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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 11 4068-4072
Copyright © 1999 by The Endocrine Society

Original Studies

No Alteration in T Lymphocyte Expression of CD40 Ligand (CD154) in Individuals with or at Increased Risk for Insulin-Dependent Diabetes Mellitus1

Eric Ottendorfer, Tamir M. Ellis, Keith S. Bahjat, Michael Clare-Salzler and Mark A. Atkinson

Department of Pathology, University of Florida College of Medicine, Gainesville, Florida 32610

Address all correspondence and requests for reprints to: Dr. Mark A. Atkinson, Department of Pathology, Box 100275, University of Florida College of Medicine, Gainesville, Florida 32610. E-mail: atkinson{at}ufl.edu


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
CD40 ligand (CD40L) regulates multiple phases of the humoral and cellular immune response through binding to CD40. Previous investigations have suggested that insulin-dependent diabetes (IDDM) in both humans and nonobese diabetic mice may be strongly influenced by similar immunoregulatory molecules. As persons with or at increased risk for the disease are characterized by a number of immunological abnormalities, including that of self-reactive autoantibody production (e.g. islet cell cytoplasmic autoantibodies), we analyzed the expression of CD40L on T lymphocytes (CD3+ cells) in a series of individuals with newly diagnosed IDDM (n = 11), nondiabetic relatives of IDDM probands at increased risk for the disease (n = 21; islet cell cytoplasmic autoantibodies positive; Juvenile Diabetes Foundation titer, >=20), and healthy controls (n = 13). Both phorbol myristate acetate (PMA)-stimulated and unstimulated peripheral blood mononuclear cells from study subjects were analyzed by flow cytometry with a series of phenotypic antibody markers (CD3, CD40L, and isotype controls). The kinetics of CD3 and CD40L expression on peripheral blood mononuclear cells under PMA-stimulated and unstimulated conditions were similar in the three study groups (6, 24, and 48 h; all P = NS). Similarly, unstimulated and PMA stimulated CD40L expressions (percentage of positive cells and level) on CD3+ cells from newly diagnosed IDDM patients and persons at increased risk for the disease were similar to those in healthy controls (6, 24, and 48 h; all P = NS). These findings do not support abnormal CD40L expression as the mechanism underlying the functional defect(s) in communication between T lymphocytes and antigen-presenting cells that allows for autoantibody production or the inability of individuals to regulate antiself immunity in IDDM.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
TYPE 1 insulin-dependent diabetes mellitus (IDDM) appears to result from an autoimmune destruction of the insulin-producing pancreatic ß-cells (1). Persons with or at increased risk for IDDM are characterized by a number of phenotypic abnormalities in their humoral and cellular immune systems, including the production of autoantibodies [e.g. islet cell cytoplasmic (ICA), insulin, glutamic acid decarboxylase, and IA2 autoantibodies, etc.], increased blastogenic responses to ß-cell autoantigens, abnormal human leukocyte antigen-DR expression, and alterations in T lymphocyte activation (reviewed in Refs. 1, 2, 3).

Studies of the molecular aspects of lymphocyte activation have resulted in the identification of a number of molecules (receptor/counterreceptor or ligand pairs) thought to be crucial for the regulation of immune functions (reviewed in Refs. 4, 5, 6, 7, 8). One such pair is that of CD40/CD40 ligand (CD40L). CD40 is expressed on antigen-presenting cells (APC; i.e. B cells, dendritic cells, and activated macrophages), whereas CD40L (i.e. CD154) is preferentially observed on CD4+ T lymphocytes and mast cells (8, 9). CD40L may be a master regulator of the immune system due to its strong influence on both B and T lymphocyte activation as well as on the development of macrophage, B lymphocyte, and T lymphocyte effector functions.

