A glycoprotein endopeptidase enhances calcium influx and cytokine production by CD4+ T cells of old and young mice

Scott B. Berger1,5, Amir A. Sadighi Akha2 and Richard A. Miller2,3,4

1 Department of Biological Chemistry and 2 Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA
3 Geriatrics Center, University of Michigan, Ann Arbor, MI, USA
4 Ann Arbor DVA Medical Center, Ann Arbor, MI 48109-0940, USA
5 Present address: 5410 CCGCB, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0940, USA

Correspondence to: S. B. Berger; E-mail: bergers{at}umich.edu


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Many of the downstream signaling defects observed in aged T cells are believed to be the result of very early events involving the initial interaction between T cells and antigen-presenting cells. Recent findings suggest that this interaction is hindered by glycosylated surface macromolecules, including CD43, on the T cell surface. Treatment of CD4+ T cells by O-sialoglycoprotein endopeptidase (OSGE), which cleaves glycosylated forms of CD43, restores the ability of cells from aged mice to form immunological synapses and to express early activation markers. Here we show that OSGE enhances Ca2+ influx in T cells from CB6F1 mice, and enhances their ability to produce IL-2, IL-4, IL-5, IL-6, IL-10, IL-13 and IFN{gamma} at the mRNA level, and IL-2 and IFN{gamma} at the protein level, in the first 6 h after activation. Although OSGE has little effect on synapse formation in CD4+ T cells from young mice, our new data show that OSGE increases the production of most cytokines by young as well as old T cells. Secretion of the Th2 cytokine, IL-4, was altered only slightly by OSGE treatment, suggesting that the removal of OSGE-sensitive surface molecules may have differential effects on Th1 and Th2 cytokines. These data support a model in which O-glycosylated surface proteins inhibit CD4+ lymphocyte activation in both young and old mice, and in which such glycoproteins contribute to the age-related decline in cytokine production.

Keywords: aging, cellular activation, glycosylation, immunosenescence, signal transduction


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Aging leads to functionally important changes in the immune system, which are typically most dramatic in the T cell compartment and may be related to thymic involution, changes in T cell subset composition and changes in activation processes within the peripheral T cell pool. The age-related decline in T cell-mediated immunity leads to defects in lymphocyte proliferation (1) and production of cytokines, including IL-2 (2), thus contributing to the defective responses of elderly individuals to infectious agents (3, 4), vaccines (5) and possibly neoplasia (6).

There is now strong evidence that poor T cell proliferation and IL-2 production reflect deficiencies in the signaling cascade that leads to immune synapse formation and T cell activation. These defects include declines, with aging, in calcium signals (7), activation of the ERK and JNK pathways (8, 9) and ultimately declines in the production of mRNA for IL-2 and IL-2R components (1, 10, 11).

Recent work has suggested that many of the downstream signaling defects observed in aged CD4+ T cells are the result of very early events involving the initial interaction between T cells and antigen-presenting cells (APCs). Contact between T cells and peptide-bearing APCs induces translocation of many signaling molecules, including Cbl, Vav, LAT and talin, to the focal point of T cell–APC interaction known as the immunological synapse. The proportion of T cells able to form immune synapses declines by ~50% in mice by the age of 18 months (12), whether the stimulus takes the form of peptide-bearing APCs or anti-CD3 hybridomas used as a polyclonal stimulus. CD4+ T cells that fail to form immune synapses also fail to translocate NFAT to the nucleus (13), and typically express high levels of surface P-glycoprotein that characterize anergic CD4+ T cells in aged mice (14).

There is evidence to suggest that T cell activation may be inhibited by large, heavily glycosylated surface molecules, the most abundant being CD43 and CD45 (1518). Thus, the observation that a major subset of CD4+ cells from aged mice expresses particularly high levels of heavily glycosylated CD43 (19) suggested that hyperglycosylation of CD43 might contribute to the decline in T cell activation in aged mice. Consistent with this hypothesis, enzymatic cleavage of the large, heavily O-glycosylated forms of CD43 from the surface of the CD4+ cells by O-sialoglycoprotein endopeptidase (OSGE) was found to restore the ability of CD4+ cells from aged mice to form immunological synapses, while having no effect on synapse formation by CD4+ cells from young mice. In addition, OSGE treatment of CD4+ cells repaired the age-dependent defects in the expression of CD69 and CD25 (19) after T cell activation.

