Published online before print June 14, 2007, doi: 10.1161/CIRCRESAHA.106.147124
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© 2007 American Heart Association, Inc.
Molecular Medicine |
CTLA-4 Ablation and Interleukin-12–Driven Differentiation Synergistically Augment Cardiac Pathogenicity of Cytotoxic T Lymphocytes
From the Vascular Research Division (V.A.L, N.G., P.D., G.S., A.L.), Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School; and Department of Pathology (A.S.), Harvard Medical School, Boston, Mass.
Correspondence to Andrew H. Lichtman, MD, PhD, Department of Pathology, Brigham and Women’s Hospital, 77 Ave Louis Pasteur, NRB 752N, Boston, MA 02115. E-mail alichtman{at}rics.bwh.harvard.edu
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Abstract |
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CD8+ cytotoxic T lymphocytes contribute to viral and autoimmune myocarditis and cardiac allograft rejection. The role of cytotoxic T-lymphocyte–associated antigen (CTLA)-4 as a negative regulator of CD4+ T cells is well defined, yet CTLA-4 regulation of CD8+ T cells is less clear. We studied CTLA-4 regulation of cytotoxic T lymphocytes in a transgenic model of CD8+ T-cell–mediated myocarditis. We generated CTLA-4–/– Rag 2–/– OT-1 mice, the CD8+ T cells of which express an ovalbumin (OVA) peptide-specific, class I major histocompatibility complex–restricted T-cell receptor. CTLA-4–/–Tc12 OT-1 effectors, differentiated with interleukin-12 present, are hyperproliferative in vitro, compared with CTLA-4+/+Tc12 OT-1 controls. Transfer of low doses of CTLA-4–/–Tc12 OT-1 cells to cMy-mOVA mice, which express OVA on cardiac myocytes, causes severe myocarditis, with 99% mortality, compared with no mortality after transfer of low doses of CTLA-4+/+Tc12 OT-1 cells. High doses of CTLA-4+/+Tc12 cells cause lethal myocarditis in cMy-mOVA mice, but high doses of CTLA-4+/+Tc0 CTL, generated without interleukin-12, are hypoproliferative within the cardiac-draining lymph node and do not significantly infiltrate the heart. In contrast, CTLA-4–/–Tc0 cytotoxic T lymphocytes do proliferate in the cardiac-draining lymph node and diffusely infiltrate the heart. Nonetheless, high doses of CTLA-4–/–Tc0 cells cause only limited tissue damage, and the disease is not lethal. These data show that CTLA-4 regulates myocarditic CD8+ T cell responses and that CTLA-4 deficiency partly overcomes a differentiation block that exists when naïve CD8+ T cells are stimulated without interleukin-12. Therefore, targeting CTLA-4 solely or in conjunction with interleukin-12 could influence effector CD8+ T cell responses in therapeutically beneficial ways.
