mice. At least two sets of sections, 100 µm apart, were analyzed for each mouse, and the scoring was done blindly. In general, 20–35 islets were counted from each pancreas. Pancreata were scored for insulitis (substantial mononuclear cell islet infiltration) and peri-insulitis (mononuclear cells surrounding the islet) using criteria, as previously described (22). In MRL-Faslpr mice, spleens and brachial, axial, and inguinal lymph nodes (LN) were removed and weighed. Kidney sections were fixed in Bouin’s solution and stained with periodic acid-Schiff and hematoxylin, and glomerulonephritis (GN) was scored blindly using a 1–4 scale (23). Macroscopic assessment of skin lesions, predominantly found on the back of the neck and the ears, was perfomed.

Flow cytometry

Fluorescent dye-conjugated Abs reactive with CD4, CD25, B220, and CD8 were purchased from BD PharMingen (San Diego, CA) and eBioscience (San Diego, CA). Production of BDC 2.5 TCR-specific MHC Ag7/2.5mi and Ag7/GPI control tetramers has been previously reported (24). Soluble purified TCRs were biotinylated and tetramerized using PE-labeled streptavidin. LN cells were treated for 1 h with Fc block and 5 mg/ml avidin at room temperature, washed, then incubated with tetramers and Abs (CD4 FITC, CD8 PerCP Cy5, and B220 PerCP Cy5) for 1 h on ice. PerCP Cy5+ cells (B and CD8 cells) were excluded from the analysis. Data from live cells were immediately acquired on a FACSort and analyzed with CellQuest software (BD Biosciences, San Diego, CA). Propidium iodide was used to exclude dead cells.

Statistics

Survival was analyzed by Kaplan-Meier statistic using censored events with significance determined by log-rank (Mantel-Cox) test. The unpaired t test or Fisher’s exact test was used to compare groups, where indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increased survival of TdT-deficient female NOD mice

Female littermates from generations N4 to N8 were assessed for incidence of hyperglycemia. Beginning at 20 wk of age, female mice were examined for glucose levels and checked every 2–3 wk. After 1 year, mice with glucose levels lower than 300 mg/dl were sacrificed and considered disease survivors. In the NOD TdT+/+ group of 26 mice, 54% were hyperglycemic, while in the NOD TdT-/- group only 2 mice of 34 (~5.9%) became hyperglycemic, 1 at the final bleed at 53 wk (Fig. 1).



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  		FIGURE 1. Incidence of diabetes in NOD TdT-/- and NOD TdT+/+ mice. Twenty-five female N4-N8 NOD TdT+/+ (solid line) and 34 NOD TdT-/- (dashed line) littermates were followed for 1 year. The total number of diabetic mice was 13 (52%) for the TdT+/+ group and 2 (5.9%) for the TdT-/- group (p < 0.0001).

 
Incidence of insulitis in TdT-deficient NOD mice

Pancreatic sections were examined to determine the incidence of peri-insulitis and insulitis. Female N7 and N8 generation NOD TdT-/- (n = 23) and NOD TdT+/+ (n = 14) mice were sacrificed between the ages of 18 and 33 wk, and pancreata were removed for histological examination. The percentage of islets with insulitis and peri-insulitis for each mouse is shown in Fig. 2, and representative islets are shown in Fig. 3. The total number of islets counted in all of the TdT+/+ mice was 311, with 21% of those islets displaying peri-insulitis and 19.2% insulitis. The total number of islets counted in the TdT-/- group was 540, with 13% of them showing peri-insulitis and 7.6% with insulitis. Fisher’s exact test shows a highly significant difference between the extent of both insulitis and peri-insulitis in NOD TdT+/+ vs TdT-/- mice (p < 0.0002).



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  		FIGURE 2. Peri-insulitis and insulitis scores in individual NOD TdT-/- and NOD TdT+/+ mice. Crossbars indicate mean percentage. Percentage of peri-insulitis (A) mean ± SEM is 21.0 ± 4.5 for TdT+/+ and 13.0 ± 2.6 for TdT-/- mice (p = 0.101). The percentage of insulitis (B) mean ± SEM is 19.2 ± 5.7 for TdT+/+ and 7.7 ± 3.3 for TdT-/- mice (p = 0.066).

