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The Journal of Immunology, 1999, 163: 4160-4167.
Copyright © 1999 by The American Association of Immunologists

Cytokine Requirements for Production of a Novel Anti-CD8-Resistant CTL Population1

Esi S. N. Lamousé-Smith*, David S. Dougall{dagger} and Susan A. McCarthy2,*,{dagger},{ddagger}

* Immunology Graduate Training Program and Departments of {dagger} Surgery, and {ddagger} Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A population of CD8+ CTL can be generated in vitro in the presence of anti-CD8 mAb. Due to their apparent high avidity characteristic, these anti-CD8-resistant CD8+ CTL may have important functional in vivo roles in graft rejection, and may be important in antiviral and antitumor responses. We have previously reported that this anti-CD8-resistant subset of CD8+ CTL demonstrates functional differences from anti-CD8-sensitive CD8+ CTL. One important difference between the subsets is the markedly greater dependence of anti-CD8-resistant CTL upon exogenous cytokines for their generation in vitro. In this study, we have investigated in detail the cytokine requirements for the generation of allospecific CD8+ CTL in vitro and have found that IL-4 can augment the generation of anti-CD8-sensitive but not anti-CD8-resistant CTL, whereas IL-2 or IL-12 can augment the generation of both anti-CD8-sensitive and anti-CD8-resistant CTL. However, anti-CD8-resistant CTL require at least 10-fold higher concentrations of IL-2 than do anti-CD8-sensitive CTL. This more stringent IL-2 requirement precludes the efficient generation of anti-CD8-resistant CTL in vitro in the absence of exogenous IL-2 because they cannot produce sufficient IL-2 to meet their needs, in contrast to anti-CD8-sensitive CTL. By providing exogenous cytokines to allospecific CTL generation cultures, we further demonstrate that anti-CD8-resistant CTL can be functionally skewed to the Tc1 subset, but differ from anti-CD8-sensitive conventional CTL in that they cannot be skewed to the Tc2 subset.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Many CD8+ T cells require the activity of the CD8 molecule for its contributions to both signal transduction and avidity augmentation during development and activation (1, 2, 3). Interference with the binding of the CD8 molecule to its ligand (the {alpha}3 domain of the MHC class I molecule) can therefore inhibit the generation and functional activity of anti-CD8-sensitive CD8+ CTL (4, 5). We have previously described a unique population of CD8+ CTL that require the signaling activity but not the avidity contribution of CD8 during their in vitro induction (6, 7, 8). These anti-CD8-resistant effectors are subsequently resistant to inhibition by anti-CD8 Ab during CTL functional target lysis assays.

We proposed that these anti-CD8-resistant CD8+ CTL are a subset of high avidity CTL that are activated in vivo when Ag is limiting, or in vitro when a limiting Ag stimulus is mimicked by the introduction of anti-CD8 Ab into the induction cultures. Berzofsky and colleagues have recently reported that high avidity CTL could be induced in vitro by low concentrations of the priming Ag (9). These in vivo primed CTL could also be activated in cultures that contained high concentrations of Ag, but only in the presence of anti-CD8 Ab (10). These data are consistent with the hypothesis that in an in vitro culture environment containing high concentrations of Ag, such as in anti-MHC alloantigen MLC conditions, anti-CD8 Ab has the effect of reducing the total avidity, and therefore the total antigenic stimulus, down to a level that promotes the activation (rather than the inactivation or death) of anti-CD8-resistant pCTL3/CTL. However, we also proposed that these anti-CD8-resistant CTL are not merely high avidity clones of the conventional CTL lineage, but instead represent a distinct CTL lineage or subset. This latter proposal was based on our findings that, although anti-CD8-resistant CTL are currently phenotypically indistinguishable from anti-CD8-sensitive CTL, they exhibit both in vivo developmental differences (including critical differences in their tissue distributions) and in vitro functional differences from conventional anti-CD8-sensitive CTL (8).

CD8+ CTL are generally dependent on IL-2 for their generation and proliferation in vitro and in vivo, and are therefore often dependent on IL-2-producing CD4+ T cells for their efficient Ag-specific activation (11). Long-term lines of CD8+ CTL are usually maintained in vitro by Ag-specific stimulation in conjunction with cytokine-containing conditioned medium or rIL-2.

More recently, it has been shown that IFN-{gamma}-producing CTL can be generated in the presence of the potent type 1 cytokine IL-12 (12, 13), which has extended the evaluation and characterization of CD4+ Th1 and Th2 subsets to CD8+ T cells. It is now well accepted that conventional anti-CD8-sensitive CD8+ CTL can be skewed into either a type 1 (Tc1) or a type 2 (Tc2) profile. CD8+ T cells generated in primary MLC to alloantigens in the presence of IL-2, IL-12, and anti-IL-4 mAb exhibit Ag-specific cytotoxicity and produce the type 1 cytokines IL-2, IFN-{gamma}, and TNF-{alpha} (13, 14, 15, 16). CD8+ T cells generated in primary MLC to alloantigens in the presence of IL-2, IL-4, and anti-IFN-{gamma} mAb exhibit Ag-specific cytotoxicity and produce the type 2 cytokines IL-5, IL-10, small amounts of IL-4, and variable amounts of IFN-{gamma} (13, 14, 15, 16). Stable clones and lines of Tc1 and Tc2 cells can be generated and have been shown to retain their cytokine production phenotypes upon repeated Ag stimulation in either the presence or absence of the original skewing cytokines (14, 17, 18, 19).