Key Words: rodent • cytotoxic T lymphocytes • CTLA-4 • myocarditis
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Introduction |
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The regulation of CD8+ T-cell activation and differentiation by members of the B7 and CD28 regulatory molecules remain incompletely understood. Cytotoxic T-lymphocyte antigen (CTLA)-4 is a CD28 family member that is expressed on the surface of lymphocytes after T-cell receptor (TCR) stimulation.1 CTLA-4 and CD28 share specificity for the B7 molecules CD80 (B7-1) and CD86 (B7-2). It is well documented that CD80 and CD86 binding to CTLA-4 on CD4+ T cells results in downregulation of cell-cycle progression and interleukin (IL)-2 production.2,3 In contrast, there are conflicting studies regarding the effects of CTLA-4 ligation on CD8+ T-cell regulation.4,5 Several reports indicated that CTLA-4 blockade does not alter cytotoxic T lymphocyte (CTL) responses to lymphocytic choriomeningitis virus, mouse mammary tumor virus, or Leishmania major infection in mice.4,6,7 Yet experiments with CTLA-4–deficient class I major histocompatibility complex–restricted TCR transgenic mice indicate that CTLs are hyperresponsive to antigen on restimulation, suggesting a negative regulatory role in CTL activation.4,5,8 Furthermore, blockade of CTLA-4 with neutralizing antibody augments CTL-mediated responses against tumors in mice and humans and heightens cardiac allograft rejection.9–12
CD8+ CTLs contribute to the pathogenesis of several myocardial diseases, including Coxsackie B3–induced myocarditis,13 autoimmune myocarditis,14 and cardiac allograft rejection.11 We explored whether CTLA-4 influences CTL responses, using cMy-mOVA mice that express ovalbumin (OVA) exclusively in cardiac myocytes and develop a robust myocarditis after receiving adoptive transfer of OVA peptide-specific OT-1 CD8+ T cells.15,16
We reported previously that in vitro OT-1 effector CTLs generated in the presence of IL-12 (OT-1Tc12) cause lethal myocarditis in cMy-mOVA mice, if transferred in sufficient numbers. However, CTLs generated in the absence of added IL-12 (OT-1Tc0) are nonpathogenic in cMy-mOVA mice. We found that OT-1Tc0 cells fail to divide in vivo, do not egress from a cardiac-draining lymph node (CDLN), and have reduced cytotoxic activity.15
Because evidence supports a role for CTLA-4 in the negative regulation of CTLs against tissue-derived antigens, we hypothesized that abrogating CTLA-4 signals would enhance myocarditis and overcome the requirement for IL-12. To test this hypothesis, we compared the phenotypes of the CTLA-4–deficient and wild-type OT-1 cells and their responses in cMy-mOVA mice. Our results indicate that CTLA-4 significantly limits CTL proliferation and pathogenicity in the cMy-mOVA model, but CTLA-4 ablation does not overcome the limited pathogenicity of CTLs generated without IL-12.
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Materials and Methods |
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Mice and Cell Lines
C57BL/6 and C57BL/6 Rag2 knockout mice were purchased from Charles River Laboratories (Wilmington, MA) and Taconic (Hudson, NY), respectively. The previously described OT-1 TCR transgenic mice were kindly provided by W. R. Heath and F. Carbone (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia).17 Thy 1.1+ OT-1 mice lacking both CTLA-418 and Rag2 were generated by interbreeding relevant transgenic and knockout parental strains. C57BL/6 cMy-mOVA were previously described.15 Mice were bred in a pathogen-free facility at the Harvard Medical School in accordance with the Committee of Animal Research at the Harvard Medical School and the NIH animal research guidelines (see the online data supplement at http://circres. ahajournals.org).
Naïve OT-1 Isolation and In Vitro Generation of OT-1 Effector Cells
Isolation of naïve OT-1 cells from 8- to 16-week-old CTLA-4–/– and CTLA-4+/+ OT-1 mice and generation of effector cells was performed as described previously.15,16,19 Antigen-stimulated T cells generated in the presence of 60 U/mL recombinant mouse IL-2 (R&D Systems, Minneapolis, Minn) and 10 ng/mL recombinant mouse IL-12 (R&D Systems), are referred to as CTLA-4+/+Tc12 or CTLA-4–/–Tc12 OT-1 cells. In some cultures, no IL-12 was added, to generate CTLA-4+/+Tc0 or CTLA-4–/–Tc0 OT-1 cells.