The goals of the present study were to see if OSGE treatment of CD4+ lymphocytes could affect TCR-mediated Ca2+ influx, and restore the age-related defect in IL-2 production normally observed in aged T cells, to evaluate the effects of OSGE on production of other cytokines and to see if cytokine production by T cells from young mice would also be altered by pre-treating the responding cells with OSGE.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Specific pathogen-free male (BALB/cJ x C57BL/6J) F1 (CB6F1) mice were purchased from the National Aging Institute contract colonies. Mice were considered young at 4–8 months of age and old at 18–24 months of age. Mice were given free access to food and water. Sentinel animals were examined quarterly for serological evidence of viral infection; all such tests were negative during the course of these studies. Mice with splenomegaly or macroscopically visible tumors at the time of sacrifice were not used for experiments.

Antibodies and reagents
Antibody to mouse CD3{varepsilon} was produced from clone 145-2C11 (American Type Tissue Collection, Rockville, MD, USA) and antibody to CD28 was produced from clone 37.51. OSGE was purchased from Cedarlane Labs (Hornby, Ontario, Canada). Trizol LS reagent was purchased from Life Technologies (Grand Island, NY, USA). Colorless [{alpha}-32P] UTP was purchased from MP Biomedicals (Irvine, CA, USA).

Cell preparation, enzymatic treatment and stimulation
CD4+ T cells were obtained as previously described (20, 21). Flow cytometric analysis of a typical preparation showed it to be 90–95% positive for both CD3 and CD4. The purified CD4+ T cells (1 x 106 cells per ml in HBSS) were incubated with 50 µg ml–1 OSGE for 30 min at 37°C. Cells were then either left unstimulated or stimulated for 6 h in culture with 0.1 µg immobilized {alpha}CD3 ({alpha}CD3i), 1.0 µg soluble {alpha}CD3 ({alpha}CD3s) or 1.0 µg soluble {alpha}CD3 in conjunction with 1.0 µg soluble {alpha}CD28 ({alpha}CD3 + {alpha}CD28). Cells were cultured in RPMI 1640 with 10% fetal bovine serum at 37°C in a 10% CO2 atmosphere for 6 h prior to harvest for assessment of mRNA and protein production.

Purification of naive CD4+ T cells
Naive CD4+ T cells were obtained using the CD4+ CD62L+ T cell isolation kit purchased from Miltenyi Biotec (Auburn, CA, USA) according to the manufacturer's instructions. Briefly, CD4+ T cells were isolated by negative selection using anti-CD8a, anti-CD45R, anti-CD11b, anti-CD49b and anti-Ter-119. The purified CD4+ cells were then positively selected using microbeads conjugated to monoclonal anti-mouse CD62L. The naive subset was confirmed to be >90% pure by flow cytometry using CD4 and CD62L double staining.

Measurement of cytosolic Ca2+
Cytosolic Ca2+ measurement was performed as previously described (22). Splenic T cells (either untreated or pre-treated with OSGE), were loaded with Indo-1 AM (2.5 µM) (Molecular Probes, Eugene, OR, USA) at room temperature for 30 min, washed and then stained with anti-CD4 (clone RM4-5) and anti-CD44 (clone IM7) antibodies. The calcium mobilization assay was conducted at 37°C on a FACSVantage flow cytometer using FACSDiva software (BD Biosciences, San Jose, CA, USA). Baseline levels were collected for 20 s, after which the cells were stimulated with 5 µg ml–1 of non-cross-linked anti-CD3 (clone 145-2C11). Each assay was performed for a 5-min period. The acquired data were analyzed using FlowJo's kinetic platform (TreeStar, Ahsland, OR, USA). Each histogram displays the mean of the violet : blue ratio as a function of time with smoothing for moving average.

Ribonuclease protection assay
Total RNA was extracted using Trizol LS reagent. Aliquots of RNA representing equal cell numbers from individual animals were assessed for cytokine mRNA using the RiboQuantTM Multi-Probe RNase Protection Assay System (PharMingen, San Diego, CA, USA) according to the manufacturer's instructions. The multiprobe template set employed was mCK1, which contained DNA templates for mouse IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, IFN{gamma}, L32 and GAPDH. The mouse positive control RNA was also purchased from PharMingen. The template set was used to synthesize [{alpha}-32P] UTP-labeled probes in the presence of a GACU pool using a T7 RNA polymerase. Probes were hybridized overnight with target RNA, followed by RNase digestion and proteinase K treatment. Samples were chloroform extracted and ethanol precipitated in the presence of ammonium acetate. Dried pellets were re-suspended in 5 µl 1x loading buffer, incubated for 3 min at 90°C and then immediately placed on ice. Ribonuclease protection assay (RPA) products were resolved by electrophoresis on a 5% polyacrylamide–8 M urea sequencing gel. The gel was dried and exposed to a phosphor screen for 4 days and then evaluated using a Storm 840 Phosphorimager (Molecular Dynamics, Sunnyvale, CA, USA). The ImageQuant Software Package V5.2 (Molecular Dynamics) was utilized for densitometric analysis. To control for differences in probe intensity and exposure conditions among experiments, the density value for each cytokine band was normalized, by dividing by the density of the band for ribosomal protein L32 in unstimulated CD4+ cells from the young donor. Each day's experiment compared one young mouse to one old mouse.

