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The Journal of Immunology, 1999, 162: 689-695.
Copyright © 1999 by The American Association of Immunologists

Presence of Pentoxifylline During T Cell Priming Increases Clonal Frequencies in Secondary Proliferative Responses and Inhibits Apoptosis1

Manisha Gupta*, Anna George*, Ranjan Sen{dagger}, Satyajit Rath*, Jeannine M. Durdik{ddagger} and Vineeta Bal2,*

* National Institute of Immunology, New Delhi, India; {dagger} Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, MA 02254; and {ddagger} Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Naive T cells appear to be primed by specific Ag to differentiate into either effectors or memory cells. We have been analyzing the factors involved in this differential commitment in the priming of alloresponsive human T cells in vitro and have shown that the presence of a phosphodiesterase inhibitor, pentoxifylline (POX), during priming results in a decrease in the primary response and enhancement in the secondary proliferative response. We now show that the POX-mediated effect can be mimicked by dibutyryl cAMP. The secondary response enhancement is due to the effects of POX on the T cells rather than the APCs, because even fixed APCs can prime T cells in the presence of POX. POX affects T cells directly by increasing clonal frequency rather than the burst size of the secondary responders. The known inhibition of IL-2 production by POX is not responsible for this effect, because exogenous IL-2 supplementation does not block it. The presence of POX during priming alters the outcome of T cell activation, resulting in a lower frequency of cells expressing IL-2R{alpha} (CD25) and a decrease in their subsequent apoptosis, and this anti-apoptotic effect is consistent with the enhanced commitment of T cells to secondary responsiveness by POX.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
To generate an immune response, mature naive peripheral T cells need both cognate Ag-specific and noncognate costimulatory signals for optimal activation (1). If appropriate noncognate signals are missing, the naive cells may become anergic instead of responding (2). However, even in the presence of both signals, responding T cells make choices between alternate cytokine profiles (the Th1 and the Th2 groups), as well as becoming either immediate effector cells or quiescent memory cells (3). In recent years, the role of noncognate signals—both cell surface molecules such as the B7 family, and cytokines like IL-6, IL-10, and IL-12—in differential T cell commitment to the Th1 and Th2 pathways has been reported (4, 5, 6, 7). The role of altered peptide ligands in changing effector cell response has also been documented extensively (8). However, the regulatory signaling mechanisms controlling the differential commitment to effector vs memory cells have not been as extensively studied. In fact, even phenotypic distinctions between immediate effectors and quiescent memory cells are not reliable, although it is possible to distinguish naive from activated cells (3, 9, 10).

Following ligation of the TCR-CD3 complex with appropriate peptide-MHC ligands, TCR oligomerization leads to activation of tyrosine kinases such as lck and fyn followed by ZAP-70 (11). The CD4 or CD8 coreceptors and protein kinases associated with them also contribute to activation (12). Events further downstream involve the generation of calcium flux and calcineurin activation with transcriptional regulation by both calcineurin-dependent and -independent pathways (13), and induction of transcription factors of the rel and NFAT family involved in regulating many activation-induced T cell genes (14). Here again there is little information on the differences in signaling that are involved in changing the commitment of T cell differentiation to either the effector or the memory pathway, although differences in signaling events between naive vs formed memory T cells have been studied (15, 16, 17).

In this connection, we have been studying the effects of pharmacological agents present during T cell priming in vitro on the subsequent secondary "recall" proliferative responses mounted by these primed cells. We have shown earlier that the presence of pentoxifylline (POX)3 during priming of human T cells in vitro by HLA-mismatched PBMCs results in a decrease in primary proliferative responses but enhanced secondary proliferative responses (18). POX is a drug used extensively clinically for a variety of inflammatory and vascular disorders (19). It is a phosphodiesterase inhibitor and can induce increases in intracellular cAMP levels (19). Its effects have been studied on a wide variety of cell types including T cells, B cells, and cells of myeloid lineage (20, 21, 22). In T cells, it has been shown that the effects of POX may be mediated via modulation of induction of transcriptionally active proteins of the rel family (23). Here we explore the mechanism of action of POX responsible for the enhancement of secondary or "memory" recall responses. We find that the effect of POX is mimicked by enhancement of cAMP levels in T cells, is independent of IL-2, and is associated with alterations in the degree of apoptosis in the responding T cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
T cell priming and proliferation assays