In Vitro Assays of T-Cell Proliferation and Cytokine Secretion Assays
To measure proliferation in primary and restimulation cultures, [3H]-thymidine incorporation assays were performed in microwell plates and incorporated radioactivity was assayed by liquid scintillation. Supernatants (100 µL) from restimulation cultures were collected for cytokine analyses. Interferon (IFN)-
– or IL-2–specific ELISAs, and IFN-– and tumor necrosis factor (TNF)-
–specific Cytokine Bead assays (BD Biosciences) were performed as described.15,20
Antibodies and Flow Cytometric Analyses
All fluorescein isothiocyanate–, phycoerythrin-, and CyChrome– conjugated antibodies were obtained from BD Pharmingen for fluorescence-activated cell sorting (FACS) analysis. Stained-cell preparations were analyzed using a FACScalibur instrument and CellQuest software (Becton Dickinson, San Jose, Calif) as described previously.15,16,19 For cytoplasmic staining of perforin and granzyme (GzB), effector OT-1 cells were incubated with Golgistop (Monensin) and Golgiplug (Brefeldin A) (BD Biosciences) for 6 hours. Surface staining using anti-CD8a (Ly-2) was performed before fixation with Cytofix (BD Biosciences), and intracellular staining was done in the presence Cytoperm (BD Bioscience). The following antibodies were used: anti–GzB–allophycocyanin (APC) (Caltag clone GB11) plus APC–mouse IgG1 (Caltag) or anti-perforin Ab (clone eBioOMAK-D; Ebiosciences) plus phycoerythrin–rat IgG2a, 
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T-Cell Cytotoxic Activity Assays
Four-hour [51Cr] sodium chromate release and live/dead assays (Molecular Probes Inc, Eugene, Ore) were performed as described previously.15,19
OT-1 Adoptive Transfer and Myocarditis Studies in cMy-mOVA Mice
OT-1 cells removed from primary stimulation cultures on day 5 were injected intraperitoneally into cMy-mOVA mice as described previously.5,16,19 Doses ranged from 0.5x105 to 5x106 OT-1 effector cells. Hematoxylin and eosin staining and immunohistochemistry of heart sections and serum troponin (Tn)I analyses were performed on samples taken from mice euthanized 5 days after adoptive transfer.15
In Vivo Cell Proliferation Analysis, Carboxyfluorescein Ester Dilution, and 5-Bromodeoxyuridine Labeling
Thy 1.1+ OT-1 effector cells were labeled with carboxyfluorescein ester (CFSE) as described.15,20 Subsequently, 5x106 cells were transferred by intraperitoneal injection to Thy 1.2+ cMy-mOVA mice. The CDLNs were harvested from recipients 2 days after transfer.15 Cells were stained with phycoerythrin anti–Thy 1.1 antibody and Thy 1.1/CFSE double-positive cells were quantified by FACS. For 5-bromodeoxyuridine (BrdUrd) experiments, 1x106 day 5 OT-1 cells were adoptively transferred to cMy-mOVA mice, and 24 hours later, mice were injected with 1.5 mg of BrdUrd solubilized in PBS. Sixteen hours after the labeling, mice were euthanized, cryostat sections of heart were prepared, and BrdUrd-positive cells were identified by immunohistochemistry, using the In-Situ detection Kit (BD-Pharmingen).
Real-Time PCR
Total RNA isolation, conditions for quantitative real-time PCR, and primers sequences have been described elsewhere.15,20
Statistical Analysis
Data are presented as mean±SD of multiple samples. Analyses were performed using the Mann–Whitney test for data that was not normally distributed, as determined by the Kruskal–Wallis test, and Students t test for normally distributed data. A value of P<0.05 was considered to be significant.
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Results |
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CTLA-4 Deficiency Enhances the Proliferative Response of Differentiated Effector but Not Naïve CD8+ T Cells In Vitro
Mice deficient in CTLA-4 die of a lymphoproliferative disorder.21,22 TCR transgenic mice develop the same disorder, albeit with delayed kinetics.23 Therefore, we generated OT-1 mice that also lack Rag2. For simplicity, we will refer to the Rag2–/– OT-1 cells as either CTLA-4–/– or CTLA-4+/+. Analysis of cell surface markers indicated that naïve and in vitro stimulated CTLs from both CTLA-4–/– and CTLA-4+/+ mice were phenotypically similar (see the online data supplement).