Cytokine ELISA
The lymphocytes were stimulated as described above. The supernatants were collected 6 h after stimulation and stored at –70°C until cytokine analysis. IL-2, IL-4 and IFN{gamma} were quantified using commercial ELISA kits (BioSource International, Camarillo, CA, USA) according to the manufacturer's protocols. The minimum detectable dose was <8 pg ml–1 for IL-2, <1 pg ml–1 for IFN{gamma} and <5 pg ml–1 for IL-4.

Statistical analysis
In the case of Ca2+ assays, the peak increase above baseline was calculated for CD4+ T cells and their subsets in every sample, with the data then subjected to a two-tailed Mann–Whitney test at P = 0.05. Cytokine data were analyzed using a Friedman analysis of variance (ANOVA). Post hoc analysis was done using Wilcoxon matched-pairs tests.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Pre-treatment of CD4+ T cells with OSGE leads to enhanced Ca2+ influx
To examine the functional effect of OSGE treatment on the T cell calcium response, both untreated and OSGE-treated T cells were loaded with Indo-1 AM, and stimulated with soluble anti-CD3 antibody without further cross-linking. Figure 1 shows a representative experiment, where anti-CD3 has, as expected, led to a transient increase in cytosolic Ca2+. Consistent with previous reports, in the absence of OSGE, CD4+ memory T cells (CD4+ CD44hi) mounted a significantly lower response to anti-CD3 stimulation than their naive (CD4+ CD44lo) counterparts (23). This was true for cells derived from both young (P = 0.001) and old (P = 0.02) donors. Pre-treatment with OSGE significantly enhanced the anti-CD3-induced Ca2+ response in CD4+ cells from both young and old mice (Fig. 1). This was the case for the CD4+ T cells as a whole (P = 0.001 for the young, P = 0.02 for the old), as well as their naive (P = 0.002 for the young, P = 0.02 for the old) and memory (P = 0.0006 for the young, P = 0.02 for the old) subsets. These results underscore the ability of OSGE to enhance the anti-CD3-induced Ca2+ response in naive and memory CD4+ T cells of both young and old mice.



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Fig. 1. Effect of OSGE on the calcium response. CD4 T cell Ca2+ responses were induced by non-cross-linked anti-CD3 in young (Y) and old (O) mice, either with or without pre-treatment by OSGE. Top panel: CD4 T cells. Middle panel: CD4 naive cells, i.e. gated for low expression of CD44. Bottom panel: CD4 memory cells, i.e. gated for high expression of CD44. The break in each line shows where stimulatory antibodies were added after 20 s of baseline data collection. The graphs are representative of the data obtained from seven young and four old mice.

 
Pre-treatment of CD4+ T cells with OSGE results in enhanced IL-2 mRNA expression
Previous data from this laboratory have shown that treatment of CD4+ T cells from old mice with the CD43-cleaving endopeptidase OSGE restores their ability to form immune synapses with APCs to the level characteristic of cells from young mice (19). Because in previous work from this and other laboratories the effects of age on cytokine production in vitro depended on the strength and quality of the stimulus used (24), we evaluated three forms of CD3-dependent activation: plate-immobilized anti-CD3 (CD3i, a strong stimulus for cytokine production), soluble anti-CD3 (CD3s, a weaker stimulus) and the combination of soluble anti-CD3 and anti-CD28 antibodies, which typically leads to intermediate levels of cytokine production. IL-2 mRNA expression was examined by RPAs 6 h after activation, the earliest time point found in preliminary experiments to produce quantifiable levels of IL-2 mRNA. The top panel of Fig. 2 shows IL-2 mRNA results for cells stimulated with CD3i. Young CD4+ cells produced slightly more IL-2 mRNA than old cells under these conditions, but the difference did not reach statistical significance (P = 0.09). Treating the cells isolated from the old animals with OSGE resulted in a significant (P = 0.008) increase in IL-2 mRNA expression. CD4+ T cells from the young donors also showed enhanced (P = 0.008) IL-2 transcription after OSGE treatment. Pre-treatment of the cells with OSGE without stimulation did not result in any cytokine transcription (data not shown). There was no effect of age on IL-2 mRNA expression in cells treated with OSGE (P = 0.17). There was no significant difference (P = 0.2) in IL-2 production between T cells from young and old mice stimulated after OSGE treatment.