PBMCs from HLA-mismatched individuals were separated using density-gradient centrifugation on Ficoll-Paque (Pharmacia, Uppsala, Sweden). Stimulator PBMCs were used in titrated doses after gamma-irradiation (2000 rads). For the primary proliferative alloresponses, stimulators in graded doses were used with 1–3 x 105 responders per well in 200 µl of RPMI 1640 (Life Technologies, Grand Island, New York) fortified with L-glutamine (Life Technologies) and antibiotics, containing 10% heat-inactivated responder autologous serum. Cultures were incubated in a CO2 atmosphere for 5 days, pulsed with 0.5 µCi of [3H]thymidine (New England Nuclear, Boston, MA) for the last 8–12 h of the assay and harvested onto glass fiber filters for counting cell-incorporated thymidine (Betaplate; Wallac, Finland). Results are expressed as mean cpm ± SE for triplicate cultures.

For priming, responder and stimulator cells were mixed in 2- to 5-ml cultures in a ratio of 1:1–3:1. After 5 days of incubation, the cells were harvested, counted, and viable cells used as primed responders with gamma-irradiated allostimulators in secondary alloproliferation assays, which were pulsed with [3H]thymidine after 4 days for estimating cell proliferation. For flow cytometric analysis, cells were separated from debris by Ficoll-Paque gradient centrifugation before staining.

Dibutyryl cyclic AMP (dbcAMP), POX, aphidicolin (all from Sigma, St. Louis, MO), and rIL-2 (Boehringer Mannheim, Mannheim, Germany) were added singly or together to the cultures in various doses as indicated during priming and in primary alloresponse assays. Where fixed PBMCs were to be used as stimulators, they were fixed with 0.05% paraformaldehyde (PF; Sigma) at room temperature for 20 min, washed extensively, and used.

Clonal responder frequency and burst size analysis

To determine the frequency of allospecific precursor cells in the various responder populations, titrating numbers (from 10,000 to 200 per well) of variously primed cells were added to a constant number (1 x 105 per well) of gamma-irradiated stimulator PBMCs. Of the 48 wells plated per responder dose, those wells showing a response at least twice that of the background value were scored positive. The responder frequency was calculated by taking the number of negative wells into account (24). The response was considered clonal at the responder cell number where the proportion of positively responding wells was <37% (25). At responder cell concentrations showing clonal frequencies, the average cpm values of responding (vs nonresponding) wells were used as an estimate of the average clonal burst size of single responding T cells.

Flow cytometry

For analysis of cell surface markers, primed cell populations separated on Ficoll-Paque gradients were used. All cells used for staining showed >95% viability by trypan blue exclusion. Briefly, 1–3 x 105 cells per well were stained with primary Abs at 4°C, followed by appropriately labeled secondary reagents if required. Samples were analyzed using a Bryte flow cytometer (Bio-Rad, Hemel Hampstead, U.K.). Abs to the T cell markers CD3 (OKT3), CD4 (OKT4), and to the activation markers CD25 (IL-2R{alpha}; Immunotech, Marseilles, France) and Fas (CD95; PharMingen, San Diego, CA) were used for staining. To detect apoptosis using propidium iodide (PI; Sigma), Ab-stained cells were fixed and permeabilized with 70% ethanol, washed, and PI (10 µg/ml) added just before cytometric analysis. Data were analyzed using FlowJo software (Treestar, San Jose, CA).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Enhanced T cell priming seen in the presence of POX is due to a direct effect of the drug on responder T cells

We have shown previously that the presence of POX during a primary alloresponse in vitro results in dose-dependent inhibition of the immediate proliferative response and prepares the responder T cells for an enhanced secondary response (18). POX has a variety of effects on various cell types. To examine whether POX affects T cells directly to cause the effect we observe, or whether it works via its effects on the priming APCs, priming was done using PBMCs fixed with 0.05% PF. Fig. 1GoA shows that the primary response to PF-fixed PBMCs was much lower than that observed for unfixed PBMCs, and was not enhanced by the presence of POX. Fixed PBMCs are not effective at priming T cells for a secondary response either (Fig. 1GoB). However, T cells primed with such PF-fixed PBMCs in the presence of POX show an enhanced secondary response to unfixed PBMCs (Fig. 1GoB), suggesting that the effects of POX are mediated directly on responder T cells themselves.