We previously reported that anti-CD8-resistant CTL are extremely dependent upon exogenous cytokines (in the form of Con A SN in our earlier studies) for their generation in vitro (8). We have now investigated the precise nature of this cytokine dependence using recombinant cytokines and anti-cytokine mAbs. We have also established that anti-CD8-resistant CTL are generated only under type 1 and not under type 2 conditions in vitro, which is in contrast to their anti-CD8-sensitive CTL counterparts and demonstrates an important functional difference between these two CTL susbsets.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Mice were purchased from The Jackson Laboratory (Bar Harbor, ME) or Charles River Laboratories (Wilmington, MA) or were bred in our animal facility. C57BL/6 (B6; H-2b), B6.C-H-2bm1/ByJ (bm1; H-2Kbm1 mutant), C57BL/6-CD28tm1Mak (CD28-/-; H-2b), and B6,129-H2-Ma0/0 (MHC class II-/-; H-2b, generously provided by Diane Mathis, C.U. de Strausbourg) mice were used.

Con A SN, recombinant cytokines, and anti-cytokine mAbs

Con A SN was the 18-h supernatant from Con A-stimulated B6 spleen cells prepared as previously described (20). Recombinant murine IL-2, IL-12, and IFN-{gamma} (Genzyme, Cambridge, MA), human rIL-2 (Cetus, Emeryville, CA), and murine rIL-4 (R&D Systems, Minneapolis, MN) were used at the final concentrations indicated in the figure legends. Abs to murine IL-2 (Becton Dickinson, Bedford, MA), IL-12 (clone C17.8; Genzyme), and IFN-{gamma} (clone XMG1.2, Endogen, Woburn, MA; or clone R4-6A2, a gift from Stephen H. Gregory, University of Pittsburgh School of Medicine, Pittsburgh, PA) were used at the final concentrations indicated in the figure legends. Ab to murine IL-4 (clone 11B11, a gift from Penelope A. Morel, University of Pittsburgh School of Medicine) was used as a hybridoma supernatant at a concentration that was shown to inhibit the activation and proliferation of Th2 clones by IL-4 in vitro.

In vitro generation and functional analysis of CTL

MLC of 5 x 106 responder spleen cells and 5 x 106 irradiated (2000 R) stimulator spleen cells were established in 2 ml complete RPMI 1640 medium supplemented with glutamine, nonessential amino acids, sodium pyruvate, antibiotics, 2-ME, and 5% FCS, as previously described (7). Con A SN, recombinant cytokines, and/or anti-cytokine mAbs were included in the induction cultures where indicated. To generate anti-CD8-resistant CTL, induction cultures were supplemented with anti-CD8 mAb (Ab culture supernatant or ascites fluid of the anti-CD8{alpha} mouse IgM clone 83-12-5). All cultures were incubated at 37°C in 7.5% CO2 humidified air for 5 days. On day 5, the cultures were harvested, washed, counted, and assayed for CTL activity by their ability to lyse 51Cr-labeled splenic LPS blast target cells in a 4-h 51Cr release assay. CTL were assayed in triplicate at each of four E:T ratios. Where indicated, the effector cells were preincubated for 10–30 min at 4°C with anti-CD8 mAb before the addition of labeled targets for the 4-h lytic assay; the anti-CD8 mAb was also present throughout the 4-h assay. Percent specific lysis = 100 x (experimental release - spontaneous release)/(maximum release - spontaneous release). SD were routinely less than 8% and are omitted from the figures. Lysis of responder strain targets was routinely less than 10% and is not shown.

Generation of Tc1 and Tc2 cells

Tc1 CTL were generated as above, except that Con A SN was replaced with 35 U/ml murine IL-2, 2.5 ng/ml IL-12, and 10% anti-IL-4 mAb hybridoma supernatant. Tc2 CTL were generated as above, except that Con A SN was replaced with 35 U/ml murine IL-2, 30 ng/ml IL-4, and 10 µg/ml anti-IFN-{gamma} mAb.

FACS Abs

Anti-Fc{gamma} III/IIR Ab (clone 2.4G2; PharMingen, San Diego, CA) was used to block nonspecific extracellular binding of Abs. Anti-CD8ß-biotin (clone 53-5.8; PharMingen), anti-Leu4-biotin (Becton Dickinson), and Streptavidin-Tricolor (Caltag, Burlingame,CA) were used for cell surface FACS analysis of MLC effectors. The anti-murine cytokine Abs and their isotype controls used for intracellular analysis were all purchased from PharMingen: anti-IFN-{gamma} (XMG1.2-FITC), rat IgG1 (R35-95-FITC), anti-IL-4 (11B11-PE), rat IgG1 (R35-95-PE), anti-IL-10 (JES5-16E3-PE), and rat IgG2b (R35-38-PE).

