The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, Q.
Right arrow Articles by Shrikant, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, Q.
Right arrow Articles by Shrikant, P. A.
The Journal of Immunology, 2006, 177: 7618-7625.
Copyright © 2006 by The American Association of Immunologists, Inc.

IL-12-Programmed Long-Term CD8+ T Cell Responses Require STAT41

Qingsheng Li*, Cheryl Eppolito*, Kunle Odunsi{dagger} and Protul A. Shrikant2,*

* Department of Immunology and {dagger} Department of Gynecologic Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunological adjuvants activate innate immune cells for Ag presentation and elicitation of cytokines like IL-12 that promote T cell expansion and effector differentiation. An important but elusive aim for most immunization strategies is to produce memory T cells that provide durable immunity. Recent evidence demonstrates that the context of Ag presentation instructionally programs T cells for short- and long-term responses. However, the role and mechanisms by which cytokines like IL-12 condition CD8 T cells for long-term responses remain relatively uncharacterized. In this study, we show that brief exposure (20 h) of naive TCR-transgenic CD8 cells to IL-12 during Ag stimulation leads to transient phosphorylation of STAT4 for robust effector differentiation. Moreover, the IL-12-induced STAT4 engenders greater clonal expansion of the Ag-activated CD8 cells by regulating the expression of the transcriptional factor Bcl3- and Bcl2-related genes that promote survival of Ag-activated CD8 cells. Remarkably, the IL-12-conditioned CD8 T cells demonstrate increased sensitivity to IL-7 and IL-15, whereby they are rendered "fit" for homeostatic self-renewal as well as augmented CD4-dependent recall responses that are effective at controlling Salmonella infection in vivo. This information provides new insights into mechanisms by which IL-12 conditions CD8 T cells for long-term immunity, which is likely to benefit development of new strategies for the use of IL-12 in infectious diseases and cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD8+ T cells are ideally suited to provide protection against intracellular infection and/or cancer due to their ability to mount direct target cytolysis and production of cytokines like IFN-{gamma} (1). The conditions present during stimulation of naive CD8 T cells that program effector maturation include Ag, costimulatory molecules like B7.1, and cytokines like IL-12 (2, 3, 4). Although, it is well-appreciated that cytokines like IL-12 are critical for effector differentiation, the precise modulation mechanisms that govern the cytokine-mediated program CD8 effector maturation remain poorly understood. Most of the clonally generated effector CD8+ T cells will undergo apoptosis and a small fraction of selected cells will go on to memory formation (5, 6), the impact of cytokine-conditioned differentiation for heightened memory cell fate determination is not well-characterized. The paucity in understanding the mechanisms that regulate cytokine IL-12-mediated CD8 differentiation that results in effector and memory responses is a considerable hurdle for the development of strategies for the rational use of immunological adjuvants and/or inflammatory cytokines for durable immunity.

It is evident that most immunological adjuvants activate innate immune cells by pathogen-associated molecular patterns for TLRs leading to the generation of a costimulatory context capable of promoting T cell activation, proliferation, and differentiation that produces effector and memory T cells (7, 8, 9). However, recently chronic inflammation, typically characterized by abundant IL-12 production, has been shown to restrict CD8 T cell clonal expansion and memory generation (10). These divergent observations urge careful evaluation of the impact of IL-12 on CD8 T cell clonal expansion, survival, and long-term responses. The linear model of T cell differentiation proposes that the extent of memory generated is determined by the steady state levels of T cells produced upon clonal expansion and subsequent contraction (6, 11), thus it can be inferred that effectors with increased "fitness" for survival and self-renewal are poised for memory formation. Intrinsically, T cells with gene expression profiles that restrict apoptosis have increased fitness for survival and thus exhibit greater potential for memory generation (12, 13). The Bcl2-related genes, namely Bcl2, Bcl-xL, Bad, Bim, and Bax, have all been implicated in T cell survival (14), but their regulation by innate cytokines like IL-12 is not well-established. In addition to intrinsic survival advantage, effector cells engendered with increased ability to integrate signals from extrinsic growth factors in vivo, like IL-7 and/or IL-15, also have greater potential for self-renewal leading to memory (15, 16, 17). In fact, the loss of IL-15 reduces CD8 memory generation and IL-7R{alpha} expression on effector is considered as a marker for future memory cells (17, 18). Although, a study has reported the benefits of in vivo IL-12 administration on CD8+ T cell homeostatic expansion (19), the mechanisms by which innate cytokines like IL-12 program CD8 T cells for augmented survival, homeostatic self-renewal, and/or recall responses are not well understood.

In this study, we report that brief (20 h) IL-12 exposure during Ag stimulation induces transient phosphorylation of STAT4 which conditions CD8 cells for robust effector differentiation and engenders "fitness" for long-term responses by regulating the downstream gene programs that favor survival, capacity for self-renewal by IL-7 and IL-15, and produce heightened CD4-dependent recall responses to restrict infection in vivo. These results implicate the IL-12-induced transient STAT4 activation in the profound programming of Ag-specific CD8 T cells for long-term responses that can afford durable immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, reagents, and flow cytometry

The C57BL/6 (B6) mice (National Cancer Institute), OT-1, OT-1.PL, and OT-1/STAT4–/– mice were bred, housed, and used according to an approved protocol by the Institutional Animal Care and Use Committee of the Roswell Park Cancer Institute. IL-15-deficient B6 (tm1lmx) (B6-IL-15–/–) mice were purchased from Taconic Farms. The cytokine recombinant mouse IL-12 was provided by Dr. S. Wolf (Wyeth, Cambridge, MA); IL-7 and IL-15 were purchased from R&D Systems. The synthetic peptide "SIINFEKL" (OVAp) was synthesized at the Medical College of Georgia. Anti-IL-7R{alpha} (A7R.34) mAb was provided by Dr. S. Jameson (University of Minnesota, Minneapolis, MN). The phorbol ester PMA, ionomycin, and brefeldin A were purchased from Sigma-Aldrich. Annexin V-conjugated with FITC and propidium iodide (PI)3 were obtained from BD Pharmingen. All Abs were purchased from BD Pharmingen, except for anti-CD127 (IL-7R{alpha}) from eBioscience and anti-IL-15R{alpha} from R&D Systems. Staining for surface and intracellular Ag expression was performed as previously described (20). In brief, cells recovered from culture were restimulated with PMA (20 ng/ml) and ionomycin (400 ng/ml) for 4 h in the presence of 10 µg/ml brefeldin A, fixed, stained for cell surface protein expression followed by permeabilization, and staining for intracytoplasmic staining (ICS) Ags like IFN-{gamma}. The FACScan/FACSCalibur (BD Biosciences) were used for flow cytometry and typically 10,000 live CD8+ gated events were analyzed for cell surface and ICS evaluation with CellQuest software (BD Biosciences). Where indicated, OT-1 T cells were labeled with 5 µM carboxyfluorescein diacetate succinimidyl diester (Molecular Probes) and checked by flow cytometry before transfer.