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  		FIGURE 1. POX enhances secondary response commitment during T cell priming even with fixed APCs. A, The alloresponse of T cells to gamma-irradiated (open circles) or PF-fixed PBMCs in the presence (filled squares) or absence (open squares) of POX (100 µg/ml) is shown. B, Recall responses of cells primed with PF-fixed APCs in the presence (filled squares) or absence (open squares) of POX compared with unprimed cells (open circles) when restimulated with gamma-irradiated PBMCs are shown. Data are shown as mean ± SE of triplicate cultures. A representative example of four independent experiments is shown.

 
Enhancement in secondary alloresponse by dbcAMP

POX is a phosphodiesterase inhibitor and thus an enhancer of intracellular cAMP levels (26). It also has a variety of effects on tissue-specific transcription factor induction (27, 28) and is known to down-modulate IL-2 transcription in T cells (21, 29), although it is not clear whether these effects are mediated through increased cAMP levels. To begin characterizing the mechanism of action of POX responsible for the enhancement of secondary T cell responses, we tested the effects of a synthetic analogue of cAMP, dbcAMP, which degrades slowly in comparison to cAMP, thereby providing better bioavailability.

Fig. 2GoA shows that the presence of 1 mM dbcAMP during primary alloantigen exposure of T cells in vitro inhibits the primary proliferative response. Fig. 2GoB shows that there is a significant enhancement of the secondary response when priming is done in the presence of 100 µM dbcAMP, which does not inhibit the primary response, as well as in the presence of 1 mM dbcAMP, which does inhibit the primary response. No effect on either primary or recall responses is seen with 10 µM dbcAMP. Thus, high levels of intracellular cAMP during T cell priming is sufficient to diminish the primary proliferative response and enhance the secondary response capability.



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  		FIGURE 2. Priming in presence of dbcAMP results in a decreased primary proliferative response and enhanced secondary response. A, A decrease in primary alloresponse with increasing doses (filled circles, 0 µM; hollow circles, 10 µM; hollow squares, 100 µM hollow triangles, 1 mM) of dbcAMP is shown. B, Secondary responses of T cells treated with dbcAMP during priming to gamma-irradiated PBMCs (filled circles, unprimed; filled squares, PBMC primed; PBMC primed in the presence of dbcAMP doses as in A) are shown. Results are shown as mean ± SE of triplicate cultures and are representative of five independent experiments. Background counts were 1000–3000 cpm.

 
The presence of IL-2 during priming does not affect enhancement in priming mediated by POX

POX is known to suppress the transcription of IL-2 (21, 29). To examine whether this lack of IL-2 could account for the enhanced commitment to secondary responses we observe, exogenous IL-2 (3 U/ml) was added during priming in the presence or absence of POX. Responder cells primed in the presence of both POX and IL-2 exhibited an enhancement of the secondary response similar to that caused by the presence of POX alone during priming (Fig. 3Go). The addition of higher or lower concentrations of IL-2 (1–10 U/ml) during priming showed similar results (data not shown). These data suggest that the suppression of IL-2 transcription is not responsible for the augmentation of the secondary response by the presence of POX during priming.



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  		FIGURE 3. The presence of IL-2 in addition to POX during priming does not suppress the enhancement of secondary proliferative responses. Secondary response of T cells either unprimed (filled circles), primed with allo-PBMCs in the presence (filled squares) or absence (hollow squares) of POX (100 µg/ml), or in presence of IL-2 (3 U/ml; hollow triangles) or of both POX and IL-2 (filled triangles) is shown. Background counts were <2000 cpm. The results shown are from one of five independent experiments.

 
The enhancement of the secondary response by POX is due to an increase in the clonal frequency of proliferation-competent responders

We next estimated the frequency of the alloreactive responder cells after priming by titrating the number of primed cells added per well in the presence of fixed numbers of stimulator PBMCs. Fig. 4GoA shows that the PBMC-primed responder population shows a frequency higher than that in unprimed cells, and the presence of POX (or dbcAMP, data not shown) during priming significantly enhances the frequency of responders. This assay also allows the estimation of the ability of a single allospecific cell to give rise to progeny. The average magnitude of the proliferative response in responding wells at clonal frequency (positive wells <37%) was considered a reflection of clonal burst size. As shown in Fig. 4GoB, the average clonal proliferative response was not significantly different between POX-treated and -untreated groups, suggesting that differences in the burst size were not contributing to the enhancement in the secondary response observed with POX. Taken together, these data suggest that the clonal frequency of responders is enhanced by priming in the presence of POX.



