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The Journal of Immunology, 1998, 160: 5880-5885.
Copyright © 1998 by The American Association of Immunologists

Dipeptidyl Peptidase I and Granzyme A Are Coordinately Expressed During CD8+ T Cell Development and Differentiation1

Christopher L. Mabee, Michael J. McGuire2 and Dwain L. Thiele3

Liver Unit, Department of Internal Medicine, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dipeptidyl peptidase I (DPPI) is a granule protease that plays a requisite role in processing the proenzyme form of the CTL granule serine proteases (granzymes). This study assesses DPPI mRNA and enzyme expression during T lymphocyte ontogeny and CTL differentiation. The most immature CD3-CD4-CD8- thymocytes were found to express >40-fold higher levels of DPPI mRNA, although levels of DPPI enzymatic activity in CD3-CD4-CD8- thymocytes were only modestly higher than those seen for CD4+CD8+ or CD4+CD8- thymocytes. More mature CD8+CD4- thymocytes and CD8+ splenocytes expressed significantly higher levels of DPPI mRNA and enzymatic activity than CD4+CD8+ or CD4+CD8- thymocytes. Granzyme A mRNA expression was observed in DPPI expressing CD3-CD4-CD8- and CD8+CD4- thymocytes and was also observed in CD8+CD4- splenocytes; however, expression was not observed in CD4+CD8+ or CD4+CD8- thymocytes. Both DPPI mRNA and granzyme A mRNA expression in CD8+ T cells decreased to very low or undetectable levels during the first 48 h after allostimulation in MLCs. However, peak levels of both DPPI and granzyme A expression were observed later in the course of CD8+ T cell responses to alloantigen, with DPPI mRNA expression peaking on either day 3 or day 4 and granzyme A expression peaking at the end of a 5-day MLR. These data indicate that DPPI is expressed at all stages of T cell ontogeny and differentiation in which granzyme A mRNA is detected; consequently, DPPI appears to be available for the processing and activation of granzyme A during both CD8+ T cell development and differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dipeptidyl peptidase I (DPPI),4 formerly known as cathepsin C, is a cysteine protease that is capable of removing dipeptides from the amino-terminus of various peptides and protein substrates (1, 2, 3, 4, 5, 6). This enzymatic activity is expressed at higher levels in cytotoxic lymphocytes and myeloid cells than in lymphocytes without cytotoxic potential or cells of non-bone marrow origin (7, 8, 9). Within cytotoxic lymphocytes and myeloid cells, DPPI colocalizes within specialized granules with proteases from the families of structurally related granule serine proteases expressed in these cells (10). The results of a variety of studies indicate that DPPI plays a requisite role in the posttranslational processing and activation of these granule serine proteases by the removal of an activation dipeptide (10, 11, 12, 13). Moreover, inhibiting DPPI activity during CD8+ T cell responses to alloantigens (alloAgs) impairs both the activation of granzyme serine protease activity and the generation of cytotoxic effector function (14, 15, 16, 17).

During thymocyte development and differentiation, a number of genes encoding putative CTL effector molecules are expressed (18, 19). Granzyme A mRNA expression has been noted in both immature thymocytes devoid of CD4 and CD8 epitopes and in more differentiated CD8+CD4- thymocytes (18, 19). A multiphasic induction of granzyme genes during T cell ontogeny has been previously proposed (20, 21). While the expression of granzyme A and granzyme B mRNA has been noted during thymocyte differentiation, high levels of these enzymatic activities are expressed late in the course of CD8+ T cell responses to immune stimuli, and granzyme expression has been used as a marker of differentiated CTL effector cells (22). In contrast, the expression of high levels of DPPI has been noted in both CTL precursor and effector cells (9, 10). In a recent study assessing DPPI mRNA levels during the in vitro activation of lymphokine-activated killer function, increased DPPI gene expression was noted following IL-2 stimulation of human PBLs, suggesting that, similar to granzyme A and granzyme B expression, DPPI may be up-regulated in response to stimuli that elicit cytotoxic lymphocyte responses (23). However, other investigators have noted no significant differences in the levels of DPPI mRNA expressed in freshly isolated mouse splenocytes vs in vitro-activated murine lymphokine-activated killer cells or alloAg-activated splenocytes (24). Neither of these previous studies examined DPPI gene expression in purified populations of lymphocytes. Thus, the degree to which DPPI gene expression is regulated in CD8+ CTLs following various immune stimuli remains unclear. In addition, the stage at which DPPI expression is up-regulated in CTLs during T cell ontogeny is unknown. Our studies examine the expression of DPPI mRNA and enzymatic activity during the maturation and activation of CD8+ T cells. These data indicate that DPPI gene expression varies significantly during the course of lymphocyte ontogeny and CD8+ T cell differentiation. Moreover, the patterns of DPPI expression observed in the course of these studies suggest that DPPI expression is regulated in a manner that results in the availability of high levels of DPPI in T cells synthesizing the preprogranzymes that are reportedly the physiologic substrates of this granule protease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

