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The Journal of Immunology, 2004, 173: 4394-4401.
Copyright © 2004 by The American Association of Immunologists

A Nonclassical MHC Class I Molecule Restricts CTL-Mediated Rejection of a Syngeneic Melanoma Tumor1

Eugene Y. Chiang and Iwona Stroynowski2

Center for Immunology, Departments of Microbiology and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although CTL and polymorphic, classical MHC class I molecules have well defined roles in the immune response against tumors, little is currently known regarding the participation of nonpolymorphic, nonclassical MHC class I in antitumor immunity. Using an MHC class I-deficient melanoma as a model tumor, we demonstrate that Q9, a murine MHC class Ib molecule from the Qa-2 family, expressed on the surface of tumor cells, protects syngeneic hosts from melanoma outgrowth. Q9-mediated protective immunity is lost or greatly diminished in mice deficient in CTL, including {beta}2-microglobulin knockout (KO), CD8 KO, and SCID mice. In contrast, the Q9 antitumor effects are not detectably suppressed in CD4 KO mice with decreased Th cell activity. Killing by antitumor CTL in vitro is Q9 specific and can be blocked by anti-Q9 and anti-CD8 Abs. The adaptive Q9-restricted CTL response leads to immunological memory, because mice that resist the initial tumor challenge reject subsequent challenges with less immunogenic tumor variants and show expansion of CD8+ T cell populations with an activated/memory CD44high phenotype. Collectively, these studies demonstrate that a MHC class Ib molecule can serve as a restriction element for antitumor CTL and mediate protective immune responses in a syngeneic setting.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The highly polymorphic classical MHC class I Ags, or class Ia, have long been known to play an important role in CD8+ T cell-dependent responses against neoplastically transformed cells (1, 2, 3, 4). Their ubiquitous tissue distribution and peptide-presenting properties allow them to act as sentinels signaling tumor-specific alterations in a wide variety of transformed cells. Although enormous efforts have been dedicated to the study of MHC class Ia roles in tumor immunity, relatively little is known about a larger family of class I-related molecules, generically referred to as nonclassical, or class Ib, molecules.

Compared with the class Ia, which are usually encoded by one to three loci per haploid genome, the class Ib family comprises dozens of members in most mammals (5). Although it is difficult to provide an unambiguous and all-encompassing definition of class Ib products, many of them display the following properties that distinguish them from class Ia: low or moderate level of polymorphism, low level or tissue-specific expression, truncated cytoplasmic/transmembrane domains, and/or multiple, alternative splicing patterns (5, 6, 7, 8).

The Qa-2 Ags represent a unique subset of the murine class Ib Ags. The best-characterized member of the Qa-2 family, Q9, has striking similarities to the class Ia Ags (6, 7). The canonical, membrane-attached Q9 surface Ag is structurally homologous to class Ia, is ubiquitously expressed on somatic cells, and binds a large array on nonameric peptides with a unique motif containing histidine at position 7 (9, 10, 11, 12). An intriguing observation was made during the analysis of the Q9 expression pattern. Although primary cell lines derived from tissues of Q9-positive mouse strains were invariably positive for Q9 expression, established tumor lines from these strains were either negative for Q9 or severely Q9-deficient (10). In addition, analysis of tumor variants derived from B16 primary melanoma suggested that silencing of Q9 occurred early during this tumor’s development and preceded emergence of tumor escape variants with defects in class I Ag presentation pathway and class Ia expression (13). One interpretation of this observation was that Q9 was selectively shut down in the in vivo selected tumors because the presence of Q9 marked the newly transformed cells for immune destruction. This hypothesis parallels precedence established by the large number of studies reporting alteration of MHC class Ia expression in diverse tumors, especially in later stage/metastatic lesions with dedifferentiated (fetal-oncogenic) phenotypes (1, 2).