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Fig. 2. IL-2 mRNA levels estimated by RPAs. A total of 106 CD4+ T cells from young (Y) and old (O) mice were either treated with OSGE (Rx) or left untreated, and then stimulated with immobilized anti-CD3 ({alpha}CD3i), soluble anti-CD3 ({alpha}CD3s) or soluble anti-CD3 with the addition of soluble anti-CD28 ({alpha}CD3 + {alpha}CD28). RPAs were performed by hybridizing total RNA from equal input cell numbers to mCK1 multiprobe templates (BD Biosciences). The gels were then exposed for 4 days to a phosphor screen and images were obtained and quantified as described in Methods. The numbers at the top of each panel show P-values (Wilcoxon matched-pairs tests). These values reflect, from left to right, comparisons of (i) young OSGE-treated to young untreated controls, (ii) young controls to old controls and (iii) old OSGE-treated to old untreated controls. Data are expressed in arbitrary units. The graphs display the data obtained from at least eight young and eight old mice. P-values for the ratio of young (untreated) to old (OSGE treated) are as follows: top ({alpha}CD3i) P = 0.1, middle ({alpha}CD3s) P = 0.7, bottom ({alpha}CD3i + {alpha}CD28) P = 0.02.

 
The middle panel of Fig. 2 shows the outcome of experiments using the weaker stimulus, soluble anti-CD3. Under these conditions, age led to a significant decline in IL-2 mRNA production (P = 0.01). OSGE treatment led to significant increases in IL-2 mRNA production for both young (P = 0.02) and old (P = 0.04) mice. After OSGE treatment, the difference between young and old T cells in IL-2 production is reduced, and no longer statistically significant (P = 0.07). The bottom panel of Fig. 2 presents analogous results using CD3 + CD28. With this stimulus, T cells from young mice produced 50% more IL-2 mRNA than old mice. This difference was not statistically significant (P = 0.07), although young donors produced more IL-2 mRNA than the cells from the old donors tested in seven out of eight experiments. OSGE treatment led to significant increases in IL-2 production for both young (P = 0.05) and old (P = 0.03) mice, and there was no difference between OSGE-treated young and old T cells in the IL-2 production (P = 0.78). Thus, regardless of the strength of the stimulus, OSGE treatment increases IL-2 mRNA production by CD4+ cells of both young and old mice, restoring the responses of old cells to levels similar to those produced by untreated cells from young donors.

Pre-treatment of CD4+ T cells with OSGE results in enhanced transcription of IL-4, IL-5, IL-6, IL-10, IL-13 and IFN{gamma}
In order to determine if OSGE treatment also enhanced mRNA transcription for other cytokines, CD4+ T cells were treated with OSGE, stimulated with CD3i and evaluated for IL-4, IL-5, IL-6, IL-10, IL-13 and IFN{gamma} transcription (Fig. 3) at 6 h. At this early time point, only one cytokine, IL-6, showed a significant difference between old and young mice (P = 0.02). Treating the CD4+ cells with OSGE led to significant increases in the expression of all six cytokine mRNAs, bringing them to similar levels in young and old mice.



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Fig. 3. mRNA levels for IL-4, IL-5, IL-6, IL-10, IL-13 and IFN{gamma} after CD3i stimulation. A total of 106 CD4+ T cells from young (Y) and old (O) mice were either treated with OSGE (Rx) or left untreated, and then stimulated for 6 h with immobilized anti-CD3 ({alpha}CD3i). Other details are as specified in the legend to Fig. 1. Data are expressed in arbitrary units. The graphs display the data obtained from nine young and nine old mice. P-values for the ratio of young (untreated) to old (OSGE treated) are as follows: IL-4 P = 0.1, IL-5 P = 0.05, IL-6 P = 0.9, IL-10 P = 0.1, IL-13 P = 0.04 and IFN{gamma} P = 0.01.