CTLL-2 cells were obtained from the American Type Culture Collection, (Manassas, VA) and were cultured in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% FBS (Life Technologies, Gaithersburg, MD), 1 mM sodium pyruvate, 5 x 10-5 M 2-ME, 5 mM HEPES (Sigma, St. Louis, MO), 0.3 mg/ml L-glutamine, 200 U/ml penicillin G, and 10 µg/ml gentamicin. These cultures were supplemented with 25 U/ml of rIL-2 twice per week.

Mice

We obtained 5- to 8-wk-old C57BL/6J and DBA/2J from The Jackson Laboratory (Bar Harbor, ME). All animal research protocols used in this project were reviewed and approved by the University of Texas Southwestern Medical Center at Dallas Institutional Animal Care and Use Committee (Dallas, TX). Animals were 10 to 12 wk old at the time of use.

Monoclonal Ab

Anti-CD4 (GK 1.5) (25), anti-CD8 (YTS.169.4) (26), anti-NK (3A4) (27), anti-I-Ab,d,q (MS/114.15-2) (28), and anti-I-Ab (25–5–16S) (29) were prepared as culture supernatants of hybridoma cells or were staphylococcal protein A-purified from hybridoma culture supernatants as previously described (30). FITC-conjugated anti-CD3 (145-2CII) was purchased from PharMingen (San Diego, CA).

Cell purification and isolation

Thymectomies were performed on C57BL/6J mice by careful dissection, and organs were thoroughly rinsed twice with HBSS (Life Technologies) before dispersion into single-cell suspensions. Spleens were minced and filtered through nylon mesh. Viable thymocyte and splenocyte populations were separated by Hypaque 1083 (Sigma) density gradient centrifugation for 30 min at 1500 revolutions per min after suspension in RPMI 1640 medium supplemented with 0.3% FBS, 25 mM HEPES, 100 µM 2-ME, 200 U/ml penicillin G, and 10 µg/ml gentamicin. Thymocyte or T cell subsets were purified and isolated using magnetic cell sorting with a VarioMACS cell sorter (Miltenyi Biotec, Auburn, CA) (31). Briefly, cells were suspended in PBS buffer supplemented with 5 mM EDTA and 1% FBS and incubated for 15 min at 4°C with Ab-coated microbeads. Positive and negative selection columns were used where appropriate, and cell populations were washed with PBS following separation and isolation as directed by the manufacturer. Upon a subsequent flow cytometric analysis of splenocytes and single-positive thymocytes following enrichment with the magnetic-activated cell sorter magnetic bead isolation system, >95% purity of selected cell populations was routinely observed.

CD3-CD4-CD8- thymocytes

Thymocytes were incubated with FITC anti-CD3, anti-NK (3A4), and anti-I-Ab (MS 114.15-2 and 25–5–16S) Abs for 30 min at 4°C. Following washing, the cells were incubated with goat anti-mouse Ig, anti-FITC, anti-CD4 (L3T4), and anti-CD8 (Ly-2) microbeads as previously described, and the CD3-CD4-CD8- thymocytes were collected as the unbound fraction using a negative selection column.

CD4+CD8+ thymocytes

Thymocytes were incubated with fluoresceinated anti-CD8 (Life Technologies) and biotinylated anti-CD4 (Life Technologies) for 30 min at 4°C. Cells were washed and incubated with avidin-phycoerythrin (Life Technologies) for 30 min on ice before washing, and the selection of cells positively stained with both anti-CD8 and anti-CD4 by FACS using a FACStar (Becton Dickinson, Mountain View, CA) as previously described (16). Upon subsequent flow cytometric analysis, >95% purity of positively selected cell populations was routinely observed.