The best-characterized MHC class I-dependent anticancer pathway involves presentation of tumor Ags to conventional CD8+ CTL. Tumor Ags are, in most cases, derived from aberrantly up-regulated self proteins or mutant polypeptides (14, 15, 16), which are processed into peptides and loaded onto class I molecules via the proteasome and TAP components of the Ag processing pathway (17). Despite the conserved nature of many known self-tumor Ags and immune cross-protection observed between independently isolated tumors (14, 16), the nonpolymorphic class Ib have not yet been implicated as restricting elements for the conventional antitumor CTL. We hypothesize that this may be due to the fact that some key class Ib, such as Q9, are silenced on virtually all established tumors selected in vivo and their functions have never before been directly tested in an experimental system.

We have recently reported that restoration of TAP2-dependent Q9 expression in the MHC class I-deficient B78H1 model melanoma resulted in rejection of the Q9-positive tumor cells in C57BL/6 syngeneic animals (13). The Q9-mediated tumor protection was stronger than the antitumor effect induced by the class Ia H2-Kb molecule in the same melanoma model. Furthermore, in vivo Ab depletions indicated that one of the components of the immune response against Q9-positive melanoma was likely to involve CD8+ T cells (13). In this study we present evidence that the display of peptide-loaded Q9 on the surface of melanoma cells leads to an antitumor CTL response that confers protection against tumor outgrowth and induces strong immunological memory. To the best of our knowledge, this is the first report showing that a nonpolymorphic MHC class Ib molecule may function as a restriction element for antitumor CTL in a syngeneic host.


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

C57BL/6 (B6) mice were maintained in the microbiology animal colony at University of Texas Southwestern Medical Center (Dallas, TX). SCID (B6.CB17-Prkdcscid/SzJ), Beige (C57BL/6J-Lystbg-J/+), CD8 knockout (KO)3 (B6.129S2-Cd8atm1Mak), {beta}-microglobulin ({beta}2m) KO (B6.129P2-B2mtm1Unc), and CD4 KO (B6.129S2-Cd4tm1Mak) mice (purchased from The Jackson Laboratory, Bar Harbor, ME) and SCID Beige mice (purchased from Charles River Laboratories, Wilmington, MA) were housed under specific pathogen-free conditions in the Animal Resource Center animal colony at University of Texas Southwestern Medical Center. Adult mice, 8–10 wk old, were used for all experiments. All experiments were performed according to institutional review board guidelines.

Cell lines and transfectants

The B16 melanoma-derived B78H1 variant was provided by Dr. H. I. Levitsky (The Johns Hopkins School of Medicine, Baltimore, MD). GM-CSF-transduced B78H1 cells were provided by Dr. S. Ostrand-Rosenberg (University of Maryland Baltimore County, Baltimore, MD). B78H1 transfectants expressing cell surface Q9 alone (clone Q9.A7, designated in this study as B78H1Q9), Q9 in conjunction with TAP2 (clone Q9TAP.11, designated in this study as B78H1Q9TAP) and empty vector (B78H1 vector), GM-CSF-transduced B78H1 expressing both Q9 and TAP2 (clone GMQ9TAP.13, designated in this study as GMQ9TAP) and TAP2 alone (GMTAP), as well as Kb/TAP2-transfected GM-CSF-transduced B78H1 clone MJ1-5.7 (designated in this study as GMKbTAP) have been previously described (13, 18). All cell lines were maintained in 50% DMEM/50% RPMI 1640 supplemented with 10% FBS (Atlanta Biologicals, Norcross, GA), 10 U/ml penicillin/10 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO), 1 mM sodium pyruvate (Invitrogen Life Technologies, Gaithersburg, MD), 0.1 mM nonessential amino acids (Invitrogen Life Technologies), and 400 µg/ml G418 (Invitrogen Life Technologies). All cell lines were grown at 37°C and 5% CO2. To maintain Mycoplasma-free conditions, all cell lines were periodically cultured in medium supplemented with 10 µg/ml CellGro ciprofloxacin HCl (Mediatech, Herndon, VA).