Intracellular cytokine analysis by FACS

MLC effector cells were harvested on day 5 poststimulation, counted by trypan blue exclusion, and resuspended in fresh medium without exogenous cytokines at 1 x 106 cells/ml. Cells were incubated for 4–6 h in 24-well plates in the presence of 2.5 ng/ml PMA (Sigma, St. Louis, MO) and 25 ng/ml ionomycin (Sigma). Two to three hours before the end of the restimulation period, 100 ng/ml Brefeldin A (Sigma) was added to each well. At the end of the incubation period, cells were harvested and washed twice in 1x PBS, pH 7.1, or FACS medium (1x HBSS, 0.1% sodium azide, 0.1% BSA). Cells (5 x 105-1 x 106 per sample) were treated with anti-FcR-blocking mAb for 15 min at 4°C, then incubated for 30 min at 4°C with anti-CD8ß-biotin and washed twice in FACS medium. Streptavidin-Tricolor was then added to cells for 15 min at 4°C. Cells were then washed twice in FACS medium and resuspended at 1 x 106 cells/ml in 4% formaldehyde-containing FACS medium for 30–60 min at 4°C. Cells were washed once in FACS medium, aliquoted into 96-well plates, and centrifuged at 1200–1500 rpm to pellet cells. Cells were permeabilized by suspension in 100 µl of FACS permeabilization buffer (0.5% saponin (Sigma) in FACS medium), and incubated 10 min at 4°C. Cells were centrifuged to pellet, resuspended in 20 µl rat IgG serum (Sigma; 300 µg/ml in FACS permeabilization buffer), and incubated for 10 min at 4°C before the addition of 20 µl of the appropriate anti-cytokine mAbs diluted in FACS permeabilization buffer. Following the addition of anti-cytokine mAb, cells were incubated at 4°C for 30–60 min, then washed twice in FACS permeabilization buffer, once in FACS medium, and finally resuspended into 200 µl FACS medium. Cells were stored at 4°C for no longer than 48 h until FACS analysis. FACS analysis was done using the FACScan instrument from Becton Dickinson and analyzed using the LYSIS II software package.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Requirements for generation of anti-CD8-resistant CTL

MHC class I-allospecific CTL were generated in primary MLC of bm1 responder cells and B6 stimulator cells, which differ only at the MHC class I H-2K locus. The MLC were supplemented with an exogenous source of cytokines (Con A SN), in either the presence or absence of cross-linking anti-CD8 mAb (Fig. 1GoA). The effectors generated in each culture condition were Ag specific and cytolytic. However, in contrast to the CD8+ CTL generated in the absence of anti-CD8 mAb, the CD8+ CTL generated in the presence of anti-CD8 mAb were resistant to inhibition by anti-CD8 mAb in the target lysis assay (Fig. 1GoA), as we have previously shown (6, 7, 8). The cytolytic activity of CD8-resistant CTL is typically as strong as, or stronger than, the cytolytic activity of anti-CD8-sensitive CTL (as seen in Fig. 1Go, B and C).



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  		FIGURE 1. Generation of anti-CD8-resistant CTL in wild-type, MHC class II-/-, and CD28-/- mice. bm1 splenic responder cells (A) were stimulated with irradiated B6 splenic stimulator cells in the presence ({blacksquare}) or absence ({square}) of anti-CD8 mAb and in the presence of Con A SN. On day 5, effectors were tested for lytic activity against B6 target blasts in the presence (dotted lines) or absence (solid lines) of anti-CD8 mAb. Splenic responder cells from MHC class II-/- mice (B) and CD28-/- mice (C) were stimulated with irradiated bm1 spleen cells in the presence (filled symbols) or absence (open symbols) of anti-CD8 mAb and in the presence of Con A SN. On day 5, effectors were tested for lytic activity against bm1 target blasts in the presence (dotted lines) or absence (solid lines) of anti-CD8 mAb. The following effector designations are used in all figures: -/-, no anti-CD8 mAb in MLC, no anti-CD8 mAb in target lysis assay; -/+, no anti-CD8 mAb in MLC, anti-CD8 mAb in target lysis assay; +/-, anti-CD8 mAb in MLC, no mAb in target lysis assay; +/+, anti-CD8 mAb in MLC, anti-CD8 mAb in target lysis assay.

 
We recently reported evidence that anti-CD8-resistant CTL may represent a distinct CTL subset or lineage, and not simply high avidity clones of the conventional CD8+ T cell lineage (8). To investigate the requirements for the in vivo development of anti-CD8-resistant pCTL and the in vitro activation of these pCTL into effector CTL, we analyzed several related parameters. Since we had recently demonstrated that anti-CD8-resistant pCTL are dependent on Th cell-derived cytokines for their activation into CTL in vitro (8), we initially tested whether such anti-CD8-resistant pCTL are also dependent on CD4+ Th cells for their development and/or survival in vivo. To do so, we analyzed splenic responder cells from MHC class II-null mice, which are largely devoid of CD4+ T cells (21), in our standard MLC conditions (Fig. 1GoB). Both anti-CD8-sensitive and anti-CD8-resistant CTL were generated from both the wild-type and the mutant mice, indicating that CD4+ T cells are not required during either the in vivo development or the in vitro induction of either type of CD8+ MHC class I-allospecific CTL. This survival capacity of CD8+ T cells in the absence of CD4+ T cells in vivo may reflect a recently reported process by which maintenance of mature naive CD8+ T cells in vivo is controlled by exposure to self MHC class I on peripheral tissues, perhaps constituting a continuous positive selection process for mature T cells (22).