CD4+CD8- and CD8+CD4- thymocytes

Thymocytes were incubated with anti-CD4 (L3T4) or anti-CD8 (Ly-2) microbeads. The unbound fraction following selection with anti-CD4 microbeads was then incubated with anti-CD8 (Ly-2) microbeads and positively selected as CD4-CD8+ cells. The unbound thymocytes following initial selection with anti-CD8 microbeads were then incubated with anti-CD4 (L3T4) microbeads and positively selected as CD4+CD8- cells.

Isolation of CD8+ C57BL/6J splenocytes

Splenocytes were incubated with anti-CD8 (Ly-2) microbeads, and positively selected CD8+ T cells were isolated.

Generation of in vitro-activated CD8+ B6 anti-H-2d-specific CTLs

CD8+ CTLs were activated in MLCs containing both 40 x 106 C57BL/6J (H-2b) responder spleen cells and 10 x 106 irradiated (1500 cGy) T cell-depleted DBA/2J (H-2d) stimulator spleen cells in 20 ml of culture medium. Before irradiation, the DBA/2J cells were separated from other cellular elements by Hypaque 1083 (Sigma) density centrifugation. The cells were also depleted of T cells by incubation with HO 13.4 anti-Thy-1 Ab (32) (1:30 dilution) for 30 min on ice and subsequent treatment with rabbit complement (1:6 dilution) for 50 min at 37°C. The cells were passed through 30-µM nylon filters and irradiated with 1500 cGy. Each culture was suspended in 20 ml of complete media. AlloAg-activated cells were harvested on days 1 through 5 and positively enriched for CD8+ CTLs using anti-CD8 (Ly-2) microbeads (Miltenyi Biotec).

Protein, DPPI, and N-{alpha}-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) esterase (granzyme A) enzymatic activity assays

The protein concentration was assessed by the bicinchoninic acid method using BSA as the standard as previously described (10). Assay reagents were purchased from Pierce (Rockford, IL). DPPI activity was assayed by hydrolysis of glycylphenylalanyl-ß-naphthylamide (Sigma) as previously described. BLT esterase activity was assayed by hydrolysis of BLT (Calbiochem-Behring, La Jolla, CA) as previously described (10, 33). The results are given as the mean ± SE of the mean for triplicate determinations.

Generation of DPPI, granzyme A, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probes for semiquantitative PCR

Those primers (M41) (5'-CACAACTTTGTGAAGGCCATCAAT-3') and (B34) (5'-TCATCAGTTCCTCTGCGG-3') that had been used previously to amplify a 830-base pair (bp) product of mouse DPPI cDNA (33) were used to establish an RT-PCR assay for DPPI mRNA expression. These primers are complementary to sequences in the 4th and 7th exon of the mouse DPPI gene (24); consequently, the primers yield distinctive m.w. products when used to amplify genomic DNA. Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA). A 177-nucleotide bp internal probe was obtained from the previously cloned mouse DPPI cDNA (34) by digestion with BamHI and EcoRI (Life Technologies) and gel purification. The sequence of granzyme A mRNA was analyzed, and a 5' primer from the N terminus (5'-CACGGTTGTTCCTCACTCAAGACC) and a 3' primer from the C terminus (5'-TCGGCGATCTCCACACTTCTCTCCACC) were synthesized (Integrated DNA Technologies). cDNA encoding a portion of mouse granzyme A was obtained by RT-PCR using CTLL-2 RNA as a template and cloned in the PCR 3.1 vector. The cloned cDNA was verified as mouse granzyme A by sequence analysis. A 372-nucleotide bp internal probe was obtained by digestion with AvaII and BglII (Life Technologies) and gel purification. The cloned murine G3PDH sequence was analyzed, and a 5' primer from the N terminus (5'-ACCACAGTCCATGCCATCACTGC) and a 3' primer from the C terminus (5'-CCACCACCCTGTTGCTGTAGCC) were synthesized (Integrated DNA Technologies). cDNA encoding a 450-nucleotide bp segment of mouse G3PDH was obtained by RT-PCR using CTLL-2 RNA as a template and cloned in the PCR 3.1 vector as described above. The cloned cDNA was verified by sequencing and used as a probe following gel purification as previously described (34).