Antibodies

The following Abs were purchased from BD Pharmingen (San Diego, CA): anti-Qa-2 mAb 1-1-2, FITC-anti-CD4 mAb H129.19, PE-anti-CD44 mAb Pgp-1, PerCP-anti-CD8a mAb 53-6.7, allophycocyanin-anti-CD3e mAb 145-2C11, Fc block anti-mouse CD16/CD32 mAb 2.4G2, and appropriately labeled isotype control Abs. FITC-goat anti-mouse IgG (Cappell Laboratories, Durham, NC) was used as secondary Ab for Qa-2 staining. Anti-CD8a mAb 53-6.7 (BD Pharmingen) and anti-CD4 mAb GK1.5 (BD Pharmingen) were used for in vitro blocking assays. For blocking of class I MHC molecules, anti-Qa-2 mAb 20-8-4 (19), anti-Qa-2 mAb M46 (20), and anti-Kb mAb Y3 (21), purified from hybridomas or ascites as previously described (11), were used. Anti-asialo-GM1 used for in vivo depletion of NK cells was purchased from WAKO (Richmond, VA).

Flow cytometry

Up to 1 x 106 cells were washed in staining buffer (PBS with 1% FBS and 0.1% sodium azide) and pelleted. For cells harvested from spleens and lymph nodes, blocking for FcRs was performed by incubating cells with 1 µg anti-CD16/CD32 mAb in 50 µl staining buffer for 15 min at 4°C, followed by washing. A saturating amount of primary Ab(s) was added in a volume of 100 µl, vortexed, and incubated for 30 min at 4°C. Excess unbound Ab was removed by washing with staining buffer. A saturating dilution of secondary Ab was added if required and incubated at 4°C for 20 min. Cells were washed twice, resuspended in 300 µl staining buffer, and filtered through 35-µm pore size nylon mesh. A total of 1 x 104 events gated on live cells were collected on a FACScan or FACSCalibur (BD Biosciences, Palo Alto, CA). Fluorescence compensation was performed when the samples were analyzed by multicolor flow cytometric analysis. Data were analyzed using CellQuest version 3.1f software (BD Biosciences).

Tumor experiments

Tumor challenge experiments were performed as previously described (13). Briefly, cells for injection were harvested from in vitro culture and washed three times in serum-free HBSS. Unless otherwise indicated, 1 x 105 live tumor cells were injected s.c. in a volume of 200 µl of HBSS into the right rear flank. Mice were monitored daily. Mice were considered tumor bearing when the tumor was palpable and measured at least 3 x 3 mm. Caliper measurements across two axes of the tumor were recorded every other day to determine tumor growth rate. Animals were killed when the tumor burden showed physical signs of being excessive to avoid pain and suffering. For rechallenge experiments, mice that had completely rejected initial challenge with GMQ9TAP (tumor-free after day 100) were injected in the opposite flank with 1 x 105 live tumor cells. The number of mice used in each experiment is indicated in the corresponding figure legend. Data are represented as Kaplan-Meier plots. To compare growth rates, statistical analysis was performed using Student’s t test. NK cell-depleted mice were generated by i.p. injection of anti-asialo-GM1 (0.2 ml of a 1/20 dilution) 2 days before, 1 day before, and the day of tumor inoculation, followed by twice weekly injections, as previously described (13).