CTLA-4–/–Tc12 and CTLA-4+/+Tc12 cells proliferated identically during primary stimulation, as measured by [3H]-thymidine incorporation (Figure 1A). However, on restimulation, CTLA-4–/–Tc12 cells incorporated significantly more [3H]-thymidine compared with CTLA-4+/+Tc12 cells, 300 000 versus 91 000 counts per minute, respectively (Figure 1B). We measured IL-2 from antigen-restimulated OT-1 cells and found a trend toward higher IL-2 production in CTLA-4–/–Tc12 cultures compared with CTLA-4+/+Tc12 cultures (Figure 1C).
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CTLA-4 Deficiency Does Not Alter Cytotoxic or IFN- Secretory Functions of CD8+ T Cell In Vitro
Granule exocytosis- and contact-dependent killing of target cells is the major mechanism by which CTLs protect against infections and tumors and cause tissue damage in autoimmune disease and allograft rejection.24 We examined cytotoxic function of CTLA-4–/–Tc12 and CTLA-4+/+Tc12 OT-1 cells against SIINFEKL-pulsed EL4 target cells in vitro and found no significant difference between the 2 cell populations (Figure 2A and 2B).
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Activated CTLs also secrete inflammatory cytokines which help to promote clearance of intracellular pathogens.25 We measured IFN- secretion by restimulated OT-1 cells and found moderately higher concentrations of this cytokine in culture supernatants of CTLA-4–/–Tc12 cells compared with CTLA-4+/+Tc12 cells; 1150 pg/mL and 975 pg/mL, respectively (Figure 2C). Additionally, TNF- levels were similar, measuring 1420 and 1573 pg/mL for CTLA-4–/–Tc12 and CTLA-4+/+Tc12 cells, respectively (Figure 2D).
CTLA-4 Deficiency Markedly Enhances Cardiopathogenicity of CTL
To determine whether CTLA-4 influences CTL responses in vivo, we used the cMy-mOVA model of myocarditis.15 After adoptive transfer of 1x106 OT-1Tc12 effectors, cMy-mOVA animals do not survive longer than 6 days. We titrated the number of transferred cells and determined that 5x104 cells is a nonlethal dose (data not shown). Subsequently, we transferred 5x104 CTLA-4–/–Tc12 and CTLA-4+/+Tc12 effector cells into cMy-mOVA mice. Recipients did not survive longer than 10 days following low-dose adoptive transfer of CTLA-4–/–Tc12 cells. In contrast, mice receiving CTLA-4+/+Tc12 cells were healthy longer than 30 days (Figure 3A). To determine whether enhanced lethality of CTLA-4–deficient CTLs correlated with myocardial damage, we measured cardiac TnI levels in serum collected 5 days after adoptive transfers. TnI levels were almost 8 times higher in recipients of CTLA-4–/–Tc12 cells compared with recipients of CTLA-4+/+Tc12 cells (Figure 3B).
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Histological samples were evaluated following adoptive transfer. Inflammatory cell infiltration was much more extensive within the hearts of the mice receiving CTLA-4–/–Tc12 cells compared with CTLA-4+/+Tc12 cells (Figure 3C, top panels, and 3D); the mean scores were 3.11±0.23 versus 1.5±0.36, respectively. Because histological scoring does not distinguish between endogenous and transferred leukocyte infiltration, we performed immunohistochemistry to identify transferred Thy 1.1+ OT-1 cells in the hearts of the Thy 1.2+ recipient mice. Significantly more CTLA-4–/–Tc12 than CTLA-4+/+Tc12 cells were found in hearts (Figure 3B, bottom).