In vitro T cell stimulation and functional assays

Cells harvested from lymphoid organs were enriched for CD8+ T cells by negative selection (Cedarlane Laboratories). The selection resulted in 90–95% CD8+ cell purity and, in all experiments, the cells used were ≥95% V{alpha}2+ cells and ≤4% CD44high as determined by flow cytometry. The adherent cell lines BOK (MEC.B7.SigOVA: expressing H-2Kb, OVAp, and B7.1) and MEC II (expressing H-2Kb, no Ag as the control for BOK), were used to present Ag to naive OT-1 cells as previously described (21). The BOK cells (1 x 105 cells/ml) were reacted with (2.5 x 105 cells/ml) OT-1 cells. The nonadherent cells harvested after 20 h with or without IL-12 (2 ng/ml) were transferred to new wells containing medium and were reharvested for evaluation at indicated time points. In some experiments, OT-1 cells were harvested 72 h after the initiation of culture, and viable cells (1 x 105 cells/ml) were recultured with indicated doses of IL-7 or IL-15 and pulsed with 1 µCi/well [3H]thymidine for the final 12 h of culture. The proliferation was determined at 72 h after reculture with IL-7 or IL-15 by [3H]TdR incorporation. The cytolytic ability of the cells harvested after various stimulatory conditions was analyzed by using a standard 4-h chromium release assay as previously described (20). The 51Cr-labeled EL4 cells pulsed with 10 nM "SIINFEKL" peptide were used as target cells, whereas irrelevant peptide "SIIYREGL"-pulsed EL4 cells served as an Ag control.

Evaluation of percent recovery

The cell viability was assessed by trypan blue dye exclusion analysis by microscopic evaluation. The ratio between viable cells, added and harvested 72 h after culture, was multiplied by 100 for determining percent recovery.

RT-PCR

The total RNA extracted from OT-1 T cells at 72 h after culture was reverse transcribed and amplified by using primers pairs designed based on sequences listed in the GenBank database and as shown in Table I. The results shown were typically obtained after 30~32 cycles of amplification and beta-actin was used as an internal control for normalization.


View this table:
[in this window]
[in a new window]
  		Table I. PCR primers

 
Western blot analyses

Purified CD8+ cells were obtained after culture at indicated time points, lysed, and an equal amount (20 µg/sample) of total cellular protein was subjected to Western analysis by sequential staining with antiphosphorylated STAT4 (Zymed Laboratories) and total STAT4-specific Abs (Santa Cruz Biotechnology). The Ab was detected by amplification with ECL reagents (Amersham Biosciences) and visualization by exposure to x-ray film.

Evaluating CD8+ T cell homeostatic self-renewal and recall responses

The wild-type (WT) and (STAT4 knockout (KO)) OT-1 (CD8{alpha}+/Thy1.1+) T cells reacted with BOK cells for 20 h with or without IL-12 (2 ng/ml) were transferred to new wells and harvested at 72 h. Live 2 x 106 OT-1 cells were adoptively transferred (i.v.) into Thy1.2+ congenic B6 recipients that were either irradiated (700 rad) (for lymphopenia-induced homeostatic expansion) or intact (for recall responses). The homeostatic self-renewal was determined by enumerating the numbers of OT-1 T cells in the spleen 5 days after adoptive transfer by staining and flow cytometry. The requirements for IL-7 and IL-15 was established by transfer of IL-12-conditioned OT-1 T cells into IL-15–/– B6 recipients that were administered anti-IL-7R{alpha} (1 mg of the clone A7R34/injection) i.p. every second day between days –2 and 5 after adoptive transfer. To evaluate the capacity of in vitro-conditioned OT-1 cells for recall responses, mice recipient of in vitro-stimulated OT-1 cells were analyzed on day 10 or 66 (before rechallenge) and 4 days after s.c. challenge (on day 14 or 70) with 5 µg of chicken OVA protein admixed with an equal volume of IFA (Sigma-Aldrich) for the numbers of OT-1 cells by flow cytometry. The fold increase in the number of OT-1 cells was calculated by dividing the numbers of OT-1 cells detected before and after immunization. To evaluate long-term protection, recipients of adoptively transferred naive, unconditioned, or IL-12-conditioned OT-1 T cells were challenged with Salmonella-OVA (1 x 106) on day 66 (22) and sacrificed on day 70, their spleens (1 gram) were harvested, minced, and serial dilutions were plated on MacConkey agar plates. The CFU per gram of spleen were determined after incubation overnight at 37°C. The number of OT-1 T cells in the spleen and the lymph nodes of infected mice were determined by flow cytometry. For some OT-1-recipient animals, CD4+ cells were depleted by administration of 500 µg of anti-CD4 Ab (clone GK1.5) every alternate day between days 55 and 70.