Cytotoxicity assays

Syngeneic tumor-reactive CTL were generated as follows: B6 mice were given an initial challenge of 1 x 105 live tumor cells injected s.c. in a volume of 200 µl into the right rear flank. Mice that were tumor-free after 100 days were then rechallenged with the same tumor load in the same manner, followed by at least two boosting immunizations with live tumor cells given 2–4 wk apart. Splenocytes were harvested and cultured in 24-well plates at a concentration of 5 x 106 cells/well with stimulator cells. Stimulators were generated by preparing GMQ9TAP cells at a concentration of 5 x 107 cells/ml in PBS and incubation at 37°C for 1 h in the presence of 50 µg/ml mitomycin C. Stimulators were washed three times with culture medium, then plated at 2 x 106 cells/ml/well. Cultures were maintained in complete DMEM (DMEM supplemented with 10% FBS, 50 µM 2-ME (Sigma-Aldrich), 0.1 mM nonessential amino acids, 50-fold diluted essential amino acids (Invitrogen Life Technologies), 1 mM sodium pyruvate, 10 mM HEPES (Invitrogen Life Technologies), and 10 U/ml penicillin/10 µg/ml streptomycin) supplemented with 10 U/ml IL-2 (provided by Dr. M. Bennett, University of Texas Southwestern Medical Center) and incubated in a humidified incubator at 37°C in 10% CO2 for 5–7 days. For CTL assays, effectors were collected, and cell viability was determined using a standard trypan blue exclusion technique. Three-fold serial dilutions of effector cells were made in round-bottom, 96-well plates, resulting in a final volume of 100 µl. For in vitro Ab blocking experiments, effectors were incubated for 30 min with 1 µg/well anti-CD8{alpha} mAb 53-6.7 or anti-CD4 mAb GK1.5. Target cells were labeled by incubating 2 x 106 cells in a volume of 200 µl with 150–200 µCi of Na51CrO4 for 1 h at 37°C and 5% CO2. For in vitro Ab blocking of class I MHC on target cells, target cells were incubated with saturating amounts of Qa-2- or Kb-reactive mAb during Na51CrO4 labeling. Labeled targets were washed three times with medium. 51Cr-labeled target cells (1000–5000) in 100 µl of medium were added to each well, and the plates were incubated at 37°C and 5% CO2. After incubation for 4 h, 100 µl of the supernatant was removed from each well and transferred to Skatron macrowell tubes (Molecular Devices, Sunnyvale, CA). Radioactivity was counted in a Micromedic gamma counter. Data are expressed as the percent specific release, calculated as follows: ((experimental release – spontaneous release)/(maximum release – spontaneous release)) x 100. Maximum release was determined by incubating target cells with 100 µl of 1% SDS.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Q9-mediated antitumor effects are detectable over a wide range of melanoma doses

We have previously reported that Q9 expressed in class I MHC-negative, TAP-proficient and GM-CSF-secreting B78H1 melanoma cells conferred significant protection (>50% survival compared with 0% survival of Q9-negative controls) against tumor outgrowth in syngeneic B6 mice (13). The protective effect was observed with independently derived Q9 transfectants and was studied in mice injected s.c. with a set dose of 1 x 105 live melanoma cells. To examine whether Q9-induced rejection is dependent on tumor burden, endogenous GM-CSF synthesis, and/or TAP-mediated peptide loading of Q9, we performed titration experiments with a series of B78H1 derivatives constructed and characterized in detail in our previous studies (13, 18). They included TAP-negative B78H1 cells transfected with empty vector (B78H1 vector) or Q9 cDNA driven by CMV promoter (B78H1Q9) as well as TAP-positive, Q9-positive cells (B78H1Q9TAP). In addition, GM-CSF-transduced, TAP- and Q9-positive (GMQ9TAP) and TAP-positive, but Q9-negative (GMTAP), variants of the melanoma cells were used.

Age-matched groups of B6 mice were injected s.c. in the hind flank with titrated doses of melanoma derivatives. The onset of tumor growth was recorded as the day a palpable solid tumor mass, defined by caliper measurements of 3 x 3 mm, was detected at the site of injection. The results diagrammed in Fig. 1 lead to several conclusions. First, they establish that the minimal lethal dose, defined as the tumor burden of Q9-negative cells (B78H1 vector and GMTAP) resulting in 100% of mice developing tumors, corresponds, under our experimental conditions, to a dose of 1 x 104 cells. Second, they demonstrate that at this dose, the Q9-positive derivative B78H1Q9TAP is significantly protective, because only 20% of mice succumb to tumors. In contrast, TAP-negative derivative B78H1Q9, which expresses ~10-fold reduced levels of surface Q9 compared with B78H1Q9TAP (18) (data not shown), grows out in 100% of mice and recapitulates the phenotype of B78H1 vector control. This outcome indicates that the TAP pathway responsible for peptide loading of Q9 (22) is essential for antitumor effects of the transfected Q9. Finally, titrations of Q9-positive and -negative variants secreting GM-CSF reveal that the presence of this cytokine in the tumor milieu amplifies Q9-mediated rejection of melanoma and allows detection of the protective effect at a dose (1 x 105 cells) that is 10-fold higher than the minimal lethal dose. In subsequent experiments we used this high dose of live GM-CSF transduced melanoma variants for tumor take analyses as well as the less immunogenic B78H1TAP variants for studies of immunological memory.