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  		FIGURE 4. Priming in the presence of POX increases the secondary precursor frequency but not the clonal burst size. A, An increase in the frequency of T cells either unprimed or primed with allo-PBMCs in the presence or absence of POX (100 µg/ml) is shown. B, Mean cpm ± SE in positive-scored wells (hatched bars) and in negative-scored wells (hollow bars) for the same three groups are shown. The results are representative of four independent experiments.

 
CD25 is an early activation marker associated with alloprimed cells

The phenotype of CD4 T cells activated during priming in vitro was examined next using various markers associated with T cell activation. Abs against CD25 (IL-2R{alpha}), CD26, CD45RO, CD69, and gp240 (3, 30, 31) were used, but except for CD25 none of the other markers gave consistent and reliable results (data not shown). CD25 expression on CD4 T cells in the responder populations was examined in a two-color flow cytometric analysis at two separate time points—at the end of six days of priming and after two further rest days in culture after washing and resuspension in fresh medium without any stimuli. Most CD25-expressing cells in the primed cultures were CD4 T cells. Only a very small proportion of CD4 T cells showed CD25 expression in the unprimed population (Fig. 5Go, A and D). The frequency of CD25 expression increased in the PBMC-primed responders (Fig. 5GoB), and this percentage was lower if POX (100 µg/ml) was present during priming (Fig. 5GoC). However, this pattern altered during 2 days of rest in culture, and while the frequency and level of CD25 expression went down in the population primed without any drugs (Fig. 5GoE), cells primed in the presence of POX showed increased frequency of CD25 expression although with some decrease in the level of expression (Fig. 5GoF). Similar data were observed if dbcAMP was present during priming (data not shown). Data from many such independent experiments are summarized in Fig. 6Go. It can be seen that the proportion of CD25+ cells was reproducibly lower if POX was present during priming with allo-PBMCs. However, after 48 h of rest in culture, the frequency of CD25+ cells declined in the populations primed in the absence of POX, while it went up in the group primed under POX cover.



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  		FIGURE 5. CD25 expression on CD4 cells in primed populations before and after a 2-day rest is altered by POX. Responder cells, either unprimed (A and D) or PBMC primed in the absence (B and E) or presence of POX (100 µg/ml) (C and F), were stained for CD4 and CD25 either immediately at the end of 6 days of priming (A, B, and C) or after two further rest days in culture without any stimuli (D, E, and F). The frequencies of cells in various quadrants are indicated as percentages. The data are representative of four independent experiments.

 


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  		FIGURE 6. CD25+ cell frequency is lower to begin with but increases over a rest period in populations primed under POX cover. Data from four independent experiments staining for CD25 were used to calculate the CD25+ cell frequencies (mean ± SE) in populations primed with allo-PBMCs in the presence or absence of POX (100 µg/ml) as shown, either immediately after 6 days of priming (hollow bars) or after two further days of rest (hatched bars).

 
This relative increase in the CD25+ CD4 T cells in the POX-treated group during 48 h of rest could be due to one of the two possibilities. One is that the withdrawal of POX from the culture during the rest period results in derepression of proliferation of allospecific T cells, while the other is that T cells alloprimed under POX cover are surviving far better in culture compared not only to unprimed bystander T cells but also to T cells primed in the absence of POX. To examine these possibilities, we added aphidicolin, a DNA polymerase inhibitor, to inhibit cell proliferation during the rest period. Aphidicolin was titrated for inhibition of primary alloproliferative responses (data not shown), and, based on that titration, a dose of 1 µg/ml of aphidicolin was used to inhibit proliferation during the 2-day rest period. Fig. 7Go shows a flow cytometric analysis of such an experiment, in which the frequency of CD25+ cells during the rest period without aphidicolin (Fig. 7Go, B and E, respectively) was the same as in presence of aphidicolin (Fig. 7Go, C and F, respectively). Thus, the presence of POX during priming induces higher levels of CD25 with slower kinetics of down-modulation.