Statistical analysis

For statistical analysis, the unpaired Student t test was applied.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-12 conditions STAT4-dependent effector differentiation of CD8+ T cells

The presence of IL-12 during Ag stimulation provides a critical third signal for CD8+ T cell effector differentiation without affecting proliferation (23). To further characterize the ability of IL-12 to program differentiation of naive OT-1 T cells for effector and memory responses, we first tested the ability of naive OT-1 T cells to respond to IL-12 addition early during Ag stimulation. As shown in Fig. 1, A and B, and previously reported (24, 25), OT-1 cells—reacted with adherent Kb/SIINFEKL- and B7.1-expressing embryonic fibroblasts (BOK) in the presence or absence of IL-12 (2 ng/ml) for 1 or 4 h—express levels of IL-12Rbeta2 on the cell surface that are undetectable by Ab staining and flow cytometry. Moreover, addition of IL-12 does not affect Ag-driven OT-1 proliferation determined by CFSE dilution (Fig. 1B). Previously, BOK cells have been effectively used to determine the role of Ag and costimulatory molecule derived signals for instructing naive CD8+ T cell differentiation (21). To determine whether IL-12 alters expression of activation markers CD44, CD62L, V{alpha}2, and CD69 on Ag-activated CD8 cells, we analyzed OT-1 cells that were activated with Ag in the presence or absence of IL-12 by Ab staining and flow cytometry. As shown in Fig. 1C, IL-12 addition produces no effects on early activation markers (L-selectin, CD44, CD69, and TCR V{alpha}2). Surprisingly, IL-12 addition to the cultures rapidly (30 min) induces STAT4 phosphorylation in OT-1 cells, which is optimal at 4 h (Fig. 1D) and returns to baseline levels by 48–72 h (Fig. 1E), thus demonstrating the ability of IL-12 to program differentiation of Ag-stimulated OT-1 T cells via the transient STAT4 induction.


Figure 1
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 1. IL-12 induces transient STAT4 phosphorylation in Ag-stimulated OT-1 CD8+ T cells without affecting early activation or proliferation. A, Expression of IL-12Rbeta2 on CD8+V{alpha}2+ gated OT-1 T cells stimulated with BOK in the presence or absence of IL-12 (2 ng/ml) for 1 or 4 h. B and C, CFSE-labeled naive OT-1 T cells were stimulated with BOK in the absence or the presence of IL-12 (2 ng/ml) for 20 h and analysis was performed at 24 h (for CD69 and TCR V{alpha}2) or 72 h (for CFSE dilution and CD44, CD62L). D and E, The CD8+V{alpha}2+CD44low sorted OT-1 cells reacted with BOK cells in the presence or absence of IL-12 (2 ng/ml) were harvested at 0 h (naive), 0.5, 4, 12, 24, 48, and 72 h after stimulation, the total cell extracts were serially subjected to Western blotting analysis with anti-pSTAT4 and total STAT4 Abs. D, Immunoblot with anti-pSTAT4 and total STAT4 in BOK reacted OT-1 cells in the absence or presence of IL-12 for 4 h. E, Fold increase in the ratio of pSTAT:STAT. The phospho-STAT/total STAT levels were determined by densitometry analysis (NIH Image software), and fold increase relative to Ag only stimulated CD8+ T cells at 0.5 h (equalized to 1) is shown. The results shown are representative of three independent experiments.

 
To verify the requirement of IL-12-induced STAT4 activation on CD8+ T cell effector differentiation, WT OT-1 or STAT4-deficient (STAT4 KO) OT-1 cells were cultured with BOK in the presence or absence of IL-12 (2 ng/ml) for 20 h, transferred to medium alone (no Ag and/or cytokine), and harvested at 72 h to evaluate the programmed effector maturation. As expected, the IL-12-conditioned OT-1 cells produced robust effector functions at 72 h as evidenced by ICS IFN-{gamma} staining (Fig. 2A) and CTL activity (Fig. 2B), which was completely abrogated in the absence of STAT4 (Fig. 2, A and B). Moreover, the ability of IL-12 to produce robust OT-1 effector differentiation required not only STAT4 but also TCR ligation with its cognate Ag as OT-1 T cells stimulated with MEC II cells (H-2Kb+ fibroblast without SIINFEKL expression) and IL-12 produced no detectable effector maturation (Fig. 2, C and D).


Figure 2
View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 2. IL-12 requires STAT4 for CD8+ T cell effector differentiation. Naive WT or STAT4-deficient (STAT4 KO) OT-1 T cells were stimulated with BOK in the absence or the presence of IL-12 (2 ng/ml) for 20 h, transferred to new wells with no Ag or cytokine and harvested at 72 h after initial stimulation. A, The ICS IFN-{gamma} production was determined by flow cytometry. B, The percent-specific lysis in a 4-h standard chromium (51Cr) release assay. C and D, OT-1 T cell stimulated with fibroblast without cognate Ag expression (MEC II) and IL-12 produced no detectable effector maturation. Naive OT-1 T cells were stimulated with MEC II or BOK for 20 h in the absence or the presence of IL-12 (2 ng/ml) and transferred to new wells with no Ag or cytokine were harvested at 72 h. C, OT-1 T cells were gated and analyzed for the production of IFN-{gamma} (ICS). D, OT-1 T cells were analyzed for CTL activity. The result shown is representative of three independent experiments.

 
IL-12 requires STAT4 to regulate gene programs that favor survival of Ag-activated CD8+ T cells

A key hallmark of cell-mediated immunity is its potential for affording durable protection due to the ability of T cells to form memory. The instructional conditioning of naive CD8 for memory requires progressive regulation of the Ag-activated cells for sustenance by affecting their survival. To determine whether Ag and IL-12 conditioning that produced robust effector maturation would also regulate OT-1 survival, the impact of IL-12 on OT-1 cell clonal expansion was evaluated. Clearly, IL-12-conditioned OT-1 cells showed a higher percentage of viable cells recovered after 72 h of culture (Fig. 3A). Based on our observation that IL-12 does not augment OT-1 proliferation (Fig. 1B) and a previous report (25), we reasoned that IL-12 enhanced OT-1 clonal expansion by reducing apoptosis via STAT4-mediated regulation of apoptosis-related gene programs. Indeed, IL-12 required STAT4 to restrict OT-1 apoptosis as demonstrated by the increased percentage of OT-1 cells that were negative for PI and/or annexin V, markers associated with apoptotic cell death (Fig. 3B, lower left quadrants) at 72 h, however, it is noteworthy that in the absence of STAT4, the OT-1 cells for both annexin V and PI staining were increased as compared with the unconditioned WT OT-1 cells that were largely positive for PI, but not annexin V (Fig. 3B, upper left quadrant). These observations suggest that in the absence of STAT4, there is induction of apoptosis which does not progress to cell death and loss in OT-1 clonal population, but in the absence of IL-12 conditioning the Ag-activated OT-1 T cells undergo nonapoptotic cell death resulting in reduced clonal numbers. The enhanced survival produced by IL-12-induced STAT4 was associated with increases in the expression of Bcl3 and antiapoptotic genes Bcl2, Bcl-xL, but not survivin, along with decreases in the expression of proapoptotic genes Bad and Bim, but not Bax (Fig. 3C). Remarkably, the IL-12- and STAT4-conditioned survival advantage was lost in Bcl3 KO CD8 T cells (Fig. 3D), indicating the essential role for STAT4-induced Bcl3 in the IL-12-conditioned augmented survival and suggesting the potential role of Bcl3-mediated Bcl2-related gene expression in tuning the intrinsic balance to favor CD8 T cell survival.