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  		FIGURE 1. B78H1 melanoma cells expressing Q9 are rejected by syngeneic mice. B6 mice (five mice per group) were challenged s.c. with live B78H1 derivatives at various doses, as indicated. Class I-deficient lines, B78H1 vector and GMTAP, were used as controls for tumor take in syngeneic mice. The Q9-positive lines used were wild-type B78H1 transfected with Q9 (B78H1Q9), B78H1 cotransfected with both Q9 and TAP2 (B78H1Q9TAP), and GM-CSF-transduced B78H1 transfected with both Q9 and TAP2 (GMQ9TAP). Cell surface expression levels of B78H1Q9TAP and GMQ9TAP, as determined by Ab staining with Q9-specific mAb and flow cytometric analysis, were comparable, with surface Q9 expression on B78H1Q9 being ~10-fold lower (18 ) (data not shown). Tumor growth was monitored for >100 days, and the percentage of mice with palpable tumor was recorded as a Kaplan-Meier plot (data shown to day 50 for B78H1 vector and B78H1Q9 to clarify differences).

 
CD8+ T cells play a key role in Q9-mediated tumor rejection

To study in vivo effector mechanisms during the Q9-mediated tumor rejection, we initially infused mice with depleting Abs against CD8 molecules on CTL and against asialo-GM1 on NK cells (13). These initial depletion experiments revealed that both types of effectors collaborate in the rejection of the model melanoma. Because the parental Q9-negative B78H1 as well as Q9-positive variants are sensitive to NK cells (18) and because there is no indication that Q9 is recognized as an activation signal for innate immune effectors (18), we hypothesize that NK cells reduce the initial tumor burden of Q9-negative as well as Q9-positive melanoma in a Q9-independent fashion. This interpretation is consistent with the data in Fig. 1, which show that a threshold number of GMTAP cells (>1 x 103) is needed to observe outgrowth of this inoculum. Thus, a smaller tumor burden (≤1 x 103 cells) appears to be successfully eliminated by host antitumor, MHC-independent pathways.

As mentioned, rejection of the high doses of melanoma tumor is Q9-dependent and CD8+ cell-dependent. The structural similarities between Q9 and class Ia (9, 11) suggest that involvement of Q9 in tumor rejection could be rationalized by Q9-restricted antitumor CTL responses. In this study we tested this proposition.

Tumor challenge experiments were performed in B6 mouse strains deficient in various lymphocyte functions: SCID mice lacking functional T, B, and NKT cells due to defects in DNA repair and rearrangement of genes coding for Ag-specific receptors (23); Beige mice with a severe deficiency in NK cell and CTL killing functions due to defective granule formation (24); CTL-deficient mice carrying disruptions of CD8 gene (CD8 KO) (25) or {beta}2m ({beta}2m KO) (26); and CD4+ T cell-deficient mice carrying disrupted CD4 sequences (CD4 KO) (27). As a control, we monitored the outgrowth of Q9-positive and -negative melanoma derivatives in SCID Beige combined immunodeficient mice that lack lytic functions of most the effectors of adaptive as well as innate immunity. Tumor take experiments in SCID Beige animals confirmed that the observed growth differences between various tumors are attributable to their interactions with immune effectors and not to other nonimmune factors. The results shown in Fig. 2 demonstrate that the kinetics of GMQ9TAP growth parallels those of GMTAP in all immunodeficient mice except CD4 KO mice, where the protective effect of Q9 is not diminished and thus appears to be less sensitive to the immune abnormalities of this strain (28). The results point to the conclusion that CD8+ T cells, which are deficient or defective in CD8 KO, {beta}2m KO, SCID, and Beige mice, are critically involved in Q9-mediated rejection of melanoma cells.