CD28 provides a critical costimulatory signal during activation of naive T cells, leading to enhanced cytokine production (especially IL-2), proliferation, and avoidance of anergy in responding T cells (23, 24). In many cases, provision of an exogenous source of cytokines can circumvent the requirement for a CD28-mediated costimulatory signal. We therefore also tested whether CD28 costimulation is required by anti-CD8-resistant pCTL/CTL. We analyzed splenic responder cells from CD28-null mice (25) in our standard cytokine-supplemented MLC conditions (Fig. 1GoC). Both anti-CD8-sensitive and anti-CD8-resistant CTL were generated from both the wild-type and the mutant mice in the presence of exogenous cytokines (but neither was generated in the absence of exogenous cytokines; data not shown), indicating that CD28-dependent signals are not required during either the in vivo development or the in vitro induction of either type of CD8+ MHC class I-allospecific CTL. Thus, by these criteria, the two subsets of CD8+ CTL are indistinguishable.

One important criterion by which the two subsets are readily distinguishable is their dependence on exogenous cytokines during in vitro induction. We had previously shown (8), and reillustrate in this work as a reference point for subsequent figures, that although anti-CD8-sensitive CTL can routinely be generated in absence of Con A SN (albeit sometimes with weaker functional activity than those generated in the presence of Con A SN), anti-CD8-resistant CTL are almost never generated in the absence of Con A SN (Fig. 2GoA). In this and all subsequent figures, specific lysis is shown for only the anti-CD8-sensitive (-/-) and anti-CD8-resistant (+/+) CTL effector assay conditions (see legend to Fig. 1Go).



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  		FIGURE 2. Effect of anti-cytokine mAbs on anti-CD8-resistant CTL generation. bm1 splenic responder cells were stimulated with irradiated B6 stimulator cells for 5 days in the presence (filled symbols) or absence (open symbols) of anti-CD8 mAb, and in the presence of medium (squares), Con A SN (circles), or Con A SN plus anti-murine cytokine mAbs (diamonds). On day 5, effector cells were tested for lytic activity against B6 target cell blasts in the presence (filled symbols) or absence (open symbols) of anti-CD8 mAb. Only -/- (anti-CD8-sensitive CTL) and +/+ (anti-CD8-resistant CTL) effector groups are shown. The concentrations of anti-cytokine mAbs used were: 25% Con A SN (A), 10 µg/ml anti-IL-12 mAb (B), 10 µg/ml anti-IFN-{gamma} mAb (C), 10% hybridoma culture supernatant anti-IL-4 mAb (D), and 2 µg/ml anti-IL-2 mAb (E).

 
To investigate the dependence of anti-CD8-sensitive and anti-CD8-resistant CTL on specific cytokines for their functional generation, we began by adding neutralizing anti-cytokine mAbs to MLC supplemented with heterogeneous exogenous cytokines in the form of Con A SN. Neutralizing concentrations of anti-IL-12 mAb, anti-IFN-{gamma} mAb, and anti-IL-4 mAb failed to inhibit the generation of either anti-CD8-sensitive or anti-CD8-resistant CTL (Fig. 2Go, B–D). Thus, no one of these cytokines is strictly required for the generation of anti-CD8-sensitive or anti-CD8-resistant CTL in vitro in the presence of other cytokines contained in the Con A SN. In contrast, neutralizing concentrations of anti-IL-2 mAb completely inhibited the generation of both anti-CD8-sensitive and anti-CD8-resistant CTL (Fig. 2GoE). Thus, IL-2 appears to be the dominant or most downstream cytokine in Con A SN required for the induction and/or proliferation of both CTL types.

To investigate whether any cytokines can individually replace Con A SN during the generation of anti-CD8-sensitive and anti-CD8-resistant CTL, we added each of four cytokines to MLC to generate MHC class I-allospecific CTL. The generation of both anti-CD8-sensitive and anti-CD8-resistant CTL was augmented by IL-2 and IL-12 individually, but not by IFN-{gamma} (Fig. 3Go, A–C). Both IL-2 and IL-12 are known to be potent inducers of CTL generation (12, 26, 27), although IL-12 was not as effective as IL-2 in generating CTL of either type in our studies. When anti-IL-2 mAb was included in cultures containing IL-12, the generation of both anti-CD8-sensitive and anti-CD8-resistant CTL was profoundly inhibited (data not shown), suggesting that IL-12 produced by APCs or supplemented exogenously leads to downstream induction of cytokines, including IL-2 necessary for the activation and proliferation of T cells. Although IFN-{gamma} is produced by CD8+ CTL, Th1 cells, and NK cells and regulates the production of IL-12 by macrophages and dendritic cells in some cases, IFN-{gamma} has not been shown to drive the generation of CD8+ CTL themselves (13), which is consistent with its lack of effect in our studies.



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  		FIGURE 3. Effect of cytokines on anti-CD8-resistant CTL generation. bm1 splenic responder cells were stimulated with irradiated B6 stimulator cells for 5 days in the presence (filled symbols) or absence (open symbols) of anti-CD8 mAb, and in the presence of medium (squares) or recombinant murine cytokines (triangles). On day 5, effector cells were tested for lytic activity against B6 target cell blasts in the presence (filled symbols) or absence (open symbols) of anti-CD8 mAb. Only -/- (anti-CD8-sensitive CTL) and +/+ (anti-CD8-resistant CTL) effector groups are shown. The concentrations cytokines used were: 100 U/ml IL-2 (A), 1 ng/ml IL-12 (B), 100 U/ml IFN-{gamma} (C), and 30 ng/ml IL-4 (D).