Figure 3
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 3. IL-12 requires STAT4 to promote intrinsic OT-1 survival. WT or STAT4 KO OT-1 T cells stimulated with BOK cells for 20 h with or without IL-12 (2 ng/ml) were harvested at 72 h. A, The percentage of viable cell recovery, as described in Materials and Methods, was determined. Data are expressed as mean ± SE from three independent experiments set up in triplicate. *, p < 0.05; **, p < 0.01. B, OT-1 cells were reacted with annexin V and PI and ~10,000 gated CD8+V{alpha}2+ cells were evaluated by flow cytometry. Numbers in the quadrants indicate the percentage of OT-1 cells. The results shown are representative of three independent experiments. C, Total RNA isolated from cells was evaluated for indicated pro- and antiapoptotic gene expression profile by RT-PCR. D, CD8+ T cells from B6 or Bcl-3 KO B6 were stimulated with anti-CD3 (1 µg/ml) mAb and anti-CD28 (0.5 µg/ml) and analyzed at 72 h for percentage of viable cell recovery. The data shown are representative of three independent experiments with identical results.

 
IL-12 conditions STAT4-dependent homeostatic fitness

The growth factors IL-7 and IL-15 are important extrinsic factors that regulate CD8+ T cell survival and are shown to be critical for CD8 memory generation (16, 17). To determine whether IL-12-conditioned OT-1 T cells are programmed for increased sensitivity to extrinsic growth factors IL-7 and IL-15 that promote homeostatic self-renewal, we first determined the relative levels of IL-7 and IL-15 receptor expression on unconditioned or IL-12-conditioned OT-1 T cells at 72 h. The IL-12-conditioned OT-1 T cells had higher levels of IL-7R{alpha}, CD122, CD132 expression and marginally higher IL-15R{alpha} expression, which was substantially reduced in STAT4-deficient OT-1 T cells (Fig. 4A). The STAT4-dependent increases in the signaling components of the IL-7 and IL-15 receptors rendered IL-12-conditioned OT-1 T cells more sensitive to varying doses of IL-7 and IL-15, as evidenced by their increased proliferation (Fig. 4B) and clonal expansion (Fig. 4C). Interestingly, the IL-7 mediated re-expression of KLF2, which is thought to regulate cell senescence (15) in Ag-activated OT-1 T cells and is thus considered to be important for homeostatic self-renewal, was also markedly enhanced in IL-12-conditioned OT-1 T cells in a STAT4-dependent manner (Fig. 4D). These observations collectively suggest that IL-12-conditioned OT-1 cells had greater "fitness" for self-renewal, an essential attribute for memory formation. To directly test whether IL-12 conditions OT-1 cells for greater homeostatic self-renewal, we adoptively transferred unconditioned or IL-12-conditioned OT-1 T cells into irradiated B6 recipients and on day 5 evaluated their clonal frequency by enumerating OT-1 cells detected in vivo. The IL-12-conditioned OT-1 T cells maintain a considerably larger frequency than unconditioned OT-1 cells (Fig. 5), which depended on endogenous IL-7 and IL-15 in vivo (Fig. 5; WT; Ag plus IL-12 plus IL-15–/–/IL-7R{alpha}) and STAT4 in the IL-12-conditioned OT-1 cells (Fig. 5), thus indicating that IL-12 conditions CD8+ T cells via STAT4 gene programs that engender fitness for survival as well as self-renewal by increasing their ability to assimilate growth factor signals.


Figure 4
View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 4. IL-12 sensitizes CD8+ T cells for response to IL-7 and IL-15 via STAT4. WT or STAT4 KO OT-1 T cells stimulated with BOK in the presence or absence of IL-12 (2 ng/ml) for 20 h were transferred to new wells, harvested and evaluated at 72 h. A, Surface phenotype of live-gated CD8+/V{alpha}2+ cells by flow cytometry. B and C, Response to indicated doses of IL-7 or IL-15 after reculture for an additional 72 h. B, Proliferation (cpm; [3H]thymidine uptake from 60 to 72 h) and C, percentage of viable cell recovery (% recovery). Data are expressed as mean ± SE from three independent experiments set up in triplicate (*, p < 0.05; **, p < 0.01). D, The KLF2 mRNA expression in IL-12-conditioned or unconditioned OT-1 cells was determined by RT-PCR analysis of total RNA. The relative level of KLF2 mRNA expression was determined by densitometry and normalization to beta-actin. The numbers indicate the fold increases in normalized KLF2 mRNA relative to naive T cells (equalized to 1). The result shown is representative of three independent experiments.

 

Figure 5
View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 5. IL-12 enhances STAT4-dependent "fitness" for homeostatic self-renewal. Live OT-1 (2 x 106) cells that were either unconditioned ((WT) Ag) or IL-12 conditioned ((WT) Ag+IL-12) or IL-12-conditioned STAT4-deficient OT-1 cells ((STAT4 KO) Ag+IL-12) were transferred into irradiated B6 or B6-IL-15–/– mice treated with anti-IL-7R{alpha} mAb (day 0) ((WT) Ag+IL-12 (IL-15–/–+anti-IL-7R{alpha})). The OT-1 cells were enumerated by flow cytometry on day 5, and data shown are the mean ± SE from three independent experiments with five mice in each experimental group (*, p < 0.05; **, p < 0.01).