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  		FIGURE 2. Rejection of Q9-expressing B78H1 melanoma is dependent on CD8+ T cells. CD8+ T cell-deficient SCID Beige, SCID, Beige, CD8{alpha} KO, and {beta}2m KO mice as well as CD4+ T cell-deficient CD4 KO mice were given an s.c. injection of 1 x 105 live GMTAP ({circ}) or GMQ9TAP ({blacksquare}) B78H1 derivative cells. Five mice were used for each group, except for SCID Beige mice, where four mice were used in each group. Tumor take was recorded as a Kaplan-Meier plot.

 
Q9-restricted antitumor CTL are raised in mice challenged with Q9-expressing B78H1 melanoma

Because tumor challenge experiments in CD8+ T cell-deficient hosts implicated CTL as vital components of the protective response, we assessed whether Q9-restricted antitumor CTL were generated in immunized mice. To determine whether the putative antitumor CTL recognize and kill tumor targets in the context of Q9 Ag, in vitro CTL cytotoxicity assays were performed (Fig. 3A). Splenocytes from mice surviving the initial GMQ9TAP tumor challenge (>100 days postchallenge) were restimulated in vitro with mitomycin C-treated GMQ9TAP cells. After 5–7 days in culture, cells were harvested and tested for their ability to lyse Q9-positive (GMQ9TAP and B78H1Q9TAP) and Q9-negative (GMTAP) B78H1 targets. Significant killing of melanoma targets expressing Q9 was detectable, whereas no killing of Q9-negative GMTAP or H2-Kb-positive GMKbTAP cells (13) was observed, demonstrating that recognition was Q9-specific.



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  		FIGURE 3. Generation of CTL from mice challenged with Q9-positive B78H1 melanoma. A, Lytic effector cells are Q9-restricted. Syngeneic mice that were tumor-free 100 days after the initial challenge with live GMQ9TAP cells were boosted with at least two subsequent rounds of immunization. Splenocytes were harvested and restimulated in vitro with mitomycin C-treated GMQ9TAP cells in the presence of IL-2 to yield effector cells used in 51Cr release assays. Q9-positive targets were GMQ9TAP and B78H1Q9TAP; Q9-negative targets were GMTAP and GMKbTAP. Error bars for data points (<10% of the values shown) are not shown for clarity. The data shown are representative of six experiments. B, Cytotoxic effectors are generated against Q9-expressing B78H1 regardless of GM-CSF production. In vitro restimulation was performed using mitomycin C-treated B78H1Q9TAP cells. Results are representative of four experiments. C, Recognition of target-expressed Q9 Ags by effector cells is required for lysis. GMQ9TAP target cells were preincubated with anti-Q9 mAbs 20-8-4 or M46, irrelevant class I MHC Kb-specific mAb Y3, or without blocking Ab before mixing with effector cells. Data were collected at an E:T cell ratio of 200:1 from four experiments and are shown as the mean ± SD. Similar results were seen at a 67:1 E:T cell ratio (not shown). D, Killing of Q9-expressing B78H1 targets can be blocked with anti-CD8 mAb. Effector cells were preincubated with anti-CD8 mAb or anti-CD4 mAb or were not preincubated with blocking Ab before mixing with GMTAP or GMQ9TAP targets. Data were collected at an E:T cell ratio of 200:1 from six experiments and are shown as the mean ± SD. Similar results were seen at a 67:1 E:T cell ratio (not shown).