In terms of human autoimmune disease and CD40/CD40L expression, an enhanced baseline and prolonged expression of CD40L have been reported in persons with systemic lupus erythematosus (10), whereas analysis of CD40L on T lymphocytes from persons with rheumatoid arthritis revealed no such patterns (11). Recent reports have associated the expression of immunoregulatory molecules (e.g. CD95, B7–1, and B7–2) with both alteration in immune function as well as susceptibility to IDDM in human and animal models of the disease (7, 12, 13, 14, 15). Also, in the NOD mouse model of IDDM, anti-CD40L therapy prevents the development of disease (16). As previously indicated, persons with IDDM as well as those at increased risk for the disease display abnormal humoral immune reactivities through their display of islet cell-reactive autoantibodies. As CD40L may play a key role in the regulation of such processes, we thought it crucial to investigate whether altered expression of such molecules might be associated with IDDM. These issues form the subject of this investigation.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patients

Blood samples were obtained from 45 individuals involved in our ongoing studies of the natural history of IDDM (17), including 11 newly diagnosed IDDM patients (age ± SD, 15.8 ± 12.9 yr; 8 men and 3 women). Diabetes was diagnosed according to National Diabetes Data Group criteria (18). Samples were collected from patients within 30 days of the onset of initial insulin therapy. In addition, samples were obtained from 21 ICA autoantibody positive first degree relatives (age, 17.0 ± 10.3 yr; 14 men and 7 women) of the IDDM subjects used in this study. Thirteen autoantibody-negative healthy volunteers (age, 24.2 ± 4.8 yr; 7 men and 6 women) with no family history of IDDM participated in establishing normal control ranges for these studies. Informed consent was obtained from each subject and/or their parents as approved by the University of Florida institutional review board.

Autoantibodies

ICA were determined by indirect immunofluorescence using unfixed, snap-frozen human pancreas (17). Our laboratory is a regular participant in international autoantibody workshops designed for assay standardization and proficiency.

Monoclonal antibodies

Fluorescein- and phycoerythrin-labeled monoclonal antibodies were obtained from PharMingen (San Diego, CA) and included CD3 (clone UCHT1), CD40L (clone TRAP1), and isotype-specific fluorochrome-matched control monoclonal antibodies (clone MOPC-21).

Lymphocyte phenotyping

Peripheral blood mononuclear cells (PBMC) were harvested from whole blood using Ficoll density separation (19). Briefly, PBMC were distributed into 96-well tissue culture plates (1 x 105 cells/well) in RPMI 1640 (1% Pen-Strep, 1% HEPES, 3% human AB+ serum, and 1% L-glutamine) alone or containing 5 ng/ml of phorbol myristate acetate (PMA) and 500 ng/ml ionomycin. After incubation (37 C) for 6, 24, or 48 h, the cells were labeled with the monoclonal antibodies (CD3, CD40L, and anti-mouse IgG1k) according to the manufacturer’s recommendations (30 min, 24 C, dark). After labeling, the cells were washed with buffer (phosphate-buffered saline, 1% BSA, and 0.1% sodium azide), fixed with 0.5% formaldehyde (20 min, 4 C), washed, and maintained in buffer (4 C) until analysis. Flow cytometry was performed on a FACScan using Lysis II software (Becton Dickinson and Co., San Jose, CA). Instrument settings were optimized daily using single stained cell suspensions labeled with CD3 fluorescein or CD3 phycoerythrin. Data from at least 1 x 104 cells/sample were analyzed.

Statistical analysis

Data are presented as the mean + SD as well as in scatter plot form. Testing for significance was performed using ANOVA.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Previous studies by other investigators have established the kinetics and level of CD40L expression on T lymphocytes under PMA or anti-CD3 stimulation (10, 20). Such agents bypass the requirement for antigen-specific triggering afforded by engagement of T cell receptors with antigen and the subsequent molecular events associated with activation of T lymphocytes.

Using these previously optimized parameters of time (i.e. maximum of 6 h with down-regulation by 48 h) and PMA dose for examination of CD40L, the kinetics and level of CD40L and CD3 expression were evaluated in vitro on PBMC obtained from individuals within the three study groups after 6, 24, or 48 h in culture. Figure 1Go illustrates a representative fluorescence analysis of CD3 and CD40L expression on PBMC at 6 h under unstimulated (A) and stimulated (B) conditions. As shown in Fig. 2Go (A and C), the percentage of CD3+ PBMC was essentially unaltered by PMA stimulation (6 h expression for combined values of the three groups: unstimulated, 54.1 ± 10.4; stimulated, 59.6 ± 11.4; 24 h: unstimulated, 53.7 ± 10.3; stimulated, 52.6 ± 16.3; 48 h: unstimulated, 52.7 ± 10.8; stimulated, 49.0 ± 17.1; all P = not significant). Comparative analysis of the proportion of CD3+ PBMC at 6, 24, and 48 h under unstimulated (Fig. 2AGo) or stimulated (Fig. 2CGo) conditions revealed no differences among the three study groups (all P = NS).