 
IL-12 conditions STAT4-dependent CD8+ recall responses

The ability of T cells to mount effective short- and long-term recall responses is indicative of their capacity to afford durable immunity. To test whether the brief IL-12-induced STAT4 phosphorylation was sufficient to condition OT-1 T cells for long-term responses, we stimulated OT-1 T cells with BOK in the presence or absence of IL-12 (20 h), rested up to 72 h without Ag or cytokine and then adoptively transferred (2 x 106) into intact Thy1- congenic recipients. The recipients were challenged s.c. with IFA/OVA (5 µg) on day 10 or 66 after adoptive transfer. The number of OT-1 T cells detected on days 10 and 66 before Ag rechallenge was determined by flow cytometry and used to calculate the fold expansion of OT-1 T cells detected 4 days after Ag rechallenge in vivo. The recipients of IL-12-conditioned OT-1 T cells had significantly larger numbers of OT-1 cells after Ag rechallenge in the short-term (day 10) (Fig. 6A), as well as in the long-term (day 66) (Fig. 6B); this increase in clonal population was due to an increase in the fold expansion (numbers in parentheses) realized by Ag rechallenge. The ability of IL-12-conditioned OT-1 T cells for the observed augmented recall response required STAT4 (Fig. 6), implicating the IL-12-induced transient STAT4 activation in conditioning CD8+ T cells for sustenance. Recent reports implicate the CD4+ and CD8+ T cell collaboration for durable immunity (26). To determine whether IL-12-conditioned long-term CD8+ recall responses were independent of CD4+ T cells, we rendered some recipients of IL-12-conditioned OT-1 T cells deficient for CD4+ T cells between days 55 and 70 (<2% CD4+ reactivity in the lymph node/spleen; data not shown) by Ab-mediated depletion, this strategy maintains equivalent survival of the adoptively transferred IL-12-conditioned OT-1 T cells and permits revealing the requirement for CD4 T cells during the Ag rechallenge. The various groups of recipients were i.v. challenged with 1 x 106 of an attenuated strain of OVA-expressing Salmonella typhimurium (SL1344) on day 66 after adoptive transfer (22). Four days after challenge, numbers of OT-1 cells detected and Salmonella CFU were determined (Fig. 7). The IL-12-conditioned OT-1 showed significantly better recall responses, as determined by rapid increase in OT-1 T cell numbers and the fold expansion (numbers in parentheses) by 4 days of Ag challenge. Importantly, the IL-12-conditioned OT-1 recall response was effective at reducing the Salmonella burden (Fig. 7). Surprisingly, the conditioned responses required CD4+ T cells, as demonstrated by the loss of clonal expansion and efficacy to Salmonella challenge in CD4+ T cell-depleted recipients, thus indicating that IL-12-treated CD8+ T cells were conditioned for long-term response by empowering them for collaborations with CD4+ T cells in vivo.


Figure 6
View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 6. IL-12 conditions STAT4-dependent recall CD8+ responses. Unconditioned or IL-12-conditioned WT and STAT4 KO OT-1 T cells were transferred (2 x 106 cells) into nonirradiated B6. On day 10 or 66 after transfer, recipients were s.c. challenged with IFA-OVA (5 µg), and 4 days later spleen cells were harvested and analyzed for frequency of OT-1 T cells by flow cytometry. A, Short-term secondary challenge (day 10->day 14) and (B) long-term secondary challenge (day 66->day 70). Numbers in parentheses indicate the fold expansion of OT-1 T cells obtained by normalizing the numbers of OT-1 T cells detected after Ag rechallenge with those detected before in each experimental group (details in Materials and Methods). Data are expressed as mean ± SE from three independent experiments with five mice in each experimental group. **, p < 0.01.

 

Figure 7
View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 7. IL-12 conditions CD4+ T cell-dependent effective CD8+ T cell memory response. Unconditioned (Ag) or IL-12-conditioned (Ag+IL-12) WT OT-1 T cells were transferred into unirradiated (intact) B6 recipients. Some recipients were rendered CD4-deficient by GK1.5 mAb administration (500 µg) between days 55 and 70. On day 66, recipients were infected with 1 x 106 Salmonella-OVA i.v. A, Frequency of OT-1 T cells in the spleens 4 days after Salmonella challenge was determined by flow cytometry. The numbers in brackets are the average fold expansion of OT-1 T cells after challenge. B, The CFU (log10 CFU) of Salmonella-OVA detected in the spleen 4 days after challenge. Data are expressed as mean ± SE from two independent experiments with four mice in each experimental group. Results are representative of two independent experiments with four mice in each group. *, p < 0.05; **, p < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The "autopilot" model for CD8+ T cell differentiation dictates that instructions received during Ag stimulation are sufficient to program naive T cells for autonomous clonal expansion and differentiation (27). The ability of IL-12 to program CD8+ effector differentiation has been previously noted (23, 25), however, the underlying molecular mechanisms are not well understood. Moreover, the ability of IL-12 to condition CD8+ T cells for long-term responses remains uncharacterized. In our study, we have exploited a previously established in vitro system that offers the advantages of providing a constant source of Ag and cell surface-associated costimulatory signal (21) to test the impact of IL-12 on programming Ag-activated CD8+ T cells for effector and memory differentiation and better understand the molecular pathways regulating the differentiation process. Clearly, stimulation with Ag induces activation of CD44high, CD62Llow, V{alpha}2low, as well as proliferation, but does not induce robust effector differentiation (Figs. 1, B and C, and 2, A and B). The presence of IL-12 during Ag stimulation (20 h) conditions effector differentiation without affecting activation phenotype and proliferation (Figs. 1, B and C, and 2, A and B), these observations support the notion that IL-12-induced robust CD8 T cell effector differentiation is independent of the cell division rates, in line with the previous observations reported by Curtsinger et al. (23). Importantly, IL-12 is unable to produce effector differentiation in the absence of Ag stimulation (Fig. 2, C and D) and induce cell division (data not shown), thus indicating the need for IL-12-mediated signals to integrate with TCR-generated proliferation signals for effector maturation. Although these results do not rule out the contributions of cell division on T cell effector differentiation, as suggested previously (28), they encourage studies to better understanding the differential integrative potential of Ag strength and cytokine signaling in regulating CD8 proliferation and its role in effector maturation. In our studies, we have consistently observed that the presence of IL-12 (20 h) during Ag stimulation profoundly alters Ag-induced CD8+ T cell responses (Figs. 1–7), these observations were initially perplexing, due to our inability to detect IL-12Rbeta2 expression on naive OT-1 cells by flow cytometry (Fig. 1A), as previously reported (24). However, careful evaluation of IL-12Rbeta2 mRNA expression reveals rapid induction of this gene in Ag as well as Ag plus IL-12-treated OT-1 cells (data not shown), suggesting that low levels of the IL-12Rbeta not detected by flow cytometry could mediate IL-12 signals during the 20 h of stimulation. The observation that IL-12 addition during Ag stimulation rapidly induces (30 min) phosphorylation of STAT4, which returns to baseline by 48–72 h poststimulation (Fig. 1E), lends further support to the argument that low levels of IL-12Rbeta2 expression on naive OT-1 T cells are able to signal and impact naive OT-1 T cell response to Ag stimulation. Although possible, it is highly unlikely that other constitutively expressed receptors mediate the IL-12-induced STAT4 activation, this remote possibility can be tested by conducting further studies in IL-12Rbeta-deficient OT-1 T cells in the future. These observations imply that early and transient STAT4 activation may function as an intermediary in IL-12-induced OT-1 T cell effector differentiation (Fig. 2, A and B), and the loss of effector functions in STAT4–/– OT-1 cells demonstrates its critical role in imprinting the IL-12-induced differentiation program.