 
Similar results were seen when B78H1Q9TAP cells were used in the in vitro re-stimulation (Fig. 3B). Significantly, the CTL generated in response to in vivo GMQ9TAP challenge and subsequent in vitro restimulation with B78H1Q9TAP cells killed B78H1Q9TAP and GMQ9TAP targets to a similar degree, indicating that endogenous GM-CSF production and/or secretion by stimulator and target cells does not shape CTL killing patterns. Thus, it is unlikely that Q9-restricted CTL recognition is dependent on peptides derived from GM-CSF or GM-CSF-induced proteins presented in the context of Q9, or that GM-CSF secreted by target cells has a pleiotropic effect on CTL. Instead, we favor the interpretation that antitumor Q9-restricted CTL recognize melanoma-derived Ag(s) in the context of Q9.

To document that in vitro killing by CTL involves recognition of Q9 on tumor target cells, we performed Ab blocking experiments with anti-Q9 (Fig. 3C) and anti-CD8 (Fig. 3D) serological reagents. Blocking of Q9 with two different anti-Q9 mAbs, 20-8-4 and M46, effectively rendered CTL unable to kill GMQ9TAP. In contrast, blocking of GMQ9TAP targets with the control mAb Y3, specific for the class Ia MHC molecule Kb, had no effect on CTL recognition of these targets. To confirm that CD8+ CTL are responsible for the cytolytic activity observed in the in vitro killing assays, the effector cells were preincubated with anti-CD8 mAb 53-6.7 and, as a negative control, with anti-CD4 mAb GK1.5. The lytic activity against GMQ9TAP targets was abolished in the presence of anti-CD8 mAb, but not in the presence of anti-CD4 mAb, providing support for the idea that Q9-expressing targets are killed by CTL in the in vitro assay.

Protective immunity generated against Q9-positive melanoma results in immunological memory

To validate the finding that rejection of Q9-positive melanoma occurs via an adaptive and Q9-specific immune response, we performed a series of experiments summarized in Fig. 4. First, naive B6 mice received simultaneous injections of 1 x 105 GMQ9TAP cells in the right hind flank and an equal burden of either B78H1Q9TAP or GMTAP in the opposite flank. This approach was designed to determine whether the immune response against GMQ9TAP under our experimental conditions would also protect against the less immunogenic Q9-positive B78H1Q9TAP or the control Q9-negative GMTAP tumors. The control GMTAP cells injected concurrently with GMQ9TAP exhibited growth kinetics nearly identical with those of GMTAP cells injected alone (Figs. 4A and 1), demonstrating that the response against GMQ9TAP is specific and does not eliminate Q9-negative melanoma. In contrast, B78H1Q9TAP exhibited delayed tumor outgrowth in about half the mice (Fig. 4A). Significantly, the six mice in which tumor outgrowth of B78H1Q9TAP was not delayed developed, at a later time, GMQ9TAP tumors on the opposite flank (Fig. 4B). This correlation suggests that early outgrowth of B78H1Q9TAP may be due to the suboptimal CTL response against GMQ9TAP, whereas delayed outgrowth of B78H1Q9TAP becomes possible in mice that generated strong cross-reactive CTL response and rejected GMQ9TAP. Although B78H1Q9TAP cells are restrained in their growth in mice rejecting GMQ9TAP, the response generated under these conditions is incapable of fully eradicating the less immunogenic and still expanding B78H1Q9TAP tumors. If this interpretation is correct, then vaccination of syngeneic mice with highly immunogenic Q9-bearing GMQ9TAP before the subsequent challenge with B78H1Q9TAP should lead to more efficient generation of Q9-restricted memory CTL and more potent rejection of a weakly immunogenic melanoma derivative.



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  		FIGURE 4. Immune responses elicited against GMQ9TAP challenge confer protection against B78H1Q9TAP challenge. A, Primary immune response generated against GMQ9TAP challenge confers protection against simultaneous B78H1Q9TAP challenge. Mice were challenged with 1 x 105 live GMQ9TAP cells in the left flank and either GMTAP ({circ} and •) or B78H1Q9TAP ({square} and {blacksquare}) cells in the right flank. Fourteen mice were used in the GMTAP challenge; 15 mice were used in the B78H1Q9TAP challenge. Tumor growth