Semiquantitative RT-PCR

Cell populations (1.0 x 106) were individually pelleted and treated for 15 min at 15 to 30°C with TRIzol reagent (Life Technologies). Total RNA was isolated per the manufacturer’s recommendations. The isolated RNA was solubilized in 30 µl of RNase-free sterile water at 70°C for 10 min. First strand cDNA was synthesized by RT reaction with 1 µg of starting RNA in a 22 µl reaction volume. Following denaturing and annealing with 1 µl of oligo(dT) (Life Technologies) (0.5 µg/µl), the reaction was equilibrated and incubated at 45°C for 1 h with 1 µl of Superscript (Life Technologies) RT (200 U/µl). Specimens were immediately removed from the thermal controller and put on ice. A total of 1 U of RNase H (Sigma) was added to each specimen; specimens were subsequently incubated at 37°C for 30 min. In triplicate reactions, first-strand cDNA derived from 0.25 µg RNA served as the template for the amplification of DPPI, granzyme A, and G3PDH. Each reaction was amplified in a final volume of 50 µl containing 1.5 U Taq polymerase, 1x PCR buffer, 2.5 mM MgCl2, and 0.25 mM deoxynucleoside triphosphate. Mineral oil was placed over the samples to serve as a vapor barrier. The amplification sequence consisted of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and elongation at 72°C for 1 min for 27 cycles. The initial denaturation and annealing steps were conducted in the absence of Taq polymerase and deoxynucleoside triphosphate, which were added during the first incubation at 72°C. On the 28th cycle, the elongation step at 72°C was extended to 5 min. The PCR products were immediately blotted by a Nytran max-strength membrane using a Minifold II (72-well) slot-blot system (Schleicher & Schuell, Keene, NH). The nucleic acid was fixed to the membrane in a vacuum oven (15–20 pounds per square inch) for 1 h at 82°C. Membranes were pretreated with 25 ml Express Hyb hybridization solution (Amersham, Arlington Heights, IL) for 1 h at 65°C in a rocker-incubator. A total of 25 ng of previously isolated template DPPI probe was denatured at 100°C for 5 min and diluted to a volume of 45 µl sterile water on ice. The probe was synthesized in the presence of 50 µCi of [{alpha}-32P]deoxyCTP by incubating at 37°C for 10 min with the Rediprime random primer labeling system (Amersham). The reaction was stopped by adding 5 µl of 0.2 m EDTA. The radiolabeled probe was denatured at 100°C for 5 min and immediately added to the hybridization buffer to obtain a final concentration of 1 ng/ml and was incubated at 65°C for 1 h with gentle rocking. The membranes were removed from the hybridization solution and washed with two low-stringency washes for 10 min with 2x SSC plus 0.1% SDS at room temperature and with two high stringency washes for 10 min with 1x SSC plus 0.1% SDS at 65°C. Quantitative ß emission was assessed using the Ambis computer-controlled multiwire proportional counter (Ambis, San Diego, CA) and Ambis core software (version 4.0) on a Dell 486 computer (Austin, TX) using 720-min counting periods and a 1.6 x 3.2-mm detection plate. The membranes were subsequently stripped of the radioactive probe by two incubations in 95°C water baths for 10 min. The procedure was repeated with probes for granzyme A and G3PDH.

In preliminary experiments, varying quantities of RNA (range: 0.1–1.0 µg) from both CTLL-2 cells, a cell line expressing high levels of DPPI mRNA, and mouse brain, a tissue expressing very low levels of DPPI (33), were reverse transcribed and amplified for varying numbers of cycles (range: 25–35 cycles) before transfer to nylon membranes and hybridization with [{alpha}32P]-labeled DPPI or G3PDH probes. We found that cDNA that had been generated from 0.25 µg of total RNA isolated from either CTLL-2 or mouse brain amplified through 28 cycles yielded detectable but submaximal levels of DPPI and G3PDH PCR products (Fig. 1GoA, additional data not shown). Following the determination of the range of PCR cycles through which DPPI and G3PDH DNA accumulated exponentially, additional studies were performed in which PCR products were resolved by electrophoresis in 0.8% agarose gels (Fig. 1GoA) and transferred to Nytran membranes by capillary action. Hybridization with DPPI- and G3PDH-specific probes demonstrated that >80% of the radioactivity was bound to full-length dsPCR products (Fig. 1GoB). Further studies showed similar specificity for the granzyme A probe. The quantities of probes bound by PCR amplicons were assessed using the Ambis as previously described (Fig. 1GoC). In subsequent studies, the quantities of RT-PCR-amplified DPPI and granzyme A products were normalized to quantities of RT-PCR-amplified G3PDH mRNA. A single RNA isolate from CTLL-2 cells was reverse transcribed; this cDNA was included in each assay to control for interexperimental variability in the specific activity of the [{alpha}-32P]-labeled probes that were bound by the RT-PCR products of each mRNA examined.