Two distinct models for CD8 memory generation are proposed. The linear model suggests a small fraction of clonally expanded effector cells are selected for survival and destined for memory (5, 11), whereas recent data suggests that memory differentiation can occur without undergoing effector maturation (29). In either case, the mechanisms by which the cytokine instructs T cell memory remains unclear. It is well-accepted that the ability of Ag-activated T cells to survive is critical for the generation of long-term T cell responses (6, 28). The survival of the expanding pool of Ag-reactive T cells requires favorable balance between pro- and antiapoptotic proteins. The antiapoptotic Bcl2-related proteins, Bcl2, Bcl-xL as well as the I{kappa}B-related transcriptional factor Bcl3, along with the proapoptotic factors Bad, Bax, and Bim, have all been implicated in regulating T cell survival (14, 30, 31). Additionally, extrinsic signals provided by growth factors, such as IL-7/IL-15 have been shown to promote CD8+ T cell survival and self-renewal, essential for memory formation (32, 33). Evidently, IL-12 promotes T cell survival by regulating the intrinsic apoptotic pathway (34, 35), whereas one study has demonstrated that IL-12 administration in vivo enhances self-renewal of CD8+ T cells in response to IL-7/15 (19). Our results show that IL-12-conditioned OT-1 T cells produce considerable large population after 72 h of in vitro culture, primarily due to reduction in apoptosis (Fig. 3B) and not by increased proliferation (Fig. 1B). The survival advantage conditioned by IL-12 treatment is associated with STAT4-induced increased antiapoptosis (Bcl2, Bcl3, and Bcl-xL but not survivin) and decreased proapoptosis (Bad and Bim but not Bax) gene expression in OT-1 T cells (Fig. 3C). In agreement with a previous report (34), the deficiency of Bcl3 reverses the augmented cell recovery produced by IL-12, thus indicating the essential role for Bcl3 in the IL-12 conditioned survival. The IL-12/STAT4-dependent induction and silencing of the anti- and proapoptotic genes belonging to the Bcl-2 family may appear to be redundant, in lieu of the fact that Bcl3-deficient CD8 T cells do not demonstrate increased survival, but these observations may in fact reflect the intermediary role for STAT4-dependent Bcl3 expression in transcriptional regulation of Bcl2-related gene expression profile that favors increased survival. This possibility needs to be formally tested, as the implied role for Bcl3 in altering transcriptional programming and functional outcomes of Ag-activated CD8 T cells may produce new information on the role of Bcl3 in CD8 memory formation.

The novel observation that IL-12-conditioned OT-1 T cells are more sensitive to the growth factors IL-7 and IL-15 by virtue of their STAT4-dependent increases in IL-7{alpha}, IL-15R{alpha}, CD122, and CD132 receptor expression (Fig. 4A), demonstrating the profound influence innate cytokines exert on CD8 T cell differentiation, that not only produces effector maturation but also empowers them for increased survival and self-renewal (Fig. 4, B and C) (16, 17). The augmented KLF2 re-expression upon IL-7 treatment of IL-12-conditioned OT-1 cells (Fig. 4D) is indicative of the ability of IL-12 treatment to promote CD8 memory formation, as previous studies have shown the role of KLF2 in induction of quiescence, required for transitioning effector cells to memory (36). It is noteworthy that our observation of IL-12-conditioned augmented KLF2 expression by IL-7 is in contrast to the reported ability of IL-12 to neutralize the IL-7-augmented KLF2 gene re-expression in Ag-activated CD8+ T cells (37), this discrepancy may arise due to the sequential addition of IL-12 and IL-7 in our studies, compared with the simultaneous treatment used in the previous study (37). The mechanisms underpinning the differential KLF2 expression by the timing of IL-12 and IL-7 addition are not entirely clear, but careful studies aimed at identifying their interrelatedness may yield insights to benefit the use of IL-12 for homeostatic replenishment. The demonstration that the IL-12-induced transient STAT4 activation can profoundly condition CD8 cells for longevity and self-renewal lends support to the notion that innate immune cell- derived cytokine signals play an important role in CD8+ T cell effector and memory differentiation.