 


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  		FIGURE 3. Histology of pancreata from NOD TdT-/- and NOD TdT+/+ mice. H&E-stained islets from age-matched mice. A, Representative islet from NOD TdT-/- mice showing no infiltration. B, Representative islet from NOD TdT+/+ mice showing insulitis.

 
Analysis of CD4+CD25+ regulatory T cells in TdT-deficient NOD mice

Decreased incidence of diabetes and islet infiltration in NOD TdT-/- mice may be a reflection of an increased number of CD4+CD25+ T cells that regulate autoreactive T cells in the periphery (25). We analyzed spleen and thymus of age-matched NOD TdT-/- and TdT+/+ female littermates between the ages of 5 and 9 wk for the presence of these cells by flow cytometry. CD4+CD25+ T cell populations in all animals comprised ~3% of the thymocytes and splenocytes (Table I). Thus, these experiments revealed no increase in the population of CD4+CD25+ regulatory T cells in NOD TdT-/- mice.


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  		Table I. CD4+CD25+ T cell populations in NOD TdT-/- and NOD TdT+/+ micea

 
Analysis of BDC 2.5 T cells in TdT-deficient NOD mice

Another possibility for the decreased levels of hyperglycemia and pancreatic infiltration in NOD TdT-/- mice is the absence of certain diabetogenic T cells involved in mediating disease pathology. BDC 2.5 cells are a CD4+ clone of islet-specific cells that can accelerate diabetes upon transfer into young NOD mice. Using an Ag7 MHC tetramer specific for the diabetogenic BDC 2.5 TCR (Ag7/2.5mi), male and female TdT-/- and TdT+/+ NOD mice between the ages of 4 and 9 wk of age were compared for the presence of the BDC 2.5 T cell clonotype (Fig. 4). The results from flow cytometry experiments on pancreatic LN showed similar frequencies between the aged-matched NOD TdT-/- and TdT+/+ mice (Table II). These data suggest there is no significant decrease in the frequency of the T cell population reactive with the BDC 2.5 mimetope tetramer in NOD TdT-/- mice.



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  		FIGURE 4. FACS profile of pancreatic LN cells stained with the Ag7/2.5mi tetramer. Pancreatic LN cells from NOD TdT+/+ and NOD TdT-/- mice were stained with Abs and tetramer, and were gated on CD4+ cells. Ag7/2.5mi tetramer-staining CD4 cells are shown in the box. A total of 0.25 and 0.24% of CD4+ cells was stained, respectively, in the NOD TdT+/+ and NOD TdT-/- mice.

 

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  		Table II. Analysis of CD4+ T cells staining with the Ag7/2.5mi tetramer in pancreatic LN of NOD TdT-/- and NOD TdT+/+ micea

 
Increased survival of TdT-deficient MRL-Faslpr mice

Our initial study on TdT-/- MRL-Faslpr mice was conducted on littermates from mice from the N2-N5 generations (19). We have now made fully congenic 13th generation MRL-Faslpr TdT-/- mice. MRL-Faslpr TdT-/- mice (n = 24) and MRL-Faslpr TdT+/+ mice (n = 38) were analyzed for survival for at least 10 mo. The median age of survival for MRL-Faslpr TdT+/+ mice was 33 wk, which is older than that in our previous study, but is similar to the current median age of death of our main MRL-Faslpr colony. Similar to the previous study, MRL-Faslpr TdT-/- mice were observed to have a significantly greater rate of survival. In this current study, 58% of the MRL-Faslpr TdT-/- mice were alive at 10 mo compared with only 18% of the MRL-Faslpr TdT+/+ mice (Fig. 5).



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  		FIGURE 5. Life span of MRL-Faslpr TdT-/- and TdT+/+ mice. Male and female N13 MRL-Faslpr TdT+/+ (solid line) and MRL-Faslpr TdT-/- (dashed line) mice were followed for 44 wk. The Kaplan-Meyer plot was generated from 38 TdT+/+ and 24 TdT-/- mice. Eighty-two percent of the MRL-Faslpr TdT+/+ mice were dead by 44 wk, whereas only 42% of the MRL-Faslpr TdT-/- had died by the same time point (p = 0.0034).