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  		FIGURE 1. Semiquantitative RT-PCR assays of DPPI mRNA. A, 0.25 µg of RNA from CTLL-2 and mouse brain were reverse transcribed, and cDNA was amplified with DPPI- and G3PDH-specific primers by PCR for 28 cycles. DPPI primers produced a 830-bp product (lanes 1 and 2), and G3PDH primers produced a 450-bp product (lanes 5 and 6). Lane 4 displays the electrophoretic patterns of a 100-bp ladder (Life Technologies) marker DNA including prominent 600-bp, 1500-bp, and 2000-bp bands. B, the 0.8% agarose gel from A was transferred to a Nytran membrane by capillary action, the DNA was fixed to the membrane in a vacuum oven, and DPPI- and G3PDH-specific [{alpha}-32P]deoxyCTP radiolabeled probes were used to quantitatively assess mRNA expression using the Ambis computer-controlled ß emission counter. C shows quantitatively the number of cpm/mm2 detected by the Ambis counter during a 12-h scan.

 

   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DPPI and granzyme A mRNA expression in thymocytes and splenocytes

The expression of DPPI and granzyme A mRNA was assessed in various subsets of thymocytes and splenocytes as described in Figure 2Go. Both DPPI mRNA and granzyme A mRNA expression were detected in CD3-CD4-CD8- thymocytes, while the expression of neither DPPI mRNA nor granzyme A mRNA was detected in CD4+CD8+ thymocytes. CD8+ thymocytes were found to express higher levels of both DPPI mRNA and granzyme A mRNA than CD4+ thymocytes. Although the expression of granzyme A mRNA was modestly reduced in CD8+ spleen cells, this cell population continued to express detectable levels of granzyme A mRNA as well as high levels of DPPI mRNA.



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  		FIGURE 2. Expression of mRNA in thymocytes and spleen cells. Highly purified CD3-CD4-CD8- (DN Thy), CD4+CD8+ (DP Thy), CD4+CD8- (CD4+ Thy), and CD8+CD4- (CD8+ Thy) thymocytes as well as CD4+ or CD8+ spleen cells were isolated and assessed by RT-PCR for DPPI, granzyme A, and G3PDH mRNA. The results represent the means ± SEM of values obtained from three independent experiments.

 
DPPI enzymatic activity in thymocytes

Whole cell lysates from each thymocyte and spleen cell population were assayed for DPPI activity. As described in Figure 3Go, CD3-CD4-CD8- thymocytes were found to express DPPI enzymatic activity at a level that was about threefold less than that detected in peripheral CD8+ splenocytes. Progressively lower levels of DPPI activity were detected in CD4+CD8+ thymocytes and CD4+CD8- thymocytes. CD8+CD4- thymocytes were found to express higher levels of DPPI than CD4+CD8- thymocytes, with even higher levels of DPPI expressed in CD8+ splenocytes.



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  		FIGURE 3. Expression of DPPI enzymatic activity in thymocytes and spleen cells. Highly purified thymocyte and spleen cell populations as described in Figure 2Go were lysed and assessed for hydrolysis of the DPPI substrate glycylphenylalanyl-ß-naphthylamide. DPPI-specific activity is expressed as units of activity per mg protein (U/mg) with 1 U = 1 nmol ßNA released per minute. The results represent the means ± SEM of values obtained from three independent experiments.

 
DPPI and granzyme A expression in alloAg-activated CD8+ cells

In vitro alloAg-activated CD8+ CTLs were generated in a 5-day MLC. Cells were harvested daily for 5 days, and highly purified CD8+ CTLs were selected as described in Materials and Methods and assayed for DPPI and granzyme A mRNA expression (Fig. 4Go) and enzymatic activity (Fig. 5Go). In contrast to the readily detectable levels of DPPI and granzyme A mRNA noted in freshly isolated CD8+ spleen cells before allostimulation, the levels of both types of mRNA were greatly reduced or undetectable after 1 to 2 days in MLCs. In contrast to granzyme A mRNA expression, which reappeared on day 3 of MLC and continued to increase through day 5 of activation, DPPI mRNA levels were up-regulated earlier and peaked by day 3 or day 4 of culture as noted in the two representative experiments outlined in Figure 4Go.