 
Similar experiments were conducted using CD3s (Table 1) and CD3 + CD28 (Table 2) as stimuli. The tables present median ratios and P-values for each cytokine tested. The first column shows the P-values of the performed ANOVAs. The second shows the effects of OSGE on young cells; the third column tabulates age effects; the fourth column shows effects of OSGE on cells from old donors; the fifth column shows age differences after OSGE treatment; and the sixth compares the production of cytokines by untreated cells from young donors and OSGE-treated cells from old mice. The young : old median ratios were above 1.0 for all but 1 of the 14 comparisons, although they did not always reach statistical significance. OSGE treatment of T cells from old donors resulted in an increase in the production of each of the cytokine mRNAs tested, both for CD3s and CD3 + CD28. Most of these effects reached statistical significance, with P < 0.05 in 12 of the 14 cases. OSGE treatment of young CD4+ cells also resulted in higher levels of mRNA for each of the cytokines for both stimuli, reaching statistical significance in 8 of the 14 comparisons. In cells treated with OSGE, there was no age-dependent difference in cytokine production with the exception of IL-5, in which treated old cells produced more cytokine than treated young cells (P = 0.04). These results suggest that OSGE treatment increases production of most cytokine mRNAs in young and old mice and that the treatment corrects any age-dependent declines in production of cytokine mRNA early after stimulation.


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Table 1. Effect of aging and OSGE treatment on cytokine mRNA levels induced by soluble anti-CD3 antibody

 

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Table 2. Effect of aging and OSGE treatment on cytokine mRNA levels induced by soluble anti-CD3 and anti-CD28 antibodies CD3 + CD28 stimulation RPA results for seven cytokines with and without OSGE treatment

 
Pre-treatment of CD4+ T cells with OSGE results in enhanced IL-2 and IFN{gamma} protein secretion
Data shown in Fig. 2 establish that OSGE leads to increased production of IL-2 mRNA. To see if IL-2 protein production is enhanced to a similar degree, OSGE-treated CD4+ cells were cultured for 6 h, and supernatant cytokines were quantified by ELISA. The results are shown in the left-hand column of Fig. 4. Differences between young and old mice were small at this early time point, and reached statistical significance only for the CD3 + CD28 stimulus. In the CD3s data, young donors produced more IL-2 than the cells from the old donors tested in parallel in 10 out of 11 experiments. OSGE treatment of cells from young or old animals resulted in a significant increase in IL-2 secretion regardless of stimulus or donor age. Pre-treatment of the cells with OSGE without stimulation did not result in any cytokine protein secretion (data not shown).



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Fig. 4. IL-2, IFN{gamma} and IL-4 protein secretion of CD4+ T cells from young and old mice using three different forms of stimulation. A total of 106 CD4+ T cells from young (Y) and old (O) mice were either treated with OSGE (Rx) or left untreated, and stimulated with immobilized anti-CD3 ({alpha}CD3i), soluble anti-CD3 ({alpha}CD3s) or soluble anti-CD3 with the addition of soluble anti-CD28 ({alpha}CD3 + {alpha}CD28). The supernatant was collected after 6 h of stimulation and analyzed for IL-2, IFN{gamma} and IL-4 by ELISA (Biosource International) as described in Methods. These values reflect, from left to right, comparisons of (i) young OSGE-treated to young untreated controls, (ii) young controls to old controls and (iii) old OSGE-treated to old untreated controls. The graphs display the data obtained from at least eight young and eight old mice.

 
The data on IL-2 production are not only consistent with models in which OSGE repairs specific age-related defects in T cell activation pathways but also with models in which OSGE increases cytokine production independent of the direction of the age-specific effect. Many (25, 26), though not all (27), studies have found an increase with age in the production of IFN{gamma} in vitro by CD4+ T cells, although production of IFN{gamma} in response to mycobacterial PPD in vivo shows a clear age-related decline (28). Under our in vitro conditions (Fig. 4), aged CD4+ cells produced significantly more IFN{gamma} than young cells when exposed to stronger stimuli (CD3i and CD3 + CD28), though the difference was not seen using CD3s. OSGE treatment increased IFN{gamma} production significantly in both young and old CD4+ cells using each of the three varieties of stimulation. These data are thus consistent with the idea that OSGE increases production of multiple cytokines, regardless of the direction of the age effect on cytokine production pattern.