CTLA-4–/–Tc12 OT-1 Cells Are Hyperproliferative In Vivo
Because CTLA-4–/–Tc12 cells proliferate more vigorously than CTLA-4+/+Tc12 cells in vitro, we hypothesized that the enhanced pathogenicity of the CTLA-4–/–Tc12 cells in vivo was related to hyperproliferation. We therefore evaluated whether there were differences in the proliferative capacities of CTLA-4–/–Tc12 and CTLA-4+/+Tc12 cells after adoptive transfer by comparing CFSE dye dilution of Thy 1.1+ OT-1 cells. We previously described a peribronchial CDLN that drains the heart and is a site of OT-1 proliferation in cMy-mOVA mice.15,26 Adoptively transferred IL-12–stimulated OT-1 cells are found within the CDLN for approximately 72 hours posttransfer and thereafter are found within the hearts. In the present study, we found that the fraction of CFSE-labeled CTLA-4–/–Tc12 cells in the CDLN that had divided after 72 hours was 9.2-fold greater than the fraction of labeled CTLA-4+/+Tc12 cells that had divided (Figure 4A and 4B). To further characterize proliferative differences between CTLA-4–/–Tc12 and CTLA-4+/+Tc12 cells, we used a BrdUrd-labeling technique.27 Sixteen hours after T-cell transfer, we found 8 times more BrdUrd-positive cells in hearts of cMy-mOVA mice that received CTLA-4–/–Tc12 cells, compared with recipients of CTLA-4+/+Tc12 cells (Figure 4C and 4D). The stained cells have the morphology of lymphocytes, and our prior experience with this model indicates that the inflammatory infiltrate is largely composed of transferred OT-1 cells and endogenous neutrophils. Nonetheless, it possible that some of the BrdUrd staining reflects endogenous inflammatory cell proliferation.
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CTLA-4 Deficiency Partially Overcomes the Block in Differentiation of Myocarditic CD8+ T Cells in the Absence of IL-12
CTLA-4+/+Tc0 cells, generated by in vitro stimulation in the absence of IL-12, are unable to proliferate nor egress from the CDLN in cMy-mOVA mice.15 Furthermore, CTLA-4+/+Tc12 cells generated in the presence of IL-12 cause myocarditis in cMy-mOVA mice if sufficient numbers of CTLs are transferred (eg, 
0.5x106), but CTLA-4+/+Tc0 cells do not.15 Given these observations, and the hyperproliferative, pathogenic phenotype of CTLA-4–/–Tc12, we hypothesized that CTLA-4 deficiency may overcome the proliferative block and low pathogenicity seen in CTLA-4+/+Tc0 cells. In response to antigen stimulation in vitro, naïve CTLA-4–/– and CTLA-4+/+ OT-1 cells proliferated comparably to one another in the absence of IL-12 (data not shown), but after antigen restimulation, the CTLA-4–/–Tc0 effector cells showed a significant proliferative response, whereas CTLA-4+/+Tc0 did not (see Figure III in the online data supplement). We therefore compared the consequences of transferring CTLA-4–/–Tc0 and CTLA-4+/+Tc0 cells into cMy-mOVA mice. As predicted, heart sections from recipients of CTLA-4+/+Tc0 showed only a few small foci of inflammatory cells confined to the papillary muscles (Figure 5A, right). In contrast, heart sections from recipients receiving CTLA-4–/–Tc0 cells showed diffused inflammatory infiltration throughout the specimen (Figure 5A, left). Transfer of 1x106 CTLA-4+/+Tc12 cells also caused severe myocarditis (not shown). Myocarditis scores in sections from recipients injected with CTLA-4–/–Tc0, CTLA-4+/+Tc0, and CTLA-4+/+Tc12 cells were 3.33, 1.63, and 3.75, respectively (Figure 5B). Interestingly, high levels of TnI could be found within serum of recipients receiving adoptive transfer of CTLA-4–/–Tc0 cells, indicating severe myocardial damage. TnI levels in cMy-mOVA mice receiving, CTLA-4–/–Tc0, CTLA-4+/+Tc0, and CTLA-4+/+Tc12 were 89, 15, and 119 pg/mL, respectively (Figure 5C). Additionally, survival experiments to evaluate pathogenicity were performed by transferring 5x106 CTLA-4–/–Tc0 and CTLA-4+/+Tc0 cells to recipients. Although CTLA-4–/–Tc0 cells clearly migrated to the myocardium and induced inflammation, the disease was not fatal. Three separate survival experiments were conducted and all recipients were alive at 2 months (Figure 5D). These data indicate that CTLA-4 deficiency enhances the ability of Tc0 cells to infiltrate and cause tissue damage; however, CTLA-4–/–Tc0 cells are less pathogenic than CTLA-4–/–Tc12 cells. Induction of even a nonlethal form of disease requires 100 times more CTLA-4–/–Tc0 cells than CTLA-4–/–Tc12 cells.