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  		FIGURE 7. The presence of aphidicolin during the 2-day rest period after priming does not abrogate the increase in frequency of CD25+ cells in responders exposed to POX during priming. Responders primed with allo-PBMCs in the absence (A, B, and C) or presence (D, E, and F) of POX (100 µg/ml) were stained for CD25 immediately after 6 days of priming (A and D) or after rest for two further days in culture in the absence (B and E) or presence (C and F) of aphidicolin (1 µg/ml). Gates shown for CD25 expression were set from appropriate negative controls (not shown), and the frequencies of CD25+ cells are indicated as percentages. The data are representative of three independent experiments.

 
Delayed apoptosis of activated T cells may contribute to the augmentation of secondary response by POX

The difference between the kinetics of CD25 expression over 2 days of rest culture prompted the question of whether this was linked to effects of POX on the survival of T cells activated during priming. Almost all CD25+ cells generated during priming were CD4 T cells (Fig. 5GoA). Therefore, responder populations primed variously were stained in a two-color flow cytometric analysis for CD25 and Fas, because Fas engagement is involved in activation-induced T cell death (32, 33). The frequency and level of Fas expression in the CD25+ population was unchanged in the presence (Fig. 8Go, B and D) or absence (Fig. 8Go, A and C) of POX during priming. The frequency went down in both the groups at the end of the 2-day rest period (Fig. 8Go, A and B, day 0; C and D, day 2). On the other hand, two-color flow cytometric analysis for CD25 and PI for apoptosis showed that the bulk of the T cells activated during priming die by apoptosis during the subsequent 48 h (Fig. 9Go, A and C), so that PI staining shows a greatly reduced DNA content in them (34). However, if priming was done in the presence of POX, the proportion of apoptotic cells in the CD25+ population does not increase significantly over the 48-h rest period (Fig. 9Go, B and D). Thus, POX clearly inhibits apoptosis in primed T cells.



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  		FIGURE 8. Presence of POX during T cell priming does not alter the pattern of CD95 expression on the CD25+ T cells. Flow cytometric analysis for CD25 and CD95 expression on T cells primed in the absence (A and C) or presence (B and D) of POX (100 µg/ml) on day 0 (A and B) and day 2 (C and D) after priming is shown. Data are representative of two independent experiments.

 


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  		FIGURE 9. The presence of POX during T cell priming decreases apoptosis of primed T cells. T cells primed with allo-PBMCs in the absence (A and C) or presence (B and D) of POX (100 µg/ml) were stained with anti-CD25 and PI either immediately after the 6-day priming period (A and B) or after 2 days of further rest (C and D). The PI staining profiles of gated CD25+ cells are shown. Data represent two independent experiments.

 
It must be noted that the cells being analyzed here were all viable cells isolated on Ficoll-Hypaque gradients and excluding trypan blue efficiently. The viable cell numbers recovered from these cultures were consistent with the inhibition of primary proliferative responses by POX. Thus, unprimed cultures yielded 0.25 ± 0.07, PBMC-primed populations had 0.38 ± 0.05, while cultures primed under POX cover gave 0.27 ± 0.06 million viable cells per million input cells. Clearly, the presence of POX did not cause any nonspecific effects on cell viability in these cultures.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
T cell activation eventually leads to the generation of two separate types of functionally differing progeny populations: effector and memory (3, 10, 17). Effector cells may be end-stage T cells as are plasma cells in the B cell lineage (31, 35), and, if still alive, they secrete cytokines or deliver cytotoxic signals immediately upon antigenic re-exposure rather than undergoing proliferation (31, 36). In contrast, memory cells give rise to more memory cells as well as to fresh effector progeny upon reactivation (37, 38). While the immediate consequences of T cell activation have been extensively analyzed (38, 39), signaling pathways regulating such physiological and more long-term results of activation are less well understood. Part of the problem is the difficulty of phenotypic distinction between effector and memory T cells in the experienced T cell compartment, which does have some phenotypic markers such as CD44 (9) or CD45 isoforms (3) distinguishing it from naive T cells, although the stability of even that distinction is uncertain and controversial (3, 37). In the absence of clear phenotypic identification of effector and memory T cells, indirect methods are resorted to in trying to dissect the factors and mechanisms involved in regulating the balance between effector and memory T cell commitment (17, 31, 37, 38).