Pre-treatment of CD4+ T cells with OSGE has inconsistent effects on IL-4 secretion
To determine if the enhancement of cytokine secretion after pre-treatment with OSGE was limited to the Th1 response, CD4 cells were pre-treated with OSGE, stimulated and tested for early IL-4 protein secretion. The results are shown in the right-hand column of Fig. 4. At this early time point, CD4+ cells from old mice secreted less IL-4 than cells from young mice regardless of stimulation; being significant at P < 0.05 in each case. OSGE treatment of T cells from old donors led to a modest (20 to 40%) increase in IL-4 production, which was significant (P < 0.01) for two of the three stimuli tested. The effect of OSGE on young CD4+ cells, however, was inconsistent, showing either a small increase or small decrease depending on the stimulus used.

OSGE enhances IL-2 production in CD4+ naive T cells
All the cytokine data presented thus far have been obtained from experiments performed on populations of CD4+ T cells that included both naive and memory cells. In order to determine if the effects mediated by OSGE were independent of the naive to memory T cell shift normally observed in the aged T cell compartment (29), we tested the effect of OSGE treatment on IL-2 secretion in naive CD4+ T cells isolated from young and old animals. Naive cells were purified based on CD62L expression. The results are shown in Fig. 5. IL-2 production by purified naive CD4 cells was somewhat lower than the levels seen in earlier experiments using CD3 and CD28 to stimulate unseparated populations of CD4 cells (see Fig. 4). This discrepancy may be the result of differences in the purification techniques or in relative numbers of APCs. Purified naive CD4+ T cells showed no effect of age (P = 0.9) on IL-2 production after {alpha}CD3 + {alpha}CD28 stimulation. OSGE treatment led to a significant enhancement of IL-2 production in the naive cells from both the young (P = 0.03) and old mice (P = 0.03).



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Fig. 5. Naive CD4+ T cell IL-2 protein production. A total of 106 naive (CD62L+) T cells from young (Y) and old (O) mice were either treated with OSGE (Rx) or left untreated, and stimulated with soluble anti-CD3 with the addition of soluble anti-CD28 ({alpha}CD3 + {alpha}CD28). The supernatants were collected after 6 h of stimulation and analyzed for IL-2 by ELISA (Biosource International) as described in Methods. The stated P-values reflect, from left to right, comparisons of (i) young OSGE-treated to young untreated controls, (ii) young controls to old controls and (iii) old OSGE-treated to old untreated controls. The graphs display the data obtained from six young and six old mice.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
T cell activation is initiated by T cell–APC interaction. This results in the congregation of accessory and positive signaling molecules such as LFA-1 and CD28 at the immunological synapse and the dynamic active exclusion of large, heavily glycosylated proteins including CD43 and CD45 that may interfere with TCR–MHC interaction (30). Previous work has shown age-dependent defects in T cell signaling that involve very early cytoskeletal defects and precede peptide recognition by the TCR (20). Our lab has recently demonstrated that many CD4+ T cells from old mice express unusually high levels of the glycosylated forms of the bulky glycoprotein CD43 (19). In addition, T cells from old donors showed a decline in the association of CD43 with the cytoskeletal matrix and a decreased ability to exclude CD43 from the immunological synapse (19). It was shown that treatment of CD4+ lymphocytes with OSGE, which cleaves the peptide backbone of proteins, including CD43, at sites of O-glycosylation, restored defects in the translocation of signaling molecules such as talin and CD3{varepsilon} to the immunological synapse, and also restored full expression of the early activation markers CD69 and CD25, which are normally diminished in T cells from old mice (19). Previous work, however, did not show whether aged CD4+ T cells exposed to OSGE treatment would regain the ability to secrete IL-2 and other age-sensitive cytokines at levels characteristic of T cells from young mice.

In the present study, we assessed the effect of OSGE pre-treatment on the Ca2+ response, and the induction of cytokine mRNA and protein. The acquired data reproduce the laboratory's previous finding that memory CD4+ T cells mount a significantly lesser Ca2+ response to anti-CD3 stimulation than their naive counterparts (23). Furthermore, we were able to prove that pre-treatment with OSGE can enhance the anti-CD3-induced Ca2+ response in both naive and memory T cells from both young and old donors.

Because strong stimuli, such as immobilized antibodies to CD3, can sometimes overcome age-related defects in T cell activation (31), we evaluated OSGE effects in the context of three forms of T cell-specific activation: immobilized anti-CD3 (CD3i); soluble anti-CD3 (CD3s), a weaker stimulus, and the combination of soluble anti-CD3 with anti-CD28 (CD3 + CD28), which is designed to mimic the two signals received by T cells from peptide-bearing APCs. We measured cytokines and cytokine mRNAs very early after T cell stimulation (6 h), to avoid the potential complications of cytokine uptake and cytokine effects on T cell activation. This time point was the earliest at which cytokine production could be reliably quantified.