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  		Figure 1. Expression of CD40L and CD3 on untreated (A) and PMA-treated (B) PBMC after 6 h of culture in a representative control subject. A, Percentages: CD3-/CD40L- (21.6), CD3+/CD40L- (73.4), CD3-/CD40L+ (0.5), and CD3+/CD40L+ (4.5). B, Percentages: CD3-/CD40L- (19.2), CD3+/CD40L- (32.1), CD3-/CD40L+ (0.9), and CD3+/CD40L+ (47.8).

 


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  		Figure 2. Kinetics of CD3 and CD40L expression on PBMC from control ({circ}), increased risk ({blacksquare}), and newly diagnosed IDDM ({blacktriangleup}) subjects. Points represent the group mean (±SD) in unstimulated (A and B) and PMA-stimulated (C and D) conditions.

 
In contrast, PMA stimulation induced up to a 17-fold increase in the percentage of CD40L+ PBMC in all three study groups (Fig. 2Go, B and D; 6 h expression for combined values of the three groups: unstimulated, 2.2 ± 2.1; stimulated, 38.4 ± 9.27; 24 h: unstimulated, 2.5 ± 2.2; stimulated, 27.1 ± 13.4; 48 h: unstimulated, 3.1 ± 4.2; stimulated, 29.5 ± 15.8; all P < 0.0001). The maximal percentage of CD40L+ cells was observed at 6 h, with significant declines occurring relative to values obtained at the 24- and 48-h periods (P < 0.001 and 0.002, respectively). However, comparative analysis of the proportion of CD40L+ total PBMC at 6, 24, and 48 h under unstimulated (Fig. 2BGo) or stimulated (Fig. 2DGo) conditions revealed no differences among healthy controls, subjects at increased risk for the disease, and newly diagnosed IDDM patients at each of the three time points in unstimulated or stimulated conditions (all P = NS).

To examine the expression of CD40L+ on T lymphocytes, the proportion of CD40L+/CD3+ cells was analyzed in these individuals using both unstimulated and stimulated conditions. As shown in Fig. 3Go (A–C), the frequency of CD3+/CD40L+ cells was similar in unstimulated conditions at each the selected time points (all P = NS). Similar to the results shown in Fig. 2Go, PMA stimulation resulted in a marked increase in the proportion of CD40L+/CD3+ cells (Fig. 3Go, D–F). However, under even conditions of stimulation (Fig. 3Go, D–F), similar frequencies of CD40L+/CD3+ cells were observed in the three study groups. Finally, examination of the level of CD40L expression on CD3+ cells (i.e. mean fluorescence intensity) at 6, 24, and 48 h under unstimulated or stimulated conditions revealed no differences among healthy controls, subjects at increased risk for the disease, and newly diagnosed IDDM patients (Table 1Go; all P = NS).



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  		Figure 3. Unstimulated (A–C) and PMA-stimulated (D–F) CD40L expression on CD3+ cells from control, increased risk, and newly diagnosed IDDM subjects at 6 h (A and D), 24 h (B and E), and 48 h (C and F). Bars represent the group mean (±SD).

 

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  		Table 1. Unstimulated and stimulated mean fluorescence intensity (MFI) of CD40L on CD3+ T lymphocytes from persons with or at varying levels of risk for IDDM

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Studies of the molecular aspects of lymphocyte activation have identified a number of surface ligand pairs that appear key to the process of immune regulation. These include costimulatory molecules that are widely distributed on multiple tissue types (e.g. CTLA-4, CD28, CD54, and CD58) or subject to more restricted expression on APC [e.g. CD80 (B7–1) and CD86 (B7–2)] or T lymphocytes/mast cells (e.g. CD40L) (4, 5, 6, 7, 8, 9).