 
The generation of anti-CD8-sensitive but not anti-CD8-resistant CTL was augmented in most experiments by IL-4 (Fig. 3GoD). This disparity may reflect the ability of anti-CD8-sensitive CTL to produce endogenous cytokines, such as IL-2, that synergize with exogenous IL-4 to augment CTL induction. Synergy between IL-2 and IL-4 has previously been described during the generation and/or proliferation of Th cells (28, 29) and CD8+ CTL (30, 31). We confirmed that anti-CD8-sensitive CTL could produce sufficient IL-2 for their own generation, because the addition of anti-IL-2 mAb to MLC established in the absence of exogenous cytokines abolished the generation of anti-CD8-sensitive CTL (data not shown). In contrast, anti-CD8-resistant CTL are very rarely generated in the absence of exogenous cytokines (Fig. 2GoA). Thus, anti-CD8-resistant CTL are apparently unable to produce sufficient levels of endogenous cytokines to synergize with exogenous IL-4 for their induction and/or proliferation in these MLC. This scenario is consistent with our observation that in those experiments in which anti-CD8-sensitive CTL were not generated in the absence of exogenous cytokines, exogenous IL-4 did not augment their generation (data not shown).

The cytokine blocking and addition studies in Figs. 2Go and 3Go indicate that both anti-CD8-sensitive and anti-CD8-resistant CTL generation require IL-2, but do not address whether these CTL subsets differ in their quantitative dependence on this critical cytokine. To assess the relative dependence of anti-CD8-sensitive and anti-CD8-resistant CTL on IL-2, we titered rIL-2 into the MHC class I-disparate MLC system. As shown in Fig. 4GoA, the generation of both anti-CD8-sensitive and anti-CD8-resistant CTL exhibited a dose-dependent requirement for IL-2, but generation of anti-CD8-resistant CTL required at least 10-fold more exogenous IL-2 than did generation of anti-CD8-sensitive CTL. To parallel the IL-2 titration analysis, we titered anti-murine IL-2 mAb into MLC supplemented with Con A SN. As shown in Fig. 4GoB, the generation of both anti-CD8-sensitive and anti-CD8-resistant CTL exhibited a dose-dependent sensitivity to anti-IL-2 mAb, but inhibition of anti-CD8-sensitive CTL generation required 2- to 4-fold more anti-IL-2 mAb than did inhibition of anti-CD8-resistant CTL generation. These results suggest that anti-CD8-resistant CTL require more IL-2 and/or produce less IL-2 (and therefore require more exogenous IL-2) in vitro than do anti-CD8-sensitive CTL.



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  		FIGURE 4. IL-2 dependence of anti-CD8-resistant CTL generation. A, bm1 splenic responder cells were stimulated with irradiated B6 stimulator cells in the presence (filled bars) or absence (open bars) of anti-CD8 mAb and in the presence of the indicated concentrations of IL-2. On day 5, effector cells were tested for lytic activity against B6 target cell blasts in the presence (filled bars) or absence (open bars) of anti-CD8 mAb. At the E:T ratio shown (30:1), the percent specific lysis of effectors generated in Con A SN was 38% for anti-CD8-sensitive CTL (-/-) and 44% for anti-CD8-resistant CTL (+/+). B, bm1 splenic responder cells were stimulated with irradiated B6 stimulator cells in the presence of Con A SN and in the presence (filled bars) or absence (open bars) of anti-CD8 mAb, with the indicated concentrations of anti-murine IL-2 mAb. On day 5, effector cells were tested for lytic activity against B6 target cell blasts in the presence (filled bars) or absence (open bars) of anti-CD8 mAb. An E:T ratio of 30:1 is shown.

 
To distinguish between these two possibilities, we added anti-murine IL-2 mAb to MLC to prevent utilization of any endogenously produced IL-2, and then titered human IL-2 (which is not inactivated by the anti-murine IL-2 mAb) into the cultures. We found that even when any potential differences in endogenous production of IL-2 by anti-CD8-sensitive and anti-CD8-resistant CTL were rendered irrelevant by the anti-murine IL-2 mAb, generation of anti-CD8-resistant CTL required more exogenous IL-2 than did generation of anti-CD8-sensitive CTL (Fig. 5Go). Thus, we have identified a critical functional difference between these two CTL types to complement those we had previously reported (8): a quantitative difference in IL-2 requirements that renders the anti-CD8-resistant CTL subset we have studied dependent on exogenous cytokines for induction and/or proliferation in in vitro MHC class I allodisparate MLC.



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  		FIGURE 5. Exogenous IL-2 dependence of anti-CD8-resistant CTL generation. bm1 splenic responder cells were stimulated with irradiated B6 stimulator cells in the presence (filled bars) or absence (open bars) of anti-CD8 mAb, and in the presence of the indicated concentrations of human rIL-2 or Con A SN, with or without anti-murine IL-2 mAb to block the utilization of endogenously produced IL-2. On day 5, effector cells were tested for lytic activity against B6 target cell blasts in the presence (filled bars) or absence (open bars) of anti-CD8 mAb. An E:T ratio of 10:1 is shown.