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  		FIGURE 4. DPPI and granzyme A mRNA expression following alloAg stimulation. B6(H-2b) spleen cells were cultured with irradiated, T cell-depleted DBA/2(H-2d) stimulator cells in a 5-day MLC. At the indicated time points, CD8+ T cells were isolated and assayed for DPPI, granzyme A, and G3PDH mRNA expression; two representative experiments are presented.

 


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  		FIGURE 5. Expression of DPPI and BLT esterase activities following in vitro alloAg stimulation. CD8+ B6 spleen T cells were activated in MLCs as described in Figure 4Go and then assessed for DPPI and BLT activity. These data correspond to experiment 1 only.

 
As described in Figure 5Go, in vitro-activated CD8+ CTLs displayed a reduction in DPPI enzymatic activity on day 2 of the MLC, with a subsequent up-regulation of DPPI enzymatic activity by day 3 that continued throughout the remainder of the 5-day MLC. In contrast, BLT esterase activity, a measure of trypsin-like protease activity that is mediated largely, although not exclusively, by granzyme A in CD8+ T cells (35), decreased following initial stimulation, returned to preactivation levels at day 3, and then continued to increase to much higher levels by day 5 of the MLC.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
DPPI is a lysosomal cysteine protease previously noted to play a role in the posttranslational processing and activation of members of the family of granule serine proteases or granzymes expressed in activated CTLs. The present findings indicate that the expression of high levels of DPPI antecedes or is concomitant with that of granzyme A not only during the activation of CTLs but also during the earlier stages of T lymphocyte ontogeny.

When thymocyte subsets were examined for DPPI mRNA expression and enzymatic activity, DPPI mRNA expression was found to be significantly higher in immature CD3-CD4-CD8- thymocytes and more differentiated CD8+CD4- thymocytes than in intermediate stage CD4+CD8+ thymocytes or in the corresponding CD4+CD8- population of more differentiated, single-positive thymocytes. Of interest, even though CD3-CD4-CD8- thymocytes expressed very high levels of DPPI mRNA, these cells were found to express similar or only slightly greater amounts of DPPI enzymatic activity than other thymocyte subsets expressing little or no DPPI mRNA. In addition, while DPPI mRNA levels appear to fall to very low or undetectable levels in CD4+CD8+ and CD4+CD8- thymocytes, low levels of DPPI enzymatic activity remain detectable in these cells. The disparate expression of DPPI mRNA and enzymatic activity in these thymocyte populations likely relates to differences in mRNA and protein half-life or to other posttranscriptional regulatory mechanisms.

The present findings suggest that DPPI gene expression is activated before or immediately after the migration of T cell precursors to the thymus. The concomitant expression of both DPPI and granzyme A (our observations and Refs. 18 and 19) as well as granzyme B (18, 19) at this early stage of T cell development suggests that enzymatically active forms of granzyme A and B could theoretically be produced in these cells. However, we have not found detectable levels of BLT esterase activity in lysates of double-negative thymocytes in additional studies (data not shown). These findings are in agreement with the observation by Ebnet et al. (19) that BLT esterase activity was not able to be detected by histochemical techniques in CD4-CD8- thymocytes. The absence of detectable levels of granzyme A-like enzymatic activity in these cells may be related to the relatively low levels of enzymatically active DPPI that appear to accumulate in these cells before the down-regulation of DPPI mRNA during subsequent thymic differentiation steps. Prior studies have noted that granzyme processing and activation only proceed efficiently in cells expressing relatively high levels of DPPI enzymatic activity (11). The present findings also indicate that while DPPI expression is reduced during positive and negative selection processes in the thymus, DPPI expression is up-regulated again following the acquisition of the CD8+CD4- phenotype before CD8+ T cells egress to the periphery. Granzyme A expression in the thymus follows a pattern similar to that of DPPI, with the highest level of mRNA and enzymatically active protein (additional data not shown) expression occurring in CD8+CD4- thymocytes with no apparent gene transcription during intermediate phases of thymocyte differentiation.