In this context, we have been trying to analyze factors contributing to differential commitment of T cells to immediate responses vs secondary proliferative response capabilities. We have shown previously, in a system of T cell priming in vitro using allorecognition in MHC-mismatched human PBMCs, that T cell priming for a recall proliferative response does take place even if the immediate proliferative response is blocked using the DNA polymerase inhibitor aphidicolin, and that while the phosphodiesterase inhibitor POX inhibits the immediate proliferative response, its presence during priming enhances T cell recall response capabilities (18). We have now further explored the mechanisms by which POX mediates these effects on T cell priming.

The first issue is whether the immunomodulation by POX that we observe is mediated by its effects on T cells directly, or whether it works indirectly by altering the APC functions of the allostimulator PBMCs used for priming. PF fixation of stimulator APCs leads to poor primary proliferative responses as well as to poor priming for secondary responses. POX does not affect the primary response to such PF-fixed metabolically inactive stimulators, but its presence during priming enhances the subsequent secondary response to unfixed stimulator APCs (Fig. 1Go). Thus, the direct effects of POX on T cells is sufficient to induce the effects on priming we observe, and makes the alterations in signal transduction pathways brought about by POX in T cells (19, 23, 26) directly relevant.

Several effects of POX on T cell signaling pathways have been reported (19, 21, 23). Although POX is a phosphodiesterase inhibitor, it does not necessarily follow that all its effects are a consequence of that property (23). A major consequence of inhibition of phosphodiesterase is an increase in the levels of intracellular cAMP (19). The data in Fig. 2Go suggest, prima facie, that the elevation of cAMP levels brought about by POX is likely to be sufficient to mediate the effects of POX on T cell priming, because the long-lived cAMP analogue dbcAMP exerts similar effects. However, we observe in preliminary experiments that POX does not increase the intracellular cAMP levels in T cell lines significantly, unlike dbcAMP (data not shown). This raises the possibility that although dbcAMP mimics POX in its effects on T cell priming, the precise molecular pathways involved may differ.

Elevation of cAMP levels inhibits IL-2 production by activated T cells (29), and POX itself is a potent inhibitor of IL-2 transcription (23). There are reports that blocking of IL-2 functioning by the addition of anti-IL-2 receptor Abs during T cell priming may enhance secondary response capabilities of T cells (15). Therefore, it is possible that the secondary response-enhancing effect of POX is mediated through its inhibition of IL-2 transcription. If this were the case, the effect would be expected to be blocked by the addition of exogenous IL-2 in the priming cultures. Because POX still enhances T cell priming despite the presence of IL-2 (Fig. 3Go), it is evident that the critical molecular target/s of POX/cAMP involved in enhancing T cell commitment to secondary responsiveness is not likely to be IL-2.

It can be seen from Fig. 1Go and previous data (18) that the allospecific T cells proliferate more extensively during priming in the absence of cAMP/POX. Thus, the numbers of Ag-specific T cells would be expected to be greater at the end of the 6-day period of priming in cultures primed in the absence of dbcAMP or POX. Yet, paradoxically, the bulk secondary response is greater if levels of cAMP are higher in the priming cultures. There are a number of nonexclusive possibilities to explain this. One possibility is that, although there is indeed a greater number of Ag-specific T cells in the cultures primed without POX, a greater proportion of them are end-stage effectors and cannot proliferate again in response to a secondary stimulus. A second possibility is that POX inhibits activation-induced T cell apoptosis so that while a large proportion of the responding T cells in cultures primed in the absence of POX die, the proportion of such dying cells is less in cultures primed in the presence of POX. We have attempted to examine both possibilities.

When limiting-dilution assay-based estimates of the frequency of proliferation-competent allospecific precursors in primed cultures are made, it is seen that priming in the presence of POX increases this frequency, but there is no change in the clonal burst size per precursor (Fig. 4Go). These data suggest that POX may be simply altering the balance between the commitment of responding T cells to terminal effector cells vs secondary responder cells, rather than qualitatively changing the properties of the secondary T cells generated so as to enable them to mount a larger response per cell. Similar data were obtained with dbcAMP (data not shown).