Our results show strong or suggestive evidence that OSGE treatment of CD4+ T cells increases cytokine mRNA production of all seven cytokines tested after activation with all three forms of stimulation, in most cases reaching a significant (P < 0.05) level of augmentation. This enhancement was seen in the T cells from both young and old mice and occurred whether or not the cytokine mRNA showed an age-dependent change. Our results also show that OSGE treatment of CD4+ T cells increased cytokine protein production for two Th1 cytokines. These include IL-2, which has been shown to decrease with age (31), and IFN{gamma}, for which the majority of the literature shows an age-dependent increase (27). Thus, the OSGE effect is one of enhancing cytokine secretion to a similar level in T cells from young and old donors, rather than solely correcting an age-dependent defect. The effects mediated by OSGE are not restricted to the naive or memory subset of CD4+ T cells, in that OSGE treatment enhances expression of the early activation marker CD69 (32) and enhances calcium influx (Fig. 1) in both naive and memory cells, and enhances IL-2 production in purified naive CD4 cells (Fig. 5).

Consistent with the mRNA data, we observed a significant age-dependent decline in the production of the Th2 cytokine IL-4 with each method of stimulation. The effect of OSGE treatment on IL-4 secretion was, however, quite modest, with only a 20–40% increase seen for old T cells. The effect on young T cells was even smaller and inconsistent across stimuli. It is unclear why the dramatic effect of OSGE on the production of IL-4 mRNA by young and old T cells (see Fig. 2, Tables 1 and 2) was not accompanied by a parallel effect at the protein level, but the protein data suggest that inhibition of T cell cytokine production by OSGE-sensitive surface molecules may be more critical for Th1 cells than for Th2 cells. Desialylation of lymphocyte surface gangliosides promotes cytokine production by Th2 cells (33). OSGE has little or no effect on ganglioside sialylation (our unpublished data), but it is possible that Th2 and Th1 cells differ broadly in the roles played by sialylated surface molecules in various stages of activation, and hence the influence of OSGE on IL-4 mRNA production may not lead to corresponding increases in secretion of the IL-4 protein.

A previous study (19) showed that OSGE treatment increased the proportion of aged CD4+ T cells that formed immune synapses but did not have a similar effect in cells from young mice. Our data, in contrast, suggest that OSGE-sensitive molecules inhibit the function of CD4+ cells from mice of both ages. These data suggest that OSGE-sensitive glycoproteins may interfere with more than one step in the T cell activation process, among which some are critical for synapse formation and others modulate separate age-sensitive elements of the activation cascade. It is not known whether the molecules whose cleavage permits synapse formation by old CD4+ cells are the same as those whose cleavage improves cytokine production and calcium signal generation, in cells from donors of either age. Although CD43 is known to be more heavily glycosylated in old CD4+ cells compared with young CD4+ cells (19), it is possible that changes in the glycosylation pattern, membrane distribution or cytoskeletal linkages of CD43 and other OSGE-sensitive proteins could also contribute to hampered T cell activation in aged mice.

Both CD43 and CD45 deserve further study in the context of OSGE effects on T cell activation. Together, these two glycoproteins account for ~30% of the total protein on the T cell surface (34). Both molecules are heavily O-glycosylated and therefore sensitive to cleavage by OSGE (35). The functional role of CD43 is currently controversial. The initial work on CD43 suggested that it was a negative regulator of T cell signaling. T cells from CD43 knockout mice showed a lower threshold for activation, and exhibited a hyperproliferative response upon stimulation with a variety of mitogenic stimuli (16). However, a more recent study showed that some of these previously observed hyperproliferative characteristics of CD43 knockout mice might be dependent on genetic background. After back-crossing these mice to the C57BL/6J background for six generations, there was no evidence of hyperresponsiveness associated with CD43 loss (36). Recent data have shown that only the CD43 intracellular domain may be necessary to reverse CD43 knockout hyperactivation, suggesting that steric inhibition may not be the cause of CD43 negative regulation (37). In other studies, CD43 was also shown to act as a positive co-stimulator, independent of CD28, which acts in conjunction with TCR ligation (38).