The ability of Ag-activated CD8+ cells to generate a recall response is an important hall mark of T cell memory (28). Clearly, the IL-12-conditioned OT-1 cells mount a considerably better recall response to Ag rechallenge in a STAT4-dependent manner (Figs. 6 and 7). The inability of IL-12-conditioned STAT4-deficient OT-1 T cells to mount augmented recall responses is to some extent due to poor survival (data not shown, used to calculate fold expansion), but can be largely attributed to the poor expansion realized upon Ag rechallenge in vivo (Fig. 6A, fold expansion numbers in parentheses), 4 days after IFA/OVA or Salmonella-OVA challenge (day 70) (Figs. 6 and 7), thus indicating the ability of IL-12-induced transient STAT4 activation to condition the CD8 T cells for long-term recall responses. Furthermore, the rapid kinetics of the CD8 expansion and their effectiveness against Salmonella infection indicates that IL-12 conditioning produces memory CD8 T cells that mount a robust recall responses in vivo. In the future, studies aimed at determining the impact of IL-12 conditioning on the kinetics of this recall response are planned to attain better understanding of the conditional response CD8 T cells in vivo, these insights can benefit development of new strategies to regulate CD8 memory and produce durable host immunity. Several studies have demonstrated the requirement for CD4+ help in memory CD8+ responses to infectious and/or tumor challenge (38, 39). The notion that CD4+ T cell "help" is bridged via the dendritic cell to promote CD8+ T cell memory formation (40, 41) may be indicative of the role of the APC-generated IL-12 in conditioning CD8+ memory and/or late recall responses. If this were the case, then it could be expected that IL-12-conditioned OT-1 responses would be independent of CD4+"help" for the generation of recall response in vivo. Our results show that in the absence of CD4 T cells the IL-12-conditioned recall response was abrogated (Fig. 7), suggesting that IL-12 produced by APCs is not the mechanism by which CD4+ bridges "help" for memory and goes on to demonstrate that the IL-12 present during Ag stimulation program CD8 T cells for collaboration with CD4 T cells for augmented recall responses. The mechanisms underlying the collaboration between CD4+ T cells and cytokine-conditioned CD8+ T cells in vivo are not well understood; although a role for members of the TNF family members such as CD40, OX40, TRAIL, and/or 4-1BB can be readily envisioned based on previous reports (40, 42, 43), they must be carefully evaluated in our model system in the future. All in all, our results provide several novel insights into the mechanisms underpinning IL-12-mediated programming of CD8 T cells for long-term responses, this information sheds light on the ability of innate cytokines to induce progressive differentiation of CD8 T cells that can enable durable immunity. The novel mechanisms identified in this study are likely to benefit rational use of adjuvants and cytokines in immunization strategies and provide a useful platform to study the role of specific genes in programming CD8+ T cells for effector and memory functions.


   Acknowledgments
 
We thank Dr. S. P. Schoenberger (La Jolla Institute for Allergy and Immunology, San Diego, CA) for providing the BOK and MEC II cell lines, Dr. M. Kaplan (University of Indiana, Bloomington, IN) for B6 STAT4 KO mice, and Dr. S. Jameson (University of Minnesota, Minneapolis, MN) for the anti-IL-7R{alpha} Abs. The review of this manuscript by Drs. M. Kaplan, S. Jameson, and A. Frey is appreciated.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by National Institutes of Health Grant (CA104645-02; to P.A.S.), and Cancer Research Institute Anna-Marie Kellen Clinical Investigator Award (to K.O.). Back

2 Address correspondence and reprint requests to Dr. Protul A. Shrikant, Department of Immunology, Roswell Park Cancer Institute, 322 Cancer Cell Center, Elm and Carlton Streets, Buffalo, NY 14263. E-mail address: Protul.Shrikant{at}roswellpark.org Back

3 Abbreviations used in this paper: PI, propidium iodide; ICS, intracytoplasmic staining; WT, wild type; KO, knockout. Back