 
Generation of anti-CD8-resistant Tc1 and Tc2 cells

We investigated whether anti-CD8-sensitive and anti-CD8-resistant CTL also display qualitative differences in their responses to distinct cytokine environments. We did so by examining whether anti-CD8-resistant CTL can be skewed to the Tc1 and Tc2 functional subtypes by the defined cytokine conditions that had previously been established for anti-CD8-sensitive Tc1 and Tc2 cells (13, 14). We used MLC supplemented with IL-2, IL-12, and anti-IL-4 mAb for generation of Tc1 effector cells. As shown in two representative experiments, both anti-CD8-sensitive and anti-CD8-resistant CTL were efficiently generated in this type 1 cytokine environment (Fig. 6Go). The Tc1 cytokine-producing profiles of these T cells were confirmed by intracellular FACS analysis. Fig. 7GoA shows a representative example corresponding with the CTL data shown in Fig. 6GoA, and Fig. 7GoB summarizes a series of such experiments. The CD8+ T cells generated in primary Tc1 MLC were primarily IFN-{gamma} producers. A significant number of IL-10 and IFN-{gamma} dual-producing CTL were also recovered, which may represent Tc0 or unskewed CD8+ T cells that are in transit to a Tc1 profile and are thus analogous to the Th0 subset described for CD4+ T cells (32). IL-2 is generally produced at low to undetectable levels even by activated CD8+ T cells, as determined by ELISA (13, 14, 15), and we did not detect this cytokine in anti-CD8-sensitive or anti-CD8-resistant CTL using intracellular FACS analysis (data not shown).



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  		FIGURE 6. Anti-CD8-sensitive and anti-CD8-resistant CTL generation under Tc1 and Tc2 conditions. Two representative experiments are shown in A and B for B6 splenic responder cells stimulated with irradiated bm1 stimulator cells in the presence (filled symbols) or absence (open symbols) of anti-CD8 mAb, and in the presence of type 1 cytokine conditions (IL-2, IL-12, and anti-IL-4 mAb; circles) or in the presence of type 2 cytokine conditions (IL-2, IL-4, and anti-IFN-{gamma} mAb; triangles). On day 5, effector cells were tested for lytic activity against bm1 target cell blasts in the presence (filled symbols) or absence (open symbols) of anti-CD8 mAb.

 


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  		FIGURE 7. Cytokine production by Tc1 and Tc2 cells. Effectors were generated in the presence of anti-CD8 mAb and under type 1 cytokine conditions (R-Tc1; denotes anti-CD8-resistant T cells) or type 2 cytokine conditions (R-Tc2), or in the absence of anti-CD8 mAb and under type 1 cytokine conditions (S-Tc1: denotes anti-CD8-sensitive T cells) or type 2 cytokine conditions (S-Tc2). For three-color FACS analysis, cells were stained with anti-CD8ß before fixation and permeabilization, and then stained for production of IFN-{gamma} (x-axis) and IL-10 (y-axis); cytokine production by the CD8+ T cells within these cultures is shown. For the representative experiment shown in A, the percentage of CD8+ T cells were 84% (S-Tc1), 76% (S-Tc2), 62% (R-Tc1), and 68% (R-Tc2). In B, the percentage of CD8+ T cells generated in the presence (R-Tc1 and R-Tc2) or absence (S-Tc1 and S-Tc2) of anti-CD8 mAb that produce IFN-{gamma} (filled bars), IL-10 (open bars), and IFN-{gamma} + IL-10 (hatched bars) by intracellular FACS analysis is shown (n = 8).

 
Anti-CD8-sensitive and anti-CD8-resistant CTL generated in primary MLC supplemented with either Con A SN or IL-2 were also predominated by a population of single IFN-{gamma} producers with a smaller population of IFN-{gamma} and IL-10 dual-producing cells, as detected by FACS analysis (data not shown), indicating that they had acquired the Tc1 profile. Thus, anti-CD8-sensitive and anti-CD8-resistant CTL from B6 and bm1 mice are naturally skewed to the type 1 cytokine profile when generated under our normal culture conditions.

To investigate whether this tendency toward Tc1 skewing could be reversed by the appropriate cytokine environment, we used MLC supplemented with IL-2, IL-4, and anti-IFN-{gamma} mAb for generation of Tc2 effector cells. Anti-CD8-sensitive CTL were generated in Tc2-skewing MLC in the absence of anti-CD8 mAb (Fig. 6Go). Intracellular FACS analysis of cytokine profiles demonstrated that a significant population of anti-CD8-sensitive Tc2 were generated under these conditions, as determined by the population of CD8+ T cells producing only IL-10 (Fig. 7Go). Significant numbers of CD8+ T cells producing only IFN-{gamma} were also present in these cultures, which may represent contaminating Tc1 cells. It is unlikely that these contaminating Tc1 cells were solely responsible for the cytolytic activity derived from the Tc2 cultures because there was no correlation in a series of experiments between the percentage of CD8+ T cells producing only IFN-{gamma} and CTL lytic effector activity (data not shown).