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CTLA-4–Deficient Tc0 Cells Proliferate Within the Cardiac Lymph Node
Because CTLA-4 deficiency enhanced the proliferative capacity of Tc12 effector cells, we predicted that the enhanced infiltrative and pathogenic effects of CTLA-4–/–Tc0 cells might also be attributable to deregulated proliferation. Therefore, we compared in vivo proliferation of CFSE-labeled Thy 1.1+ CTLA-4–/–Tc0 and CTLA-4+/+Tc0 cells after transfer to Thy 1.2+ cMy-mOVA mice. As shown in Figure 6A, 62% of CTLA-4+/+Tc0 cells remained CFSEhi, but only 2% of CTLA-4–/–Tc0 cells remained CFSEhi, indicating that CTLA-4 deficiency resulted in a 30-fold increase in the number of proliferating cells within the CDLN (Figure 6B).
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The Absence of IL-12 During Differentiation Results in Persistent Block in Full Differentiation in Both CTLA-4–/– and CTLA-4+/+ OT-1 T Cells
Given the unexpected result that CTLA-4–/–Tc0 cells induce a less-severe myocarditis compared with CTLA-4+/+Tc12 cells, we reasoned that CTLA-4–deficient cells still require IL-12 for complete terminal differentiation. We therefore examined the ability of CTLA-4+/+ and CTLA-4–/– cells, each differentiated with and without IL-12, to kill SIINFEKL-pulsed target cells (Figure 7A). At the highest effector-to-target ratio, CTLA-4+/+Tc0 cells had less cytotoxic activity than CTLA-4+/+Tc12 cells, consistent with previous studies.15 As predicted, CTLA-4–/–Tc0 cells also had impaired cytotoxic activity compared with CTLA-4–/–Tc12 cells (Figure 7A). Therefore, the enhanced pathogenicity of CTLA-4–deficient OT-1 cells cannot be explained by enhanced cytotoxic activity, and development of a maximally cytotoxic phenotype requires IL-12, even in the absence of CTLA-4.
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We also evaluated GzB and perforin protein expression, using cytoplasmic staining and flow cytometry (Figure 7B). The results indicate that GzB is upregulated by IL-12 treatment, in both CTLA-4–deficient and control OT-1 cells. Furthermore, CTLA-4–deficient CTLs expressed slightly less GzB and equivalent amounts of perforin compared with control CTL, further supporting the conclusion that CTLA-4 deficiency does not result in enhanced cytotoxic function. We also found GzB mRNA levels were 1.9-fold lower in CTLA-4–/–Tc0 compared with CTLA-4–/–Tc12 cells and we found a 2.6-fold reduction of GzB mRNA in CTLA-4+/+Tc0 cells compared with CTLA-4+/+Tc12 cells.
Overall, our data show that CTLA-4–/–Tc0 cells are more differentiated than CTLA-4+/+Tc0 cells, as indicated by proliferative, migratory, and pathogenic phenotypes, but CTLA-4 deficiency does not compensate for the lack of signals provided by IL-12 in the differentiation of a cytotoxic phenotype.