To examine the issue phenotypically, we have looked at the expression of cell surface molecules that are T cell activation markers. While many markers used—CD26, CD45RO, CD69, and gp240 (3, 30, 31)—did not give any consistent results in independent experiments, there are interesting alterations caused by POX in CD25 expression patterns. CD25, or IL-2R{alpha}, is expressed on activated T cells and enhances the ability of T cells to respond to IL-2 by increasing the affinity of the low-affinity IL-2Rß found on all T cells (40, 41, 42). Priming in the presence of POX decreases the phenotypic frequency of activated CD25+ T cells seen at the end of 6 days of priming (Fig. 5Go). Yet, despite this reduction in the number of activated Ag-specific T cells by POX, there is an increase in the frequency of proliferation-competent allospecific T cells (Figs. 2Go and 4Go). These data suggest that POX increases the frequency of proliferation-competent progeny generated from responding T cells. Again, similar results are seen with dbcAMP (data not shown).

The IL-2R itself is unlikely to be a major factor in the enhancement of secondary responsiveness of T cells primed under POX cover, because the presence of exogenous IL-2 during priming does neither enhance the recall capability on its own nor does it block the enhancement induced by POX (Fig. 3Go). This is particularly significant because signaling of activated T cells by IL-2 via IL-2R is critical for activation-induced T cell death (43, 44). If the role of IL-2 depletion in preventing apoptosis and permitting responder T cell survival was critical for the enhanced secondary responsiveness induced by POX, the POX-mediated enhancement should be blocked by the presence of exogenous IL-2. That this does not happen further emphasizes that the effects of POX are unlikely to be mediated through the IL-2/IL-2R pathway.

However, when considering the expression of CD25 as a marker of T cells activated during priming, it can be seen that the rate of loss of CD25+ T cells during 2 days of rest after priming is altered in populations primed under POX cover (Figs. 5Go and 6Go). In responder cell populations primed without any POX, the frequency of CD25+ cells falls rapidly over 2 days of rest. However, if priming is done under POX cover, the frequency of the CD25+ cells rises significantly over the 2-day rest period, so that the addition of aphidicolin during rest is needed (Fig. 7Go) to ensure that this is a consequence of increased survival rather than proliferation. It is also possible that a decline in the levels of CD25 on primed cells over the 48-h rest period leads to the apparent loss of CD25+ cells in the responders primed without POX cover. However, if this was a major factor in the CD25+ cell loss, the increased frequency of CD25+ cells in responders primed under POX cover would be expected to be correlated with sustained levels of CD25 expression on them, which does not appear to be the case (Fig. 5Go). Therefore, it was plausible to examine the possibility that POX mediates enhanced survival of primed T cells.

If such differential survival of responding T cells postpriming is involved in the enhancement of secondary responsiveness brought about by POX, it is most likely to be mediated by inhibition of apoptosis, because induction of the apoptotic pathway is a major outcome of T cell activation (45, 46). Apoptosis of activated T cells involves Fas (CD95)-mediated signals (34, 47, 48). When the levels of Fas are examined on CD25+-activated T cells in culture at the end of 6 days of priming, it can be seen that cells primed under POX cover show frequencies and levels of Fas expression similar to cells primed in the absence of POX (Fig. 8Go), suggesting that the induction of Fas expression during T cell priming is unaffected by POX. However, the effective induction of apoptosis is inhibited nonetheless, as shown by the high frequency of CD25+ cells staining poorly with PI (Fig. 9Go). We have noted similar effects with dbcAMP as well (data not shown). This suggests that death pathways downstream of Fas may be affected by POX.

Thus, using in vitro priming of T cells as a model system, we show that the enhancement of T cell commitment to secondary responsiveness by POX is a T cell effect that can also be mimicked by enhanced cAMP levels in T cells during priming, and that this enhancement is accompanied by reduction in the activation-induced apoptosis of primed T cells.


   Footnotes
 
1 This work was supported by grants to V.B. and A.G. from the Department of Science and Technology, Government of India. The National Institute of Immunology is supported by the Department of Biotechnology, Government of India. R.S. and J.M.D. are supported by grants from the National Institutes of Health (AI41035 and GM48691, respectively). Back

2 Address correspondence and reprint requests to Dr. Vineeta Bal, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India. E-mail address: Back

3 Abbreviations used in this paper: POX, pentoxifylline; dbcAMP, dibutyryl cAMP; PF, paraformaldehyde; PI, propidium iodide. Back

Received for publication July 8, 1998. Accepted for publication September 28, 1998.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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