 
Pathology of TdT-deficient MRL-Faslpr mice

MRL-Faslpr TdT-/- and MRL-Faslpr TdT+/+ mice aged 26–30 wk were sacrificed, and spleens and LN were weighed in each group. Spleen weight was decreased in the MRL-Faslpr TdT-/- mice compared with the MRL-Faslpr TdT+/+ animals, but not sufficiently to achieve significance (Table III). However, the difference in LN weight between the two groups was highly significant (p = 0.0004). To determine whether the spleen or LN weights increased with age, we analyzed a small group of older mice (38–42 wk, and found that spleen weights increased in the MRL-Faslpr TdT-/- mice to that of the younger MRL-Faslpr TdT+/+ mice. LN weight also increased to a value intermediate between that of the younger MRL-Faslpr TdT-/- and MRL-Faslpr TdT+/+ mice. Thus, lymphadenopathy is significantly delayed in MRL-Faslpr TdT-/- mice.


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  		Table III. Pathology results for MRL-Faslpr TdT-/- and MRL-Faslpr TdT+/+ mice

 
In the previous study, GN scores were not significantly different on the 6-mo survivors, but the fact that many of the MRL-Faslpr TdT+/+ mice had died by that time places a limitation on the interpretation of that data. In this study, mice were analyzed at various time points to determine the relative GN scores. In contrast to our previous study with N3 MRL-Faslpr TdT-/- mice, there was a notable difference in GN scores between the MRL-Faslpr TdT-/- and MRL-Faslpr TdT+/+ mice. The mean GN score for MRL-Faslpr TdT-/- mice was 1.14, whereas MRL-Faslpr TdT+/+ averaged 3.09 for the mice aged 26–30 wk (p < 0.0001) (Table III). Even in the older MRL-Faslpr TdT-/- mice, little GN was observed, and the difference in GN scores between these older TdT-/- mice and the younger TdT+/+ mice was still highly significant (p = 0.008).

Skin lesion scores were higher overall in MRL-Faslpr TdT+/+ (78% compared with 47% TdT-/- animals) by 10 mo (p = 0.0023) (Table IV).


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  		Table IV. Skin lesions in MRL-Fasfpr TdT+/+ and TdT-/- micea

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show NOD TdT-/- mice, 6–8 generations backcrossed, rarely develop diabetes (6% incidence) as characterized by hyperglycemia. These mice did show evidence of pancreatic islet infiltration, but the degree of infiltration was less severe than in NOD TdT+/+ littermates. These findings are in agreement with unpublished observations by Gilfillan and colleagues (mentioned in Ref. 18). The current data also extend previous observations of decreased incidence of various symptoms of lupus in TdT-deficient MRL-Faslpr and (NZB x NZW)F1 mice (18, 19, 20).

TdT is expressed in developing T and B cells of adult mice during Ag receptor recombination and functions to greatly diversify the complementarity-determining region 3 (CDR3) segments of the Ag binding site. Therefore, its impact on Ag specificity and affinity of the T and B cell repertoires is significant. Lupus is a characteristic Ab-mediated autoimmune disease, although T cells are also essential, presumably at least in part due to their role in somatic hypermutation and isotype switching involved in the generation of pathogenic high affinity autoantibodies (26, 27). Conversely, diabetes is a classic T cell-mediated disease, with CD8 cells being the final effectors of pancreatic {beta} cell destruction. These combined findings suggest a global ameliorating effect of TdT deficiency in animals with autoimmune syndromes.

CD4+CD25+ T cells are a well-characterized subset of regulatory cells that controls self-reactive cells (28, 29, 30, 31). They appear late in thymic development and represent ~5–10% of the CD4+ T cell population (32). These regulatory T cells have been implicated in the maintenance of peripheral self-tolerance and can delay or prevent the development of diabetes when transferred into experimental mice (25, 33). Moreover, it has been reported that NOD mice have a relative deficiency of CD4+CD25+ T cells in the thymus and spleen, suggesting a possible link between the decrease in CD4+CD25+ regulatory T cells and autoimmune disease (25). The results from the current study, however, showed comparable frequencies for CD4+CD25+ T cells in NOD TdT+/+ and TdT-/- littermates in both thymus and spleen. Thus, there is no evidence for an enhanced CD4+CD25+ regulatory component in NOD TdT-/- mice to explain the decreased disease incidence.