A wide range of immunoregulatory defects have been associated with IDDM. Many of the molecular mechanisms underlying these abnormalities are unknown and could relate directly or indirectly to the expression of CD40L. Although extensive, this list would include abnormal levels of cytokine production, defects in or aberrant activation of T lymphocytes and macrophages, inefficient communication between B and T lymphocytes, and autoantibody production. In addition, unusual CD40L expression (i.e. an enhanced baseline and a prolonged expression) on CD3+ cells has previously been associated with autoimmune disease (i.e. systemic lupus erythematosus) (10). Hence, it appeared obvious to monitor the expression of CD40L on T lymphocytes in persons with or at increased risk for IDDM.

In short, this study failed to demonstrate any alteration in timing, percentage, or frequency of CD40L expression on T lymphocytes from individuals with or at varying levels of risk for IDDM. Specifically, baseline percentages of positive cells were low (~2.5%), and markedly enhanced by PMA stimulation (maximal frequencies approximating 45%), yet no differences were observed between the study groups. The kinetics of expression were similar to previously reported studies (10, 11), with declines observed after 6 h of PMA stimulation. Similar to the analysis regarding proportion of positive cells, the kinetics of expression did not differ among the three study groups. Our study would appear in accordance with recent work investigating subjects with rheumatoid arthritis that revealed no abnormal or unusual expression differences associated with CD40L (11).

Our hypothesis on how aberrant CD40L expression, were it in fact observed, would contribute to IDDM was based upon our knowledge of its role in cellular activation and immune regulation. Specifically, a major role of costimulatory molecules involves the stabilization and coordination of interactions between APC and T lymphocytes after presentation of processed antigen. This communication process is influenced not only by the number of CD40L-bearing cells, but also by the cell surface density of costimulatory molecules. Indeed, efficient triggering of B lymphocytes is afforded by increasing the density of CD40L on the surface of T lymphocytes (21, 22). However, neither such abnormality was identified, thereby questioning their contribution as an underlying factor toward the aforementioned phenotypic immune aberrances ascribed to those with or at increased risk for IDDM.

For example, persons with IDDM as well as those at increased risk for the disease display abnormal humoral immune reactivities through their display of islet cell-reactive autoantibodies. However, we and others previously reported that individuals with very high levels of ICA posses a lower rate of progression to IDDM (23, 24, 25). In these studies, the titer of ICA did not correlate with the expression of CD40L (data not shown).

Our study can be considered (in part) contrasting recent studies associating the expression of immunoregulatory and/or costimulatory molecules (e.g. CD95, B7–1, and B7–2) with both alteration in immune function as well as susceptibility to IDDM (7, 12, 13, 14, 15). The association of other immunomodulatory molecules (e.g. activation markers) with IDDM has been conflicting and controversial, with reports of normal or elevated frequencies of human leukocyte antigen-DR, CD25, and/or CD69 expressing T lymphocytes in persons with the disorder (26, 27, 28, 29, 30, 31, 32, 33, 34). Finally, this study does not deter the potential significance of CD40L as a target in terms of providing a useful therapy for IDDM. Indeed, a number of studies suggest that anti-CD40L therapy may halt the progression of autoimmunity and transplant rejection (16, 34, 35, 36).

Future investigations should continue to address potential contributions of CD40L to IDDM through an examination of its function and activation, studies not performed herein. In addition, this investigation made no separation in terms of analyzing CD3+ T lymphocytes as a function of their CD4+ or CD8+ status. This type of analysis, in addition to analysis of CD40L expression under antigen-specific or anti-CD3 stimulation, may uncover potential differences not identified through the experiments performed as part of this investigation. Indeed, the experimental design and stimulant used (i.e. PMA) were selected for the purpose of providing maximal stimulation and to be in accordance with previous investigations of CD40L expression in autoimmune disorders.

In summary, these findings do not support abnormal CD40L expression as the mechanism underlying the functional defect(s) in communication between T and B lymphocytes that allows for autoantibody production or the mechanism underlying the inability of individuals to regulate antiself-immunity in IDDM.


   Acknowledgments
 
We thank Drs. Desmond Schatz, Andrew Muir, and Patricia Salisbury for their assistance in obtaining patient materials.


   Footnotes
 
1 This work was supported in part by grants from the NIH (DK-45342, AI/DK-39250, and AI-42288), The Juvenile Diabetes Foundation, and the S. Family/American Diabetes Association Chair for Diabetes Research. Back


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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