In contrast to the anti-CD8-sensitive CTL, CD8+ T cells activated in the presence of anti-CD8 mAb and Tc2-skewing conditions lacked lytic function (Fig. 6Go) and retained a Tc1 profile, in that a significant number of Tc2-skewed cells were not detectable (Fig. 7Go). Interestingly, there was no significant difference in the cell recovery, the percentage of CD8+ T cells (see legend to Fig. 7Go), and the expression of the activation markers CD44 and CD25 on CD8+ T cells recovered from Tc2-skewing cultures established in the presence or absence of anti-CD8 mAb (data not shown). This latter observation suggests that the CD8+ T cells recovered from Tc2-skewing and anti-CD8 mAb-containing cultures received appropriate activation signals to drive cell proliferation, up-regulation of activation-induced cell surface molecules, and finally, cytokine production. Thus, a dichotomy exists between the conventional anti-CD8-sensitive CD8+ CTL population and the novel anti-CD8-resistant CD8+ CTL population we have studied: whereas anti-CD8-sensitive pCTL can mature into lytic effector cells under both type 1 and type 2 cytokine conditions (and can also generate both Tc1 and Tc2 cells), anti-CD8-resistant CD8+ pCTL are apparently destined to mature to lytic effector cells only under type 1 cytokine conditions (and fail to generate Tc2 cells under type 2 conditions). This surprising observation supports the hypothesis that these two CTL populations are indeed functionally distinct beyond their differential requirements for the avidity contribution of the CD8 coreceptor, and suggests that differential regulation and integration of signals elicited in certain cytokine-priming environments occur, resulting in two very different outcomes for anti-CD8-sensitive pCTL and anti-CD8-resistant pCTL.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We observed in this and a previous study (8) that a novel population of anti-CD8-resistant, and presumably high avidity (6, 7), CTL are very dependent upon exogenous cytokines for their induction in vitro (Fig. 2GoA). The activation and proliferation of functional CTL both in vitro and in vivo are dependent upon several parameters, including efficient Ag presentation, costimulation, and cytokines. Cytokines produced in an autocrine fashion by responding CD8+ pCTL/CTL themselves, and/or in a paracrine fashion by CD4+ Th cells or activated APC, during CTL activation may profoundly affect the outcome of an Ag-specific response. We therefore continued our characterization of anti-CD8-resistant CD8+ CTL and compared them with anti-CD8-sensitive CD8+ CTL, by examining CTL development and generation in defined cytokine culture conditions. We have determined that there are quantitative as well as qualitative differences in the cytokine requirements of anti-CD8-resistant and anti-CD8-sensitive CD8+ CTL.

The exquisite dependence of anti-CD8-resistant CTL on exogenous IL-2 during their in vitro induction ( Figs. 2–5GoGoGoGo) was somewhat surprising, given that these CTL may represent a population of high avidity CD8+ CTL triggered early in an in vivo immune response when both Ag dose and overall cytokine levels are still low. However, we have shown that minor histocompatibility Ag-specific anti-CD8-resistant as well as anti-CD8-sensitive CD8+ CTL can be primed in vivo (8), and Berzofsky and colleagues have demonstrated that virus-specific high avidity as well as low/moderate avidity CD8+ CTL can be primed in vivo (9). In each case, the activation process would be dependent on cytokines present within the priming microenvironment, indicating that the local immune environment(s) in vivo can maintain appropriate Ag and cytokine levels to promote the activation of both anti-CD8-sensitive low/moderate avidity CTL and anti-CD8-resistant high avidity CTL. One intriguing possibility is that two distinct kinds of microenvironment exist during in vivo priming: sites with relatively low Ag levels and high cytokine levels that support priming of high avidity anti-CD8-resistant CTL, and sites with relatively high Ag levels that support priming of low/moderate avidity anti-CD8-sensitive CTL.

In the case of high avidity CTL, both we (8) and Berzofsky and colleagues (9) have shown that in vivo priming does not require the avidity-reducing effect of anti-CD8 mAb, presumably because the relevant priming microenvironment has a sufficiently low Ag level to prevent the overstimulation of these cells (10) that occurs in vitro in the absence of anti-CD8 mAb. Berzofsky and colleagues have also demonstrated that high avidity CTL induced and maintained in vitro with exogenous cytokines and low dose Ag can function in vivo after transfer. Thus, the dependence of anti-CD8-resistant CTL in vitro on exogenous cytokines and anti-CD8 mAb does not preclude either their generation or survival in vivo in the absence of these reagents, suggesting that anti-CD8-resistant CTL generated in vitro should be able to survive and function when transferred in vivo for therapeutic purposes in the absence of exogenous cytokines and mAb.