Following intrathymic selection and differentiation, single-positive lymphocytes emerge from the thymus and migrate to peripheral lymphoid organs, in which mature CD8+ T cells continue to express both DPPI mRNA and enzymatic activity. In agreement with previously reported studies (10) in which we detected immunoreactive and enzymatically active granzyme A in CD8+ spleen T cells, modest but readily detectable levels of granzyme A expression were detected in the freshly isolated spleen CD8+ T cell populations examined in the present studies. It remains unclear whether this low level of granzyme A expression is present only in a subset(s) of CD8+ T cells, such as cells that have recently emigrated from the thymus or T cells recently stimulated by Ag. However, the results of previously published studies using dipeptide ester substrates of DPPI to identify cells expressing high levels of this enzyme suggest that the majority of human peripheral blood and mouse spleen CD8+ T cells continue to express high levels of DPPI enzyme activity (15, 16).

In contrast to the similarities between the patterns of DPPI and granzyme A expression in thymocytes and spleen T cells, granzyme B mRNA is reportedly expressed equally in CD4+CD8- and CD8+CD4- thymocytes (19) but not in splenic CD8+ T cells. In additional studies (data not shown) using granzyme B-specific PCR primers and RT-PCR mRNA assay conditions similar to those that detect granzyme A and DPPI mRNA in CD8+ spleen T cells, we were unable to detect granzyme B mRNA in CD8+ spleen T cells.

When spleen T cells were stimulated with alloAg, it was observed the levels of both granzyme A and DPPI mRNA in CD8+ cells declined to very low or undetectable levels during the first 1 to 2 days of the 5-day MLC. Following an initial reduction in expression during the first 2 days of culture, DPPI mRNA expression in CD8+ T cells was induced over the next 24 to 48 h; peak levels of DPPI mRNA expression in CD8+ T cells were observed during day 3 or day 4 of activation. In contrast, granzyme A mRNA expression was highest at the end of the 5-day MLC.

These findings regarding the expression of DPPI during alloAg-induced activation of CD8+ T cells are in contrast to recent reports which suggest that DPPI mRNA is constitutively expressed in both resting and in vitro-activated mouse spleen cells (24). The discrepancies may be secondary to the use of highly purified CD8+ T cells in the present studies, while previous attempts to follow DPPI mRNA levels during the activation of mouse spleen T cells assessed mRNA levels in whole spleen cell populations and thus could not distinguish the expression by CD8+ T cells from DPPI mRNA expressed by macrophages, NK cells, or other spleen cell populations. The present findings are in agreement with prior reports that human lymphocyte DPPI mRNA expression is significantly up-regulated following in vitro activation (23).

The observation that DPPI mRNA expression and levels of enzymatic activity peak earlier in the course of CTL activation than does granzyme A expression is consistent with the proposed requisite role that DPPI appears to play in posttranslational processing and the activation of granzymes. Thus, DPPI is expressed immediately before and during periods of peak granzyme A expression during both lymphoid ontogeny and again during the Ag-specific activation of CTLs. These data are consistent with other observations suggesting that a critical level of DPPI enzymatic activity is required for the production of active granzyme B in transfected COS cells (11). Therefore, the observed temporal sequence of DPPI and granzyme A expression during alloAg stimulation is teleologically appropriate. These findings suggest that the expression of high levels of DPPI is part of a multigene activation sequence leading to the expression of high levels of granule proteases in CTLs at selected stages of T cell ontogeny and T cell immune responses.


   Footnotes
 
1 This work was supported by a National Research Service Award from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 5 K32 DK09608-02, by the American Liver Foundation Amgen Postdoctoral Research Fellowship awarded to C.L.M., and by Research Grant R01 AI-24639 from the National Institutes of Health. Back

2 Present address: Ryburn Cardiology Center, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235-8573. Back

3 Address correspondence and reprint requests to Dr. Dwain L. Thiele, Liver Unit, Department of Internal Medicine, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235-9151. E-mail address: Back

4 Abbreviations used in this paper: DPPI, dipeptidyl peptidase I; BLT, N-{alpha}-benzyloxycarbonyl-L-lysine thiobenzyl ester; alloAg, alloantigen; G3PDH, glyceraldehyde-3-phosphate dehydrogenase. Back

Received for publication December 22, 1997. Accepted for publication February 19, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Izumiya, N., J. S. Fruton. 1956. Specificity of cathepsin C. J. Biol. Chem. 218:59.[Free Full Text]
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