Received for publication May 18, 2006. Accepted for publication September 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Ahmed, R., D. Gray. 1996. Immunological memory and protective immunity: understanding their relation. Science 272: 54-60. [Abstract]
  2. Mescher, M. F., J. M. Curtsinger, P. Agarwal, K. A. Casey, M. Gerner, C. D. Hammerbeck, F. Popescu, Z. Xiao. 2006. Signals required for programming effector and memory development by CD8+ T cells. Immunol. Rev. 211: 81-92. [Medline]
  3. Davis, S. J., S. Ikemizu, E. J. Evans, L. Fugger, T. R. Bakker, P. A. van der Merwe. 2003. The nature of molecular recognition by T cells. Nat. Immunol. 4: 217-224. [Medline]
  4. Swain, S. L.. 1999. Helper T cell differentiation. Curr. Opin. Immunol. 11: 180-185. [Medline]
  5. Kaech, S. M., E. J. Wherry, R. Ahmed. 2002. Effector and memory T-cell differentiation: implications for vaccine development. Nat. Rev. Immunol. 2: 251-262. [Medline]
  6. Lanzavecchia, A., F. Sallusto. 2002. Progressive differentiation and selection of the fittest in the immune response. Nat. Rev. Immunol. 2: 982-987. [Medline]
  7. Curtsinger, J. M., J. O. Valenzuela, P. Agarwal, D. Lins, M. F. Mescher. 2005. Type I IFNs provide a third signal to CD8 T cells to stimulate clonal expansion and differentiation. J. Immunol. 174: 4465-4469. [Abstract/Free Full Text]
  8. Wuthrich, M., T. Warner, B. S. Klein. 2005. IL-12 is required for induction but not maintenance of protective, memory responses to Blastomyces dermatitidis: implications for vaccine development in immune-deficient hosts. J. Immunol. 175: 5288-5297. [Abstract/Free Full Text]
  9. Mitchell, T. C., D. Hildeman, R. M. Kedl, T. K. Teague, B. C. Schaefer, J. White, Y. Zhu, J. Kappler, P. Marrack. 2001. Immunological adjuvants promote activated T cell survival via induction of Bcl-3. Nat. Immunol. 2: 397-402. [Medline]
  10. Badovinac, V. P., B. B. Porter, J. T. Harty. 2004. CD8+ T cell contraction is controlled by early inflammation. Nat. Immunol. 5: 809-817. [Medline]
  11. Opferman, J. T., B. T. Ober, P. G. Ashton-Rickardt. 1999. Linear differentiation of cytotoxic effectors into memory T lymphocytes. Science 283: 1745-1748. [Abstract/Free Full Text]
  12. Garcia, S., J. DiSanto, B. Stockinger. 1999. Following the development of a CD4 T cell response in vivo: from activation to memory formation. Immunity 11: 163-171. [Medline]
  13. Grayson, J. M., A. J. Zajac, J. D. Altman, R. Ahmed. 2000. Cutting edge: increased expression of Bcl-2 in antigen-specific memory CD8+ T cells. J. Immunol. 164: 3950-3954. [Abstract/Free Full Text]
  14. Marrack, P., J. Kappler. 2004. Control of T cell viability. Annu. Rev. Immunol. 22: 765-787. [Medline]
  15. Schober, S. L., C. T. Kuo, K. S. Schluns, L. Lefrancois, J. M. Leiden, S. C. Jameson. 1999. Expression of the transcription factor lung Kruppel-like factor is regulated by cytokines and correlates with survival of memory T cells in vitro and in vivo. J. Immunol. 163: 3662-3667. [Abstract/Free Full Text]
  16. Schluns, K. S., W. C. Kieper, S. C. Jameson, L. Lefrancois. 2000. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat. Immunol. 1: 426-432. [Medline]
  17. Schluns, K. S., K. Williams, A. Ma, X. X. Zheng, L. Lefrancois. 2002. Cutting edge: requirement for IL-15 in the generation of primary and memory antigen-specific CD8 T cells. J. Immunol. 168: 4827-4831. [Abstract/Free Full Text]
  18. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4: 1191-1198. [Medline]
  19. Kieper, W. C., M. Prlic, C. S. Schmidt, M. F. Mescher, S. C. Jameson. 2001. IL-12 enhances CD8 T cell homeostatic expansion. J. Immunol. 166: 5515-5521. [Abstract/Free Full Text]
  20. Shrikant, P., M. F. Mescher. 1999. Control of syngeneic tumor growth by activation of CD8+ T cells: efficacy is limited by migration away from the site and induction of nonresponsiveness. J. Immunol. 162: 2858-2866. [Abstract/Free Full Text]
  21. van Stipdonk, M. J., E. E. Lemmens, S. P. Schoenberger. 2001. Naive CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation. Nat. Immunol. 2: 423-429. [Medline]
  22. Chen, Z. M., M. K. Jenkins. 1999. Clonal expansion of antigen-specific CD4 T cells following infection with Salmonella typhimurium is similar in susceptible (Itys) and resistant (Ityr) BALB/c mice. Infect. Immun. 67: 2025-2029. [Abstract/Free Full Text]
  23. Curtsinger, J. M., D. C. Lins, M. F. Mescher. 2003. Signal 3 determines tolerance versus full activation of naive CD8 T cells: dissociating proliferation and development of effector function. J. Exp. Med. 197: 1141-1151. [Abstract/Free Full Text]
  24. Trinchieri, G.. 2003. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat. Rev. Immunol. 3: 133-146. [Medline]
  25. Curtsinger, J. M., C. M. Johnson, M. F. Mescher. 2003. CD8 T cell clonal expansion and development of effector function require prolonged exposure to antigen, costimulation, and signal 3 cytokine. J. Immunol. 171: 5165-5171. [Abstract/Free Full Text]
  26. Kaech, S. M., R. Ahmed. 2003. Immunology: CD8 T cells remember with a little help. Science 300: 263-265. [Abstract/Free Full Text]
  27. Bevan, M. J., P. J. Fink. 2001. The CD8 response on autopilot. Nat. Immunol. 2: 381-382. [Medline]
  28. Kaech, S. M., R. Ahmed. 2001. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nat. Immunol. 2: 415-422. [Medline]
  29. Marzo, A. L., K. D. Klonowski, A. Le Bon, P. Borrow, D. F. Tough, L. Lefrancois. 2005. Initial T cell frequency dictates memory CD8+ T cell lineage commitment. Nat. Immunol. 6: 793-799. [Medline]
  30. Harris, M. H., C. B. Thompson. 2000. The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability. Cell Death Differ. 7: 1182-1191. [Medline]
  31. Strasser, A.. 2005. The role of BH3-only proteins in the immune system. Nat. Rev. Immunol. 5: 189-200. [Medline]
  32. Tan, J. T., B. Ernst, W. C. Kieper, E. LeRoy, J. Sprent, C. D. Surh. 2002. Interleukin (IL)-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8+ cells but are not required for memory phenotype CD4+ cells. J. Exp. Med. 195: 1523-1532. [Abstract/Free Full Text]
  33. Schluns, K. S., L. Lefrancois. 2003. Cytokine control of memory T-cell development and survival. Nat. Rev. Immunol. 3: 269-279. [Medline]
  34. Valenzuela, J. O., C. D. Hammerbeck, M. F. Mescher. 2005. Cutting edge: Bcl-3 up-regulation by signal 3 cytokine (IL-12) prolongs survival of antigen-activated CD8 T cells. J. Immunol. 174: 600-604. [Abstract/Free Full Text]
  35. Chang, J., J. H. Cho, S. W. Lee, S. Y. Choi, S. J. Ha, Y. C. Sung. 2004. IL-12 priming during in vitro antigenic stimulation changes properties of CD8 T cells and increases generation of effector and memory cells. J. Immunol. 172: 2818-2826. [Abstract/Free Full Text]
  36. Kuo, C. T., M. L. Veselits, J. M. Leiden. 1997. LKLF: A transcriptional regulator of single-positive T cell quiescence and survival. Science 277: 1986-1990. [Abstract/Free Full Text]
  37. Endrizzi, B. T., S. C. Jameson. 2003. Differential role for IL-7 in inducing lung Kruppel-like factor (Kruppel-like factor 2) expression by naive versus activated T cells. Int. Immunol. 15: 1341-1348. [Abstract/Free Full Text]
  38. Shedlock, D. J., H. Shen. 2003. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300: 337-339. [Abstract/Free Full Text]
  39. Janssen, E. M., E. E. Lemmens, T. Wolfe, U. Christen, M. G. von Herrath, S. P. Schoenberger. 2003. CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature 421: 852-856. [Medline]
  40. Schoenberger, S. P., R. E. Toes, E. I. van der Voort, R. Offringa, C. J. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393: 480-483. [Medline]
  41. Alpan, O., E. Bachelder, E. Isil, H. Arnheiter, P. Matzinger. 2004. "Educated" dendritic cells act as messengers from memory to naive T helper cells. Nat. Immunol. 5: 615-622. [Medline]
  42. Cooper, D., P. Bansal-Pakala, M. Croft. 2002. 4-1BB (CD137) controls the clonal expansion and survival of CD8 T cells in vivo but does not contribute to the development of cytotoxicity. Eur. J. Immunol. 32: 521-529. [Medline]
  43. Janssen, E. M., N. M. Droin, E. E. Lemmens, M. J. Pinkoski, S. J. Bensinger, B. D. Ehst, T. S. Griffith, D. R. Green, S. P. Schoenberger. 2005. CD4+ T-cell help controls CD8+ T-cell memory via TRAIL-mediated activation-induced cell death. Nature 434: 88-93. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
C. E. Ruby, R. Montler, R. Zheng, S. Shu, and A. D. Weinberg
IL-12 Is Required for Anti-OX40-Mediated CD4 T Cell Survival
J. Immunol., February 15, 2008; 180(4): 2140 - 2148.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, Q.
Right arrow Articles by Shrikant, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, Q.
Right arrow Articles by Shrikant, P. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS