Diabetes
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published online January 9, 2008
Diabetes 57:909-917, 2008
DOI: 10.2337/db07-1256
© 2008 by the American Diabetes Association
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
db07-1256v1
57/4/909    most recent
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Brodie, G. M.
Right arrow Articles by Green, E. A.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brodie, G. M.
Right arrow Articles by Green, E. A.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

B-Cells Promote Intra-Islet CD8+ Cytotoxic T-Cell Survival to Enhance Type 1 Diabetes

Gillian M. Brodie1, Maja Wallberg1, Pere Santamaria2, F. Susan Wong3, and E. Allison Green1

1 Department of Pathology, Cambridge Institute for Medical Research, Cambridge University, Addenbrooke's Hospital, Cambridge, U.K
2 Julia McFarlane Diabetes Research Centre and Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada
3 Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol, U.K

Address correspondence and reprint requests to Dr. E.A. Green, Department of Pathology, Cambridge Institute for Medical Research, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0XY, U.K. E-mail: allison.green{at}cimr.cam.ac.uk

Key Words: BrdU, bromodeoxyuridine • CTL, cytotoxic T-lymphocyte • DC, dendritic cell • FACS, fluorescence-activated cell sorter • IFN, interferon • PLN, pancreatic lymph node • RIP, rat insulin promoter • TNF, tumor necrosis factor • Treg, T regulatory


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVE—To determine the role of B-cells in promoting CD8+ T-cell—mediated β cell destruction in chronically inflamed islets.

RESEARCH DESIGN AND METHODS—RIP-TNF{alpha}-NOD mice were crossed to B-cell–deficient NOD mice, and diabetes development was monitored. We used in vitro antigen presentation assays and in vivo administration of bromodeoxyuridine coupled to flow cytometry assays to assess intra-islet T-cell activation in the absence or presence of B-cells. CD4+Foxp3+ activity in the absence or presence of B-cells was tested using in vivo depletion techniques. Cytokine production and apoptosis assays determined the capacity of CD8+ T-cells transform to cytotoxic T-lymphocytes (CTLs) and survive within inflamed islets in the absence or presence of B-cells.

RESULTS—B-cell deficiency significantly delayed diabetes development in chronically inflamed islets. Reintroduction of B-cells incapable of secreting immunoglobulin restored diabetes development. Both CD4+ and CD8+ T-cell activation was unimpaired by B-cell deficiency, and delayed disease was not due to CD4+Foxp3+ T-cell suppression of T-cell responses. Instead, at the CTL transition stage, B-cell deficiency resulted in apoptosis of intra-islet CTLs.

CONCLUSIONS—In inflamed islets, B-cells are central for the efficient intra-islet survival of CTLs, thereby promoting type 1 diabetes development.

Chronic inflammation defines many organ-specific autoimmune diseases. Initiated after, for example, viral infection (1), inflammation promotes transgression of immune cells from the vascular system into the target organ. Different subtypes of immune cells respond to inflammatory signals, leading to a complex milieu of effector cells involved in either destruction of the target tissue or control of the autoreactive response. Thus, therapeutic intervention of autoimmune diseases requires a detailed knowledge of the relationship between distinct immune cells when exposed to an inflamed environment.

Type 1 diabetes is an autoimmune disease in which the destruction of the insulin-producing β-cells in the islets of Langerhans is T-cell mediated (2). Type 1 diabetes development is genetically and environmentally influenced, and incidences in the developed world increase by 3% per year. The nonobese diabetic (NOD) mouse, a spontaneous murine model for type 1 diabetes, has been invaluable in deciphering its genetic, environmental, and immunological complexities (3). Studies in NOD mice revealed that before type 1 diabetes detection, islets become infiltrated with CD4+, CD8+ T-cells, dendritic cells (DCs), and B-cells, with B-cells constituting ~60–70% of intra-islet immune cells (4). Although both CD4+ and CD8+ T-cells contribute to diabetes, β-cell destruction is thought to be due to CD8+ cytotoxic T-cells (CTLs) (5,6). The high prevalence of intra-islet B-cells has driven several investigations into their role in type 1 diabetes in NOD mice. To date, these studies have focused on the role of B-cells in the priming and epitope spreading of the anti-islet CD4+ T-cell response (7–11), and a central role for B-cells in both these phenomena has been clearly established. Further, B-cell deficiency results in a substantial reduction in the inflammation and a corresponding significant drop in migration of immune cells to the islets. However, the strong association between inflammation, CD8+ T-cells, and type 1 diabetes in NOD mice necessitates investigations into whether B-cells additionally influence islet-reactive CTL activity in the inflamed islet independent of their effects on CD4+ T-cell responses.

Previously, we described a unique inflammation-based NOD model of type 1 diabetes, RIP TNF{alpha}-NOD (TNF{alpha}-NOD) mice (12). In such mice, constitutive expression of the diabetes-relevant pro-inflammatory cytokine tumor necrosis factor (TNF)-{alpha} (13,14) is restricted to the islet under control of the rat insulin promoter (RIP). TNF{alpha}-NOD mice develop type 1 diabetes rapidly with 100% penetrance of disease by 15 weeks of age. Interestingly, TNF{alpha}-NOD mice share similar characteristics to NOD mice: Diabetes development requires the NOD major histocompatibility complex (MHC) and recessive NOD alleles (15), and before diabetes, islets become infiltrated sequentially with DCs, B-cells, CD4+ T-cells, and, lastly, CD8+ T-cells (4,12). Nevertheless, unlike the NOD mouse, diabetes development is CD4+ T-cell independent, CD8+ T-cell dependent (16). TNF{alpha} has divergent roles in type 1 diabetes: Whereas neonatal transgenic expression or systemic injection of TNF{alpha} accelerates disease, similar manipulations in adult NOD mice protects (12,14,17). The mechanisms behind these divergent outcomes are not completely clear. Nevertheless, we and others (13) have documented that progression to diabetes in NOD mice is characterized by increasing levels of TNF{alpha} mRNA in the inflamed islet, suggesting TNF{alpha} may push the diabetic response in unmanipulated NOD mice. These unique features highlighted the attractiveness of using TNF{alpha}-NOD mice to decipher the intricate relationship between B-cells and anti-islet CD8+ T-cells in type 1 diabetes, particularly during chronic inflammation. Here, we show that B-cell–deficient TNF{alpha}-NOD mice have significantly reduced kinetics of disease and provide new evidence for a divergent role for B-cells depending on the stage of disease and the environment where they reside. Whereas in the pancreatic lymph nodes (PLNs), B-cells promote differentiation of CD8+ T-cells to CTL, in the islet, inflammation overrides this block. Here, B-cells provide survival signals for CD8+ T-cells to maintain high levels of aggressive CTLs. Our findings open up new therapeutic avenues to manipulate the CD8+ T-cell response to β-cells in type 1 diabetes.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and genotyping.
RIP-TNF{alpha} NOD mice (N22) and NOD-µMT–/– (N20) mice have been described elsewhere (10,12,16). TNF{alpha}-µMT–/– mice were generated by breeding male TNF{alpha}-NOD-µMT+/+ mice to female NOD-µMT–/– mice, then intracrossing TNF{alpha}-µMT–/+ male mice to female NOD-µMT–/+ littermates to create TNF-µMT–/– and TNF-µMT+/+ mice littermates for all studies described. TNF{alpha}-µMT–/– mice were screened by fluorescence-activated cell sorter (FACS) analysis of peripheral blood for B-cells using a rat anti-CD19 Ab (BD Biosciences).

NOD-mIg.µMT–/– (18) mice were crossed to TNF{alpha}-µMT+/+ mice, and the resultant heterozygous littermates were intracrossed to generate TNF{alpha}-µMT–/–, TNF{alpha}-µMT+/+, TNF{alpha}-mIg.µMT–/–, and TNF{alpha}-mIg.µMT+/+ mice.

All mice were housed in specific pathogen-free barrier conditions at Cambridge University Central Biomedical Services. All experimental procedures were conducted under U.K. Government Home Office guidelines.

Cell preparations.
Islet-residing cells were isolated after collagenase digestion of pancreata as previously described (12). Cells were isolated from lymph nodes using standard protocols. For purification of CD4+ T-cells, cell suspensions were incubated with rat anti-mouse CD8 (53-6.72) and B220 (RA3-6B2) antibodies (both BD Biosciences), followed by BioMag goat anti-rat IgG magnetic beads (Qiagen). Macrophages and DCs were depleted by incubation of the cell suspension on plastic at 37°C for 30 min. For CD8+ T-cell purification, a rat anti-mouse CD4 Ab (GK1.5; BD Biosciences) was used in place of anti-CD8 Ab. In all cases, cell purity was >96% as confirmed by FACS analysis.

For purification of intra-islet immune cells for intracellular staining, pancreatic extracts were incubated with rat anti-CD45 Ab (BD Biosciences) and CD45+ cells isolated using a MoFlo cell sorter (Dako Cytomation).

Antigen presentation assays.
A total of 3 x 105 purified T-cells were cultured with 5 x 104 irradiated islet-infiltrating antigen-presenting cells (APCs) and islet cells. The assay was pulsed with 1 µCi/well [3H]-methyl thymidine (Factor; Amersham Biosciences UK) for the last 6 h of a 4-day culture, and incorporation of radioactivity was detected using a Top Count β-counter (PerkinElmer, Beaconsfield).

Intraperitoneal injections.
For CD4+ T-cell depletion studies, mice were injected with 0.5 mg purified anti-CD4 (GK1.5; BioExpress). Control mice received 0.5 mg purified rat IgG (Jackson ImmunoResearch). First injections were given at 26, 28, and 30 days of age and then subsequently at 2-week intervals until the end point was reached (diabetic or 30 weeks old). For bromodeoxyuridine (BrdU) studies, mice were injected with 1 mg of BrdU (BD Biosciences) for three consecutive days before analysis.

Flow cytometry and intracellular staining.
All antibodies/kits were obtained from BD Biosciences unless otherwise stated. Single-cell suspensions were incubated with the appropriately flurochrome-labeled antibodies CD3 (2C11), CD4 (GK1.5), CD8 (53-6.7), CD45 (30-F11), and CD44 (IM7), following Fc receptors blockage. Foxp3 was detected using a Foxp3 staining kit (eBioscience). For BrdU detection, a BrdU detection kit was used following the manufacturer's instructions. All samples were acquired on a FACS Calibur flow cytometer (Becton Dickinson) using Cellquest software and analyzed using FlowJo software (Tree star). For apoptosis studies, an Annexin V-PE kit was used following the manufacturer's protocol.

Cytokine measurement.
Approximately 1.5 x 106 PLN cells or 1 x 106 islet T-cells were stimulated with {alpha}-CD3 and {alpha}-CD28 Ab (both BD Pharmingen) at 1 µg/ml. For the final 4 h of a 24-h incubation period, 1 µg/ml of golgi stop (BD Pharmingen) was added. The cells were stained with fluorochrome-labeled anti-CD3, CD8, interferon (IFN)-{gamma}, granzyme B (Caltag Laboratories), or the appropriate isotype control and analyzed by FACS.

Diabetes detection.
Mice were tested once weekly for glycosuria using Diastix reagent strips (Johnson & Johnson). Diabetes was confirmed after two serum glucose readings of 13.9 mmol/l 48 h apart.