There are several documented T cell clones known to accelerate or mediate diabetes in NOD animals. The BDC 2.5 is an Ag7 class II-restricted V{beta}4/V{alpha}1 CD4+ T cell clone derived from a diabetic female NOD mouse (34). These T cells accumulate as peri-islet infiltrates early on (~5 wk) in NOD mice (24, 35), and, when transferred to young NOD recipients or to nondiabetic mice, induce rapid onset of islet destruction (36, 37). In BDC 2.5 transgenic mice, CD4+ thymocytes develop and migrate to the peripheral lymphoid organs, where they rapidly infiltrate into the pancreatic islets and mediate {beta} cell destruction (35). The BDC 2.5 TCR can be identified on cells using an agonistic MHC-mimetic peptide (2.5mi) bound to a soluble Ag7 MHC molecule (24, 38). We examined NOD TdT-/- mice to determine the frequency of BDC 2.5 CD4+ T cells using the 2.5 peptide/Ag7 MHC tetramer in flow cytometry, and found no difference between the NOD TdT-/- and TdT+/+ mice. However, it must be noted that these tetramer-reactive cells are heterogeneous with regard to TCR usage, and probably cover a range of affinities and endogenous target Ags. It has been shown that immunization of NOD mice with a peptide (NRP) reactive with diabetogenic CD8+ T cells bearing highly homologous TCR{alpha} chains, but diverse TCR{beta} chains, protects these mice from disease (39). Although NRP/H-2Kk tetramer-staining cells were still present in these mice, they were shown to be of lower affinity. As in our studies, these mice still display some insulitis, but they did not progress to full diabetes, as measured by elevated serum glucose levels. Hence, the absence of high affinity self-reactive NRP/H-2Kk tetramer-staining CD8 cells precluded the onset of diabetes in that study. Based on this study, although we see similar numbers of 2.5 peptide/Ag7 (BDC 2.5) tetramer-staining cells in the NOD TdT+/+ and TdT-/- mice, we suggest that it is possible that the TdT-/- NOD mice may lack high affinity diabetogenic BDC 2.5+ T cells, thus displaying a phenotype similar to that of the NRP peptide-immunized NOD mice (39).

We also extended our earlier findings on MRL-Faslpr TdT-/- mice (19). We have now backcrossed the MRL-Faslpr TdT-/- mice for 13 generations, and have reassessed several disease parameters in these fully congenic MRL-Faslpr TdT+/+ and MRL-Faslpr TdT-/- lines established from N13 littermates, because non-MRL background genes from the limited backcross mice in our previous study could have affected the phenotypes. The N13 congenic MRL-Faslpr TdT-/- have a markedly longer life span than the MRL-Faslpr TdT+/+ mice. Our MRL-Faslpr colony has a longer life span than it did 2 years ago when our earlier results were published, but the relative increase in longevity of the MRL-Faslpr TdT-/- mice compared with the MRL-Faslpr TdT+/+ is the same, in agreement with our early studies and with another recently published study (19, 20). The incidence of skin lesions is lower in the MRL-Faslpr TdT-/- mice compared with the MRL-Faslpr TdT+/+ mice, as reported in the N3-N5 generation mice, although the current study shows a higher incidence in the MRL-Faslpr TdT-/- mice than was previously observed by us and by Molano et al. (19, 20). The only apparent difference from our previous study is the average GN scores. Previously, histology performed on the kidneys of all mice surviving at 6 mo of age revealed no significant difference between the two groups. However, many of the MRL-Faslpr TdT+/+ mice had died by that time point, while few of the TdT-/- mice had died by the 6-mo analysis. In the current study of fully congenic strains, mice were analyzed at various time points to determine the relative GN scores. MRL-Faslpr TdT+/+ mice developed GN by 29 wk, whereas age-matched MRL-Faslpr TdT-/- mice had minimal kidney pathology. We also analyzed the kidneys of 38- to 41-wk-old MRL-Faslpr TdT-/- mice, and still found that the GN characteristic of end-stage disease was minimal. Overall, these data implicate TdT deficiency as a critical resistance factor in diminishing the autoimmune disease phenotype.

We had initially predicted that the effect of TdT deficiency in lupus might be primarily due to the lack of N region-encoded arginines in CDR3 of potential anti-dsDNA B cell precursors (19). However, the impact of TdT deficiency in MRL-Faslpr mice was much broader, affecting not only all serological assays, but also the frequency of B220+ dominant-negative T cells and skin disease. We therefore suggested that TdT deficiency might have a more global impact than just reducing the frequency of B cells with specificity for dsDNA. In this study, we show that the ameliorating effect of TdT deficiency is true not only for lupus, but also for the T cell-mediated autoimmune disease diabetes present in NOD mice. Looking at these two very different disease models, including the participating lymphocytes, Ags, and organs involved, it is clear that the lack of TdT activity significantly reduces the deleterious effects of autoimmune disease in general. This effect could be due to a lack of N region diversity in T cells, B cells, or both populations. Although lupus is an Ab-mediated disease, and pancreatic destruction in diabetes is T cell mediated, lupus and diabetes each require both T and B cells for disease to occur (21, 27, 40, 41, 42). In diabetes, the primary role of B cells is APC function, while in lupus, a major role of B cells is the production of autoantibodies such as anti-dsDNA, which cause kidney damage (42, 43). However, B cells have also been shown to function as APC in lupus (44), and the repertoire of B cells will influence which Ags are captured and which peptides are therefore presented to T cells. Thus, one cannot a priori determine whether the major impact of TdT deficiency is in the T or B cell compartment, or both. Production of radiation chimeric mice should be able to answer this question.

Previous studies on T cells deficient in N region diversity due to lack of TdT activity suggest that these TCRs demonstrate innate proclivity for self MHC, as evidenced by enriched positive selection of CD3high single-positive thymocytes in TdT-deficient mice (17). Furthermore, TdT-/- CD8 T cell clones showed an increased level of peptide promiscuity, suggesting increased recognition of the MHC (16). The outcome is a T cell population that interacts with a larger number of MHC-peptide complexes and might suggest a greater opportunity for development of autoimmunity. However, our studies on TdT-deficient autoimmune-prone mice show that these in vitro results on CTL clones are not predictive of the effect of TdT deficiency in the intact mouse. Indeed, both lupus-prone strains of mice and a diabetes-prone strain of mice have a lower incidence of autoimmunity than their TdT wild-type littermates. Based on the present findings, we hypothesize that, although TdT deficiency does not interfere with the selection of at least some autoreactive clonotypes as determined by tetramer staining, the lack of N region diversity may generate either a lower precursor frequency of autoreactive cells, or a population of self-reactive cells with primarily lower affinities for self Ag. It is interesting to note that exposure of humans and mice to pathogens or adjuvants early in life, but not later, is protective against diabetes and other autoimmune diseases (45). It may be that expansion by infectious agents of the neonatal lymphocytes with their paucity of autoreactive clones may convert many of these lymphocytes into long-lived memory cells. This may alter the balance of N region-lacking vs N region-containing lymphocytes in the periphery.

Whatever the reason, it is apparent that the fetal/neonatal N region-lacking lymphocyte repertoire delays the onset of autoimmune disease and, in these two examples, also greatly decreases end-stage disease. Because regulatory T cells are not present at birth, these early TdT-lacking B and T cell repertoires are then safe for the newborn until higher level control mechanisms are in place, coincident with the onset of N region-encoded CDR3 diversity. The advantage of the greatly enhanced diversity afforded by N region-generated junctional diversity may bring with it too high a frequency of potentially pathogenic high affinity self-reactive lymphocytes for a neonate to handle, and thus it may have been necessary to counterbalance this with an early stage filled with less pathogenic N region-lacking lymphocytes. We suggest that this could be an important reason for the delayed onset of TdT activity and for the existence of an N region-lacking, followed by an N region-containing lymphocyte repertoire during ontogeny.


   Acknowledgments
 
We thank Natasha Hill for assistance with the Genescan analysis, Marcie Kritzik for photography in Fig. 3, and Elizabeth Kompfner for excellent technical support.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI29672 (to A.J.F.), DK55037 (to L.T.), AR42242 (to D.H.K.), and AR32103 and AR39555 (to A.N.T.). I.F.R. was supported by National Institutes of Health Training Grant T32-AI007244. Back

2 Address correspondence and reprint requests to Dr. Ann J. Feeney, Department of Immunology IMM-22, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: feeney{at}scripps.edu Back

3 Abbreviations used in this paper: NOD, nonobese diabetic; CDR3, complementarity-determining region 3; GN, glomerulonephritis; LN, lymph node. Back

Received for publication November 12, 2003. Accepted for publication January 26, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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