Similarly, CD45 is thought to provide both positive and negative regulatory signals in different contexts (39). CD45 is a protein tyrosine phosphatase that functions in antigen receptor signaling by dephosphorylation of the src family kinases Lck (40) and Fyn (41). This dephosphorylation of Lck and Fyn results in TCR-mediated activation by the removal of the negative regulatory tyrosine at the C terminus of these kinases (42, 43). For these reasons, T cells deficient in CD45 fail to respond to activation through antigen receptors (44). However, recent evidence suggests that CD45 can also function as a Janus kinase tyrosine phosphatase that negatively regulates cytokine receptor signaling involved in the differentiation, proliferation and antiviral immunity of hematopoietic cells (17). Thus, the effects of OSGE on CD43 and/or CD45 might reflect a mixture of pathways, some that alter the signaling function of these glycoproteins and others that involve improved T cell interaction with or access to APC after removal of the glycocalyx formed by both these molecules at the T cell surface.

A third set of potential mechanisms involves charge effects; removal of O-linked glycoproteins from T cell surfaces removes an abundance of negatively charged terminal sialic acid residues. It has been previously shown that removal of terminal sialic acid residues from the surface of B cells enhances the ability of the cells to stimulate the proliferation of allogeneic and antigen-specific syngeneic T cells (45). In addition, it has been postulated that specific sialic acid linkages, in particular the {alpha}2-6 bonds, may be more important than the simple number of sialic acid residues in the modulation of inhibitory effects on immune function (46). CD43 and CD45 both contain large amounts of terminal sialic acid residues that modulate binding of mAbs to internal determinants (47). The degree to which OSGE effects on T cell synapse formation and cytokine production reflect changed surface charge also deserves further investigation.

New data from our laboratory show that there are alterations with age in the levels, accessibility or conformation of multiple glycoproteins on the surface of CD4+ T cells (32). While some differences are due to the accumulation of memory cells with age, others are age sensitive and found exclusively in the naive subset or in both naive and memory subsets. Furthermore, analysis of CD43 and CD45 molecules shows that age alters the glycosylation of specific proteins that regulate TCR interaction with APCs (32). The molecular basis for these age-dependent changes in T cell glycosylation are currently unknown and are being investigated. Some possible explanations may include age-related alterations in endoplasmic reticulum–golgi processing, or age-dependent changes in the gene expression of glycosyltransferases and/or glycosidases responsible for T cell surface glycosylation.

Our data suggest a model in which deregulation of glycosylation pathways in T cells from old mice might lead to hyperexpression of glycosyl moieties that interfere with T cell–APC interaction, thus contributing to age-dependent defects in T cell signaling and function. According to this model, the positive effects of OSGE on T cells from young donors reflect its effects on surface glycoproteins that may, in untreated cells, prevent hyperstimulation and autoimmune responses. T cells from old donors may have a higher amount of cell surface glycosylation, leading both to diminished function in untreated cells and to a greater response to treatment with OSGE. We believe that OSGE treatment removes these glycosyl moieties, leaving behind a similar minimal level of glycosylation on the T cells from both the young and old donors. This is why in most cases we see a higher level of function of the OSGE-treated old cells as compared with the young untreated cells, and a similar amount of function when comparing young treated to old treated cells.

Based on our findings, the potential for pharmacological intervention with OSGE or enzymes with similar specificity to reverse age-dependent immune defects in the elderly may deserve further study. The use of these agents would of course be contingent on humans having similar OSGE-sensitive barriers to T cell activation, and would require that activation could be induced without undesirable autoimmune responses. Therapeutic approaches might involve either treatment of T cells ex vivo or the use of genetically engineered APCs that express OSGE at the cell surface. When these peptide-loaded APCs form an immunological synapse with the corresponding T cell, the OSGE-sensitive molecules would be cleaved from the surface of the lymphocyte, enhancing T cell response. Overall, our results further extend the possibility that glycoproteins play a crucial role in T cell immunosenescence, and could potentially provide new avenues for the restoration of immune function in the elderly.


    Acknowledgements
 
We wish to thank Gonzalo G. Garcia for advice and comments. This work was supported by NIH grants AG19619, AG00114, and the Glenn/AFAR Scholarship for Research in the Biology of Aging.


    Abbreviations
 
ANOVA   analysis of variance
APC   antigen-presenting cell
OSGE   O-sialoglycoprotein endopeptidase
RPA   ribonuclease protection assay

    Notes
 
Transmitting editor: T. F. Tedder

Received 22 September 2004, accepted 10 May 2005.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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