Statistical analysis.
Comparisons of disease frequencies were performed using the log-rank test. P values <0.01 were considered significant. For other assays, statistical significance was measured using the Mann-Whitney nonparametric test. P values <0.05 were considered significant. To illustrate differences in islet-infiltrating CD8+ T-cells in TNF-µMT+/+ versus TNF-µMT–/– mice, a nonparametric Kruskal-Wallis test was performed. The P values were corrected using a permutation test permuting the assignment of mice to groups within experiments to allow for potential differences between replicated experiments. P values <0.001 were considered significant.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
B-cells are essential for accelerating inflammation-dependent type 1 diabetes.
To assess the importance of B-cells in type 1 diabetes development under potent inflammatory conditions, TNF{alpha}-NOD mice were crossed to B-cell–deficient NOD-µMT–/– mice, and resultant TNF{alpha}-µMT–/– NOD (hereafter called TNF{alpha}-µMT–/–) mice were monitored for progression to diabetes. TNF{alpha}-µMT–/– mice had significantly decreased disease kinetics (P = 0.0001) compared to wild-type TNF{alpha}-µMT+/+ mice, although the overall penetrance of disease was the same (Fig. 1A). This observation was independent of the sex of the mouse. Nontransgenic littermates were protected from disease as previously reported (data not shown) (10,19). Thus, B-cells are essential for accelerating type 1 diabetes in the presence of a strong proinflammatory environment.


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 1. B-cell deficiency regulates TNF{alpha}-mediated diabetes. A: Diabetes development was monitored in TNF{alpha}-µMT+/+ (n = 26) and TNF{alpha}-µMT–/– mice (n = 30). The data are presented as a Kaplan-Meier survival curve, and statistical comparisons of disease frequencies were performed using the log-rank test. B: Diabetes development was monitored in TNF-mIg.µMT–/– (n = 25), TNF{alpha}-µMT–/– mice (n = 31), TNF{alpha}-µMT+/+ (n = 33), and TNF{alpha}-mIg.µMT+/+ (n = 26) mice. The data are presented as before. C: Representative experiments using CD8+ T-cells (left panel) or CD4+ T-cells (right panel) purified from a pool of the inguinal, axillary, and PLNs of TNF{alpha}-µMT+/+ ({square}) or TNF{alpha}-µMT–/– mice ({blacksquare}) and stimulated with islet-infiltrating APCs/antigens derived from either TNF{alpha}-µMT–/– or TNF{alpha}-µMT+/+ mice. As a control, T-cells were stimulated with 105 irradiated splenocytes from TNF{alpha}-µMT+/+ mice. After a 4-day culture, cell proliferation in counts per minute (cpm) was assessed by measuring incorporation of [3H]-methyl thymidine following a 6-h pulse on the last day of culture. Background counts for islets/APCs alone and T-cells alone were <200 cpm and have been subtracted from the data. The data are presented as means ± SD of triplicate cultures. The result shown was reproducible on six separate occasions.

 
B-cell enhancement of disease kinetics was not linked to the secretion of autoantibodies, as has been previously reported (20). This theory was based on our observation that crossing TNF{alpha}-µMT–/– mice to transgenic NOD-mIg.µMT–/– mice that harbor B-cells carrying a mutation in the secretory component of the Ig gene, thereby preventing Ig secretion but not membrane-anchored expression of Ig (18), efficiently restored disease acceleration in double transgenic TNF{alpha}-mIg.µMT–/– mice (Fig. 1B).

CD4+Foxp3+ Treg cell–independent mechanisms control type 1 diabetes development under chronic inflammatory conditions.
Isolated CD4+ or CD8+ T-cells from either TNF{alpha}-µMT+/+ or TNF{alpha}-µMT–/– mice efficiently responded to a broad range of islet antigen presented by intra-islet APCs from either strain with equal efficacy (Fig. 1C). Such responses were antigen specific since T-cells isolated from either strain failed to respond to irradiated splenocytes from TNF{alpha}-µMT+/+ mice. This suggested priming of the T-cell repertoire and functionality of intra-islet DCs was unimpaired by B-cell deficiency. We, therefore, hypothesized that the disease kinetics in TNF{alpha}-µMT–/– mice were linked to in situ suppression of T-cell responses by CD4+Foxp3+ T regulatory (Treg) cells (21). However, FACS analysis established no significant differences in the percentages of CD4+Foxp3+ Treg cells between TNF{alpha}-µMT+/+ and TNF{alpha}-µMT–/– mice in either the islets or the PLNs (Fig. 2A). Furthermore, the delayed kinetics of disease in TNF{alpha}-µMT–/– mice did not require functional CD4+Foxp3+ Treg cells. This finding was based on observations that administration of anti-CD4 depleting Ab to TNF{alpha}-µMT+/+ and TNF{alpha}-µMT–/– mice did not alter disease development in any mouse group with respect to the appropriate isotype control antibody–treated mice (Fig. 2B). These data suggest that immune-mediated β-cell destruction can be controlled independent of the traditional CD4+Foxp3+ Treg cell suppression.


Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 2. Foxp3+ Treg cells do not regulate T1D development in TNF{alpha}-µMT–/– mice. A: Representative histograms are shown for the percentage of Foxp3+ cells within a CD45+CD3+CD4+ gate from the PLNs or islets of 7- to 9-week-old TNF{alpha}-µMT–/– and TNF{alpha}-µMT+/+ mice. The graph displays the total number of animals analyzed for each strain, and statistical analysis was performed using the nonparametric Mann-Whitney test. B: Diabetes development was monitored in TNF{alpha}-µMT+/+ (n = 14) and TNF{alpha}-µMT–/– (n = 18) mice treated with either {alpha}CD4 Ab or IgG isotype control antibodies. Bimonthly analysis of peripheral blood lymphocytes confirmed depletion of CD4+ T-cells. The data are presented as a Kaplan-Meier survival curve.

 
Intra-islet CD8+ T-cells are significantly reduced in the absence of B-cells.
Previously we demonstrated that ablation of CD8+ T-cells protected TNF{alpha}-µMT+/+ mice from developing type 1 diabetes, whereas CD4+ T-cells played a redundant role (16), a finding that was supported by our above observations. Therefore, we focused our attention on the CD8+ T-cell population in TNF{alpha}-µMT–/– mice and TNF{alpha}-µMT+/+ mice. Flow cytometric analysis of lymphocytes from each mouse strain mice revealed a significant decrease (P = 0.0001) in the percentage of intra-islet CD8+ T-cells in TNF{alpha}-µMT–/– mice compared with TNF{alpha}-µMT+/+ mice at 8 weeks of age. This time point corresponds to the transition of activated CD8+ T-cells into CTLs (16). In contrast, we saw no differences in the percentage of intra-islet CD4+ T-cells in TNF{alpha}-µMT–/– mice compared with TNF{alpha}-µMT+/+ mice (P = 0.042) (Fig. 3A). No variation was observed in T-cell proportions within the PLNs of all mice examined (data not shown). To determine whether B-cell ablation results in a general decrease in intra-islet CD8+ T-cell accumulation, we performed time-course studies. We found no significant differences in CD8+ T-cell percentages in the islets of TNF{alpha}-µMT–/– mice compared with TNF{alpha}-µMT+/+ mice at 4 and 6 weeks of age (not shown). These data imply that during inflammation, B-cells are central for the accumulation of autoreactive CD8+ T-cells in the target tissue at the CTL generation stage of the disease process.


Figure 3
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 3. B-cells control intra-islet CD8+ T-cell accumulation at the CTL transition stage. A: Lymphocytes were isolated from the islets of TNF{alpha}-µMT+/+ and TNF{alpha}-µMT–/– mice, and the percentage of CD4+ or CD8+ cells within a CD45+CD3+ gate was determined by FACS. The graph represents all animals examined, and significance in T-cell populations between the two groups of mice was determined using the Kruskal-Wallis test. B: TNF{alpha}-µMT+/+ (•) or TNF{alpha}-µMT–/– ({blacktriangleup}) mice were injected with BrdU, and the percentage of CD8+BrdU+ T-cells in the shown tissues was determined by FACS. The dot plot shows the total number of mice analyzed per strain within one representative experiment of three. Statistical significance between the respective groups was determined using the Mann-Whitney test. C: Intra-islet CD8+ T-cells were assessed by analyzing the percentage of CD44+ cells within a CD45+CD3+CD8+ gate in TNF{alpha}-µMT+/+ compared with TNF{alpha}-µMT–/– mice. The graph shows the total number of mice examined.

 
Intra-islet CD8+ T-cell turnover and reactivation are independent of B-cells.
Using MHC class I tetramers–peptide complexes, we determined that B-cell deficiency did not hinder intra-islet accumulation of CD8+ T-cells specific for two key diabetogenic antigens: insulin and islet-specific glucose-6-phosphate catalytic subunit–related protein (IGRP; data not shown) (6,22).

We speculated B-cells to be involved in the later postpriming stage of disease. To test the requirement for B-cells at the intra-islet secondary activation/expansion phase, groups of TNF{alpha}-µMT–/– and TNF{alpha}-µMT+/+ mice were injected with the thymidine analogue, bromodeoxyuridine (BrdU), which incorporates into the DNA of proliferating cells. We initially focused on 8-week-old mice, as 8 weeks is the critical age when significant differences in intra-islet CD8+ T-cell levels are apparent in the two strains of mice. Flow cytometric analysis of BrdU+CD8+ T-cells isolated from the islets, PLNs, and control peripheral inguinal lymph nodes from each mouse strain established the greatest percentage of proliferating CD8+ T-cells to be located in the islet fraction in both TNF{alpha}-µMT–/– and TNF{alpha}-µMT+/+ mice (Fig. 3B). Interestingly, although TNF{alpha}-µMT–/– mice showed significantly decreased turnover of CD8+ T-cells in the lymph nodes compared with TNF{alpha}-µMT+/+ mice, we detected no differences in the percentage of intra-islet BrdU+CD8+ T-cells between TNF{alpha}-µMT+/+ and TNF{alpha}-µMT–/– mice (mean values 12 and 14%, respectively). Furthermore, we could detect no differences in the level of the activation/memory marker CD44 on intra-islet CD8+ T-cells between the two strains of mice (Fig. 3C). Examination of 6-week-old mice revealed an identical pattern of cell turnover (not shown). Thus, environmental influences dictate the necessity for B-cell help in the expansion of CD8+ T-cells.

B-cells promote intra-islet survival of activated CD8+ T-cells.
The above evidence that CD8+ T-cells are significantly decreased in the islets of TNF{alpha}-µMT–/– mice with respect to TNF{alpha}-µMT+/+ mice, even though activation and in situ proliferation of CD8+ T-cells are similar between the two strains of mice, led us to hypothesize that intra-islet B-cells directly or indirectly provide survival signals to activated CD8+ T-cells. This hypothesis was supported by evidence that between 7 and 9 weeks of age, the percentage of CD8+ T-cells undergoing apoptosis was significantly higher in the islets of TNF{alpha}-µMT–/– compared with TNF{alpha}-µMT+/+ mice (Fig. 4). This observation was restricted to the CD8+ T-cell population within the islets, as similar analysis of the PLNs revealed no significant differences in the survival of CD8+ T-cells between the two strains of mice. In contrast, we found no significant differences in the survival of intra-islet or PLN-derived CD4+ T-cells in TNF{alpha}-µMT+/+ and TNF{alpha}-µMT–/– mice. Thus, in the absence of B-cells, it would therefore appear that CD8+ T-cells lack a vital signal required for their prolonged intra-islet survival.


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 4. B-cells enable prolonged survival of intra-islet CD8+ T-cells. Intra-islet T-cells from TNF{alpha}-µMT–/– mice and TNF{alpha}-µMT+/+ mice were stained with anti-CD45, CD4, CD8, Abs, Annexin V, and 7AAD. The percentage of CD8+Annexin V+ 7AAD- and CD4+Annexin V+ 7AAD- T-cells were determined by FACS. The data shown are representative of one of four repeat experiments and shows Annexin V+ cells within intra-islet CD8+ T-cells (A), intra-islet CD4+ T-cells (B), CD8+ T-cells in PLNs (C), and CD4+ T-cells (D) in PLNs. Statistical significance was determined by the Mann-Whitney test.

 
Inflammation bypasses block in CD8+ T-cell differentiation to CTLs in the islets of TNF{alpha}-µMT–/– mice.
The above data clearly state a role for B-cells in the in situ survival of activated CD8+ T-cells. However, we wished to know if this was the only role B-cells played in type 1 diabetes. CD8+ T-cell–mediated destruction of cells requires an activated CD8+ T-cell to transform to a CTL. We therefore determined whether CD8+ T-cells exposed to an inflammatory environment in the presence or absence of B-cells had equal capacity to transform to CTL. Our data suggest that a B-cell role for the transition to CTL is dependent on the environment. IFN{gamma}+ and granzyme B+ production at the per cell basis in CD8+ T-cells was significantly reduced in the PLNs of 7- to 9-week-old TNF{alpha}-µMT–/– mice in comparison to TNF{alpha}-µMT+/+ mice (Fig. 5). Interestingly, in inflamed islets, the percentage of CD8+ T-cells that expressed IFN{gamma} or granzyme B was slightly enhanced in TNF{alpha}-µMT–/– mice compared with TNF{alpha}-µMT+/+ mice (Fig. 6A,B). Nevertheless, the absolute number of intra-islet differentiated CTLs in TNF{alpha}-µMT–/– mice was decreased between 10 and 30% with respect to the absolute numbers of CTLs in TNF{alpha}-µMT+/+ mice (Fig. 6C), a finding that is consistent with the increased apoptosis of CD8+ T-cells at this time point in TNF{alpha}-µMT–/– mice.


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 5. B-cells enable transformation of CD8+ T-cells into CTLs in the PLN. PLN-derived cells of TNF{alpha}-µMT+/+ and TNF{alpha}-µMT–/– mice were stimulated with anti-CD3 and anti-CD28 antibodies, and the percentage of CD8+IFN{gamma}+ and CD8+granzyme B+ T-cells was determined by FACS. The data shown represent one experiment of four performed, each giving identical results.

 

Figure 6
View larger version (29K):
[in this window]
[in a new window]

  		FIG. 6. Intra-islet differentiated CTLs require B-cell–derived survival signals. CD45+ hematopoietic cells were sorted from the pancreas of 8-week-old TNF{alpha}-µMT+/+ or TNF{alpha}-µMT–/– mice, and their absolute numbers were determined using a CASY automatic cell counter. After anti-CD3 and anti-CD28 antibody stimulation, CD8+IFN{gamma}+ and CD8+granzyme B+ T-cells were determined by FACS. A: Representative FACS plot for one experiment of three independent experiments showing percentage of CD8+IFN{gamma}+ and CD8+granzyme B+ T-cells. B: Dot plot showing the total number of animals examined. C: Percentage decrease in absolute numbers of CD8+IFN{gamma}+ and CD8+granzyme B+ T-cells in TNF{alpha}-µMT–/– mice with respect to TNF{alpha}-µMT+/+ mice. Two or three mice were pooled for each experiment; a total of seven mice were examined for each strain.

 
These data show B-cells have distinct roles to play in type 1 diabetes development depending on the environment: In the PLNs, they are essential for CTL generation, not survival of CD8+ T-cells; in the islet, alternative mechanisms promote CTL generation. In this latter case, B-cells are central for providing CD8+ T-cell survival signals to maintain high levels of anti-islet CTLs needed to promote type 1 diabetes progression.


   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Understanding the complex interactions of distinct immune cells that invade inflamed target tissues and promote autoimmunity will help decipher the mechanisms that lead to the destruction of the target cell and associated immunopathologies. Here we show for the first time a link between chronic inflammation, B-cells and CD8+ T-cells, and the promotion of type 1 diabetes. We show B-cells play dual roles: In the PLNs, they drive CD8+ T-cell transition to CTL; in the islet, B-cells promote survival of activated CD8+ T-cells at the CTL transition stage, thereby accelerating disease progression.

It is known that B-cells are important in type 1 diabetes development in the NOD mouse at the level of CD4+ T-cell priming to islet antigen GAD (8,9,23,24). The need for B-cells at the level of the anti-islet CD8+ T-cell, postulated to be the main effectors of β-cell destruction in both NOD mice and humans (5,25,26), is not clear and may depend on the specificity of the CD8+ T-cell (27,28). Here we used the TNF{alpha}-NOD mouse model of type 1 diabetes, where intra-islet TNF{alpha}-driven chronic inflammation resulted in CD8+ T-cell–dependent, CD4+ T-cell–independent destruction of β cells (16) to determine the importance of the B-cell–CD8+ T-cell relationship in the presence of a normal T-cell repertoire. We showed that TNF{alpha}-µMT–/– mice had significantly reduced kinetics of type 1 diabetes development compared with TNF{alpha}-µMT+/+ mice.

There are several possibilities as to why the ablation of B-cells significantly decrease the kinetics of diabetes development even in the presence of chronic inflammation: 1) failed priming of the CD8+ T-cell repertoire to islet antigen either directly or indirectly through production of Ig that enables cross-presentation of islet antigens by DCs (29); 2) failed recruitment of CD8+ T-cells to islets; 3) enhanced functionality of CD4+Foxp3+ Treg cells that control autoreactive T-cells (30–32); 4) impaired differentiation of activated CD8+ T-cells to CTLs (33); and 5) decreased survival of activated CD8+ T-cells/CTLs (34). Our data suggest the first three hypotheses do not account for the phenotype seen in TNF{alpha}-µMT–/– mice. Restoration of B-cells devoid of secretory Ig production reestablished the accelerated kinetics of disease, and islet-antigen primed CD8+ T-cells are present in the lymphatic system and respond to stimulation with islet-residing APCs, presenting a vast range of islet peptides. Interestingly, CD4+ T-cell priming is unimpaired in our model, contrasting with reports in unmanipulated NOD mice in which CD4+ T-cell priming to GAD is defective (11). This disparity likely reflects our experimental system, for which we used TNF{alpha}-exposed intra-islet APCs and a vast array of islet antigens. Nevertheless, CD4 depletion experiments showed CD4+ T-cells are redundant for disease progression. Surprisingly, CD4+ T-cells also do not contribute to suppression of disease in TNF{alpha}-µMT–/– mice, a finding that contrasts to studies by Hu et al., where depletion of B-cells in adult NOD mice delays disease, in part, by enhancing CD4+ Treg mechanisms (35). Our data highlight the unique pathway by which autoreactivity is dampened independent of CD4+ Treg cells, a finding that has potential implications for autoimmune disease linked to failed CD4+ Treg functionality.

The answer to why B-cell ablation delays diabetes development seems to lie within the CD8+ T-cell population, with two distinct mechanisms operating depending on the environment investigated. In the PLNs, B-cell deficiency affects the proliferation and differentiation of primed CD8+ T-cells but not their survival. This was evident by similarities in both the numbers of and significant decreases in both BrdU+ cells, as well as an absence of IFN{gamma} and granzyme B production by PLN-residing CD8+ T-cells in TNF{alpha}-µMT–/– mice compared with TNF{alpha}-µMT+/+ mice. However, in inflamed islets, although the ability of CD8+ T-cells to proliferate and differentiate to CTL is not dependent on B-cells, their survival is. This latter hypothesis was based on observations that at 8 weeks of age, when transition of intra-islet CD8+ T-cells to CTL occurs, there was an increased rate of apoptosis and an ~30% drop in the absolute numbers of CD8+IFN{gamma}+ and CD8+granzyme B+ T-cells in TNF{alpha}-µMT–/– mice compared with TNF{alpha}-µMT+/+ mice. Examination of CD8+ T-cell proliferation, survival, and numbers at earlier, pre-CTL differentiation stages (4–6 weeks of age) revealed no difference between the two strains of mice, suggesting this effect of B-cells on CD8+ T-cell survival is both stage and environment dependent (not shown). This inability of CD8+ T-cells to proliferate and develop into CTLs in the PLN of TNF{alpha}-µMT–/– mice may reflect impaired B-cell–DC cooperation for efficient CTL generation due to an absence in BAFF–TACI signaling between the two cell populations (33). In support of this hypothesis, depletion of B-cells in adult NOD mice results in DCs defective in inducing expansion of and IFN{gamma} production from both CD4+ BDC2.5 TcR and insulin-specific CD8+ TcR cells (35).

Nevertheless, in the islets, the proliferation and differentiation of CD8+ T-cells to CTL are not dependent on B-cells. These intriguing findings likely reflect the uniqueness of the inflamed islet environment created by TNF{alpha}, empowering residing DCs to efficiently present islet antigens to CD8+ and CD4+ T-cells and impart the necessary signals for the former to differentiate to CTLs. Indeed, both absolute numbers and activation status of intra-islet DCs were identical in the two strains of mice (not shown). This finding is not surprising considering the potency of TNF{alpha}-associated inflammation to promote DC functionality independent of conventional activation signals (16). Despite these attributes, it is clear that even in inflamed islets activated DCs alone are not sufficient to enhance progression to diabetes; B-cells are necessary to provide direct, or indirect, survival signals to responding CD8+ T-cells during their transition phase to CTLs. It is interesting that TNF{alpha}-µMT–/– mice eventually succumb to diabetes, suggesting that in chronically inflamed islets, eventually the need for B-cells is compensated by as yet unknown mechanisms. Future studies resolving the disparity in the roles B-cells play in the PLNs and islets, as well as identification of compensatory pathways that push B-cell–independent assault of β cells, are ongoing. In addition, it will be important to determine whether this B-cell–CD8+ T-cell relationship is operational in NOD mouse models that require CD4+ T-cells to promote diabetes progression.

Despite that autoantibody production to islet antigens is a strong predictor of future diabetes development in prediabetic individuals (36,37), subscribing a role for B-cells in diabetes development in humans is controversial, principally because of an absence of direct experimental evidence and documentation of a single patient with X-linked agammaglobulinemia developing diabetes (38). Thus, translating data from inbred strains of NOD mice to genetically diverse humans should be viewed with caution. Nevertheless, clinical trials using rituximab—a humanized anti-human CD20 mAb that depletes B-cells and has been shown to ameliorate T-cell–mediated rheumatic arthritis—are ongoing to test its efficacy to prevent/reverse type 1 diabetes (www.diabetestrialnet.org). Our data suggest B-cell depletion in patients with clinical manifestations of diabetes but not overt disease could significantly delay disease progression. This hypothesis has recently been substantiated by studies in NOD mice transgenic for human CD20 on B-cells, where B-cell depletion using anti-CD20 Abs during the insulitis phase resulted in significant delay in disease (35). The emergence of data from the ongoing clinical trials will be imperative for determining the importance of B-cells in human type 1 diabetes development and the validation in translation of the findings from B-cell–deficient NOD mouse models to humans.


   ACKNOWLEDGMENTS
 
This work was supported by the Juvenile Diabetes Research Foundation and the Wellcome Trust (E.A.G.). E.A.G. is a Wellcome Senior Research Fellow in Basic Biomedical Science. F.S.W. is a Wellcome Senior Research Fellow in Clinical Science. P.S. is supported by the Canadian Institutes of Health and Research and is a scientist of the Alberta Heritage Foundation for Medical Research.

We would like to thank Jason Cooper for statistical support, Anna Petrunkina-Harrison for cell sorting expertise, and Philip Spence for critical reading of the manuscript.


   FOOTNOTES
 
Published ahead of print at http://diabetes.diabetesjournals.org on 9 January 2008. DOI: 10.2337/db07-1256.

G.M.B. and M.W. contributed equally to this article.

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.

Received for publication September 5, 2007 and accepted in revised form January 4, 2008


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ehl S, Hombach J, Aichele P, Rulicke T, Odermatt B, Hengartner H, Zinkernagel R, Pircher H: Viral and bacterial infections interfere with peripheral tolerance induction and activate CD8+ T-cells to cause immunopathology. J Exp Med 187:763–774, 1998[Abstract/Free Full Text]
  2. Green EA, Flavell RA: The initiation of autoimmune diabetes. Curr Opin Immunol 11:663–669, 1999[Medline]
  3. Delovitch TL, Singh B: The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity 7:727–738, 1997[Medline]
  4. Green EA, Flavell RA: Tumor necrosis factor-alpha and the progression of diabetes in non-obese diabetic mice. Immunol Rev 169:11–22, 1999[Medline]
  5. Liblau RS, Wong FS, Mars LT, Santamaria P: Autoreactive CD8 T-cells in organ-specific autoimmunity: emerging targets for therapeutic intervention. Immunity 17:1–6, 2002[Medline]
  6. Lieberman SM, Evans AM, Han B, Takaki T, Vinnitskaya Y, Caldwell JA, Serreze DV, Shabanowitz J, Hunt DF, Nathenson SG, Santamaria P, DiLorenzo TP: Identification of the beta cell antigen targeted by a prevalent population of pathogenic CD8+ T-cells in autoimmune diabetes. Proc Natl Acad Sci U S A 100:8384–8388, 2003[Abstract/Free Full Text]
  7. Dai YD, Carayanniotis G, Sercarz E: Antigen processing by autoreactive B-cells promotes determinant spreading. Cell Mol Immunol 2:169–175, 2005[Medline]
  8. Noorchashm H, Lieu YK, Noorchashm N, Rostami SY, Greeley SA, Schlachterman A, Song HK, Noto LE, Jevnikar AM, Barker CF, Naji A: I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T-cell tolerance to islet beta cells of nonobese diabetic mice. J Immunol 163:743–750, 1999[Abstract/Free Full Text]
  9. Noorchashm H, Moore DJ, Noto LE, Noorchashm N, Reed AJ, Reed AL, Song HK, Mozaffari R, Jevnikar AM, Barker CF, Naji A: Impaired CD4 T-cell activation due to reliance upon B-cell-mediated costimulation in nonobese diabetic (NOD) mice. J Immunol 165:4685–4696, 2000[Abstract/Free Full Text]
  10. Serreze DV, Chapman HD, Varnum DS, Hanson MS, Reifsnyder PC, Richard SD, Fleming SA, Leiter EH, Shultz LD: B lymphocytes are essential for the initiation of T-cell–mediated autoimmune diabetes: analysis of a new "speed congenic" stock of NOD.Ig mu null mice. J Exp Med 184:2049–2053, 1996[Abstract/Free Full Text]
  11. Serreze DV, Fleming SA, Chapman HD, Richard SD, Leiter EH, Tisch RM: B lymphocytes are critical antigen-presenting cells for the initiation of T-cell–mediated autoimmune diabetes in nonobese diabetic mice. J Immunol 161:3912–3918, 1998[Abstract/Free Full Text]
  12. Green EA, Eynon EE, Flavell RA: Local expression of TNFalpha in neonatal NOD mice promotes diabetes by enhancing presentation of islet antigens. Immunity 9:733–743, 1998[Medline]
  13. Held W, MacDonald HR, Weissman IL, Hess MW, Mueller C: Genes encoding tumor necrosis factor alpha and granzyme A are expressed during development of autoimmune diabetes. Proc Natl Acad Sci U S A 87:2239–2243, 1990[Abstract/Free Full Text]
  14. Yang XD, Tisch R, Singer SM, Cao ZA, Liblau RS, Schreiber RD, McDevitt HO: Effect of tumor necrosis factor alpha on insulin-dependent diabetes mellitus in NOD mice. I. The early development of autoimmunity and the diabetogenic process. J Exp Med 180:995–1004, 1994[Abstract/Free Full Text]
  15. Chamberlain G, Wallberg M, Rainbow D, Hunter K, Wicker LS, Green EA: A 20-Mb region of chromosome 4 controls TNF-alpha-mediated CD8+ T-cell aggression toward beta cells in type 1 diabetes. J Immunol 177:5105–5114, 2006[Abstract/Free Full Text]
  16. Green EA, Wong FS, Eshima K, Mora C, Flavell RA: Neonatal tumor necrosis factor alpha promotes diabetes in nonobese diabetic mice by CD154-independent antigen presentation to CD8(+) T-cells. J Exp Med 191:225–238, 2000[Abstract/Free Full Text]
  17. Grewal IS, Grewal KD, Wong FS, Picarella DE, Janeway CA, Jr, Flavell RA: Local expression of transgene encoded TNF alpha in islets prevents autoimmune diabetes in nonobese diabetic (NOD) mice by preventing the development of auto-reactive islet-specific T-cells. J Exp Med 184:1963–1974, 1996[Abstract/Free Full Text]
  18. Wong FS, Wen L, Tang M, Ramanathan M, Visintin I, Daugherty J, Hannum LG, Janeway CA, Jr, Shlomchik MJ: Investigation of the role of B-cells in type 1 diabetes in the NOD mouse. Diabetes 53:2581–2587, 2004[Abstract/Free Full Text]
  19. Noorchashm H, Noorchashm N, Kern J, Rostami SY, Barker CF, Naji A: B-cells are required for the initiation of insulitis and sialitis in nonobese diabetic mice. Diabetes 46:941–946, 1997[Abstract]
  20. Silveira PA, Grey ST: B-cells in the spotlight: innocent bystanders or major players in the pathogenesis of type 1 diabetes. Trends Endocrinol Metab 17:128–135, 2006[Medline]
  21. Hori S, Nomura T, Sakaguchi S: Control of regulatory T-cell development by the transcription factor Foxp3. Science 299:1057–1061, 2003[Abstract/Free Full Text]
  22. Wong FS, Karttunen J, Dumont C, Wen L, Visintin I, Pilip IM, Shastri N, Pamer EG, Janeway CA, Jr.: Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library. Nat Med 5:1026–1031, 1999[Medline]
  23. Greeley SA, Moore DJ, Noorchashm H, Noto LE, Rostami SY, Schlachterman A, Song HK, Koeberlein B, Barker CF, Naji A: Impaired activation of islet-reactive CD4 T-cells in pancreatic lymph nodes of B-cell-deficient nonobese diabetic mice. J Immunol 167:4351–4357, 2001[Abstract/Free Full Text]
  24. Silveira PA, Dombrowsky J, Johnson E, Chapman HD, Nemazee D, Serreze DV: B-cell selection defects underlie the development of diabetogenic APCs in nonobese diabetic mice. J Immunol 172:5086–5094, 2004[Abstract/Free Full Text]
  25. DiLorenzo TP, Serreze DV: The good turned ugly: immunopathogenic basis for diabetogenic CD8+ T-cells in NOD mice. Immunol Rev 204:250–263, 2005[Medline]
  26. Wong FS, Janeway CA, Jr.: The role of CD4 and CD8 T-cells in type I diabetes in the NOD mouse. Res Immunol 148:327–332, 1997[Medline]
  27. Graser RT, DiLorenzo TP, Wang F, Christianson GJ, Chapman HD, Roopenian DC, Nathenson SG, Serreze DV: Identification of a CD8 T-cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T-cell helper functions. J Immunol 164:3913–3918, 2000[Abstract/Free Full Text]
  28. Verdaguer J, Schmidt D, Amrani A, Anderson B, Averill N, Santamaria P: Spontaneous autoimmune diabetes in monoclonal T-cell nonobese diabetic mice. J Exp Med 186:1663–1676, 1997[Abstract/Free Full Text]
  29. Harbers SO, Crocker A, Catalano G, D'Agati V, Jung S, Desai DD, Clynes R: Antibody-enhanced cross-presentation of self antigen breaks T-cell tolerance. J Clin Invest 117:1361–1369, 2007[Medline]
  30. Sakaguchi S: Naturally arising CD4+ regulatory T-cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 22:531–562, 2004[Medline]
  31. Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, Sharpe A, Bluestone JA: B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T-cells that control autoimmune diabetes. Immunity 12:431–440, 2000[Medline]
  32. Serra P, Amrani A, Yamanouchi J, Han B, Thiessen S, Utsugi T, Verdaguer J, Santamaria P: CD40 ligation releases immature dendritic cells from the control of regulatory CD4+CD25+ T-cells. Immunity 19:877–889, 2003[Medline]
  33. Diaz-de-Durana Y, Mantchev GT, Bram RJ, Franco A: TACI-BLyS signaling via B-cell-dendritic cell cooperation is required for naive CD8+ T-cell priming in vivo. Blood 107:594–601, 2006[Abstract/Free Full Text]
  34. Asano MS, Ahmed R: CD8 T-cell memory in B-cell-deficient mice. J Exp Med 183:2165–2174, 1996[Abstract/Free Full Text]
  35. Hu CY, Rodriguez-Pinto D, Du W, Ahuja A, Henegariu O, Wong FS, Shlomchik MJ, Wen L: Treatment with CD20-specific antibody prevents and reverses autoimmune diabetes in mice. J Clin Invest 117:3857–3867, 2007[Medline]
  36. Gardner SG, Gale EA, Williams AJ, Gillespie KM, Lawrence KE, Bottazzo GF, Bingley PJ: Progression to diabetes in relatives with islet autoantibodies. Is it inevitable? Diabetes Care 22:2049–2054, 1999[Abstract/Free Full Text]
  37. Wasserfall CH, Atkinson MA: Autoantibody markers for the diagnosis and prediction of type 1 diabetes. Autoimmun Rev 5:424–428, 2006[Medline]
  38. Martin S, Wolf-Eichbaum D, Duinkerken G, Scherbaum WA, Kolb H, Noordzij JG, Roep BO: Development of type 1 diabetes despite severe hereditary B-lymphocyte deficiency. N Engl J Med 345:1036–1040, 2001[Free Full Text]

Add to CiteULike CiteULike   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
db07-1256v1
57/4/909    most recent
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Brodie, G. M.
Right arrow Articles by Green, E. A.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brodie, G. M.
Right arrow Articles by Green, E. A.
Social Bookmarking
Add to CiteULike   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Diabetes Diabetes Care Clinical Diabetes Diabetes Spectrum