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Discussion |
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We investigated the role of CTLA-4 in the regulation of effector CD8+ T-cell responses by comparing the functional phenotypes and cardiac pathogenicity of CTLA-4+/+ and CTLA-4–/– OT-1 cells activated the presence or absence of IL-12. Some studies examining CTL antiviral responses in mice found no evidence of enhanced CTL responses in CTLA-4–/– mice or in mice treated with anti–CTLA-4 blocking antibody,4,6,7 whereas other studies showed a role for CTLA-4 in the regulation of CTLs.9,11 The conflicting reports may reflect multiple experimental systems used to study the impact of CTLA-4 on CTL regulation. One way to reconcile these differences is to consider that CTLA-4 regulates CTL responses against tissue antigens, as shown here, but may not have a significant impact on CTL responses against viral or other microbial antigens.
Our data show synergistic effects of CTLA-4 ablation and IL-12 in CTL differentiation and function. This is consistent with mouse studies of low-dose oral tolerance28 and in anergy induction using systemic CTLA-4 monoclonal and IL-12 injections.29 Our data show that CTLA-4 regulates cellular proliferation, whereas IL-12 promotes CTL effector differentiation. In our model, CTLA-4+/+Tc0 cells are functionally anergic, consistent with other studies showing that IL-12 is required for CTL effector differentiation.30–32 However, CTLA-4 deficiency can overcome the proliferation block and partially overcome the differentiation block because CTLA-4–/–Tc0 cells proliferate in the CDLN and enter the myocardium. Nonetheless, CTLA-4–/–Tc0 cells are not fully differentiated, as indicated by their impaired cytotoxic function in vitro, and their inability to cause lethal myocarditis, even when 100-fold more of these cells are transferred than a lethal dose of CTLA-4–/–Tc12 cells. Our findings are consistent with studies showing that regulation of CD8+ T-cell proliferation and development of cytotoxic function are not obligatorily linked, and that a lack of IL-12 signals differentially impairs cytotoxic function.33
Our data are consistent with reports that both CD4+ and CD8+ T-cell proliferative responses are kept in check by CTLA-4, albeit at different stages of activation. Importantly, this study correlates the hyperproliferative CTL response caused by CTLA-4 ablation with disease in an animal model. CTLA-4–/– CD4+ T cells display a profound increase in production of IL-2. We also observed increases in secreted IL-2 from CTLA-4–/–Tc12 cells (Figure 1C) and CTLA-4–/–Tc0 cells (see supplemental Figure III) relative to control populations.
We found reduced GzB in both CTLA-4+/+Tc0 and CTLA-4–/–Tc0 cells, compared with CTLA-4+/+Tc12 and CTLA-4–/–Tc12 cells, which is a likely reason for their impaired cytotoxic function. We did not see differences in perforin expression. Previous work suggests that CTL killing is perforin dependent, but not GzB dependent, because CTLs from GzA/GzB double-knockout mice induced [51Cr] release in target cells at levels similar to wild-type mice.34 Recent work delineates a novel connection between IL-12 and GzB regulation, which is concordant with our results.35 It is possible that IL-12 influences expression of additional granzymes and other genes necessary for cell-mediated lysis and apoptosis induction.
The use of exogenous cytokines such as IL-2 and IL-12 as adjuvants in conjunction with vaccine regiments to control melanoma and other tumors has shown promise clinically.36,37 In addition, neutralizing anti–CTLA-4 antibodies have been used in mice and clinical trials with some positive results.10,38 Our data indicate that enhancement of CTL effector function may be more profound when CTLA-4 signals are blocked in the presence of IL-12. This finding may prove significant in consideration of new combinatorial therapies designed to enhance antitumor-specific responses. Alternatively, the effects of ligation of CTLA-4 in the absence of IL-12 may be instructive for the design of protocols to limit autoimmunity and graft rejection.
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Acknowledgments |
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We thank Dr Avi-Hai Hovav for critically reviewing the manuscript and Hong Pang for excellent technical assistance.
Sources of Funding
This work was supported by the following National Institutes of Health grants: AI059610 (VL, AHL) HL072056 (AHL), AI046414 (AS).
Disclosures
None.
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Footnotes |
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Original received December 20, 2006; revision received May 25, 2007; accepted May 31, 2007.
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References |
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