In vivo priming microenvironments contain heterogenous mixtures of cytokines that may influence both the lytic function and cytokines produced by CD8+ CTL. Thus, it was important to establish whether dominant skewing cytokines (e.g. IL-12, IFN-{gamma}, IL-4) could influence the generation of anti-CD8-resistant and anti-CD8-sensitive CTL. Both anti-CD8-resistant and anti-CD8-sensitive CTL from B6 and B6-related bm1 mice exhibited a Tc1 predominance in response to alloantigenic stimulation in vitro under known Tc1-skewing conditions (Figs. 6Go and 7Go) as well as under neutral conditions (data not shown). The anti-CD8-sensitive Tc2 effectors we generated were lytic (Fig. 6Go) and many produced IL-10, but not IFN-{gamma}, as determined by FACS analysis (Fig. 7Go). In contrast, anti-CD8-resistant CD8+ T cells were limited to the Tc1 phenotype (Fig. 7Go) and generated lytic effector function only under Tc1-skewing conditions (Fig. 6Go). The CD8+ T cells generated under Tc2 conditions in the presence of anti-CD8 mAb expressed normal cell surface levels of the TCR/CD3 complex, expressed normal levels of perforin and granzyme, had proliferated and expanded in culture (data not shown), but nevertheless did not exhibit significant cytolytic function. Because perforin and granzymes are the major effector mechanism of most, but not all, CTL types (8, 33, 34), we are currently investigating the molecular basis for the lack of lytic function from anti-CD8-resistant T cells derived from Tc2-condition cultures.

In addition to their lack of lytic effector function, the expected Tc2 cytokine expression pattern was not detected in the CD8+ T cells generated in Tc2-skewing conditions and anti-CD8 mAb (Fig. 7Go). At some stage during the course of their activation, the signals required to generate lytic activity and produce IL-10 may be altered in anti-CD8-resistant T cells generated from IL-4-containing cultures. There are at least four possible explanations for these results: 1) IL-4 in the MLC inhibits the function of cross-linking anti-CD8 mAb required for the generation of cytolytic anti-CD8-resistant CTL; 2) anti-CD8 mAb in the MLC inhibits IL-4-mediated effects in the generation of type 2 T cells; 3) only low/moderate avidity pCTL (anti-CD8 sensitive) are activated in Tc2-skewing conditions, and therefore high avidity CTL (anti-CD8-resistant CTL) are not effectively generated under these conditions; and/or 4) Tc2 generation requires stronger activation signals than can be generated in the presence of anti-CD8 mAb (35, 36). Thus, we have identified a significant characteristic of anti-CD8-resistant CTL that may have implications for the in vivo effector function of this unique CD8+ CTL subset.

We used intracellular FACS analysis to analyze Tc1 and Tc2 effectors because this technique provides a significant advantage compared with the ELISA technique with respect to speed, simplicity, and the ability to selectively focus analysis on one cell subset in a mixed population of cells by combining extracellular and intracellular Ab staining. The application of three-color FACS analysis provides the ability to discriminate among populations of CTL that are activated in vitro in type 1 or type 2 skewing conditions. Similar FACS results were also reported by Cerwenka et al. (19), in which two populations existed in hemagglutinin-specific Tc2 priming cultures, one that produced IFN-{gamma} and a second that produced IL-4 only. In our Tc1 and Tc2 cultures, a population of IFN-{gamma} and IL-10 dual-producing cells was detected, which presumably represents a transitional or Tc0 population of cytokine-producing cells. We were not able to detect significant IL-4 and IL-5 in Tc2 by FACS analysis, although low levels of these cytokines have been reported to be produced by Tc2 cells when assayed by ELISA (13, 14, 15, 16, 17, 18, 19), which monitors the cytokines produced and accumulating in culture supernatant over an extended time period. The lack of detectable IL-4 or IL-5 from our in vitro derived Tc2 effectors could therefore be due to low and/or transient production by CD8+ Tc2 cells that is below the detection threshold at the time of intracellular FACS analysis.

We have presented a simple, reproducible, and efficient system to reveal a high avidity subset of CD8+ CTL in vitro. Anti-CD8-resistant CTL are perforin- but not Fas/FasL-dependent killers (8), and thus have a broad potential target range, because they are not dependent on target cells’ expression of a functional Fas-mediated apoptotic signaling pathway. Together with their high avidity characteristic, these CTL may have important functional in vivo roles in graft rejection (37, 38) and may be important in antiviral (9, 10, 39) and antitumor responses. Our model system does not depend on identification of the specific target Ags/peptides for the induction and expansion of high avidity CD8+ CTL, and may have applications in therapeutic strategies for the in vitro expansion of potent Ag-specific CTL. The cytokine requirements of this subset are therefore relevant and have important practical implications. We have demonstrated in this study that anti-CD8-resistant CTL are highly dependent upon IL-2 in vitro, and are limited and even inhibited by subtle qualitative differences in their surrounding cytokine milieu during Ag activation, implying that anti-CD8-resistant CTL may actively contribute to type 1-driven immune responses, and cross-regulate other effectors during type 2-driven immune responses.


   Acknowledgments
 
We thank Drs. Ann Stewart-Akers and Penelope A. Morel for helpful suggestions and critiques during the course of these studies.


   Footnotes
 
1 These studies were supported by grants to S.A.M. from the American Cancer Society (IM-749 and RPG-94-007-04-IM), and a 1997 UNCF/Merck Graduate Dissertation Fellowship to E.S.N.L.-S. from the United Negro College Fund and Merck Research Labs. Back

2 Address correspondence and reprint requests to Dr. Susan A. McCarthy, Department of Surgery, University of Pittsburgh, W1554 Biomedical Science Tower, Terrace and Lothrop Streets, Pittsburgh PA 15213. E-mail address: Back

3 Abbreviations used in this paper: pCTL, CTL precursor; Con A SN, Con A-induced supernatant. Back

Received for publication May 20, 1999. Accepted for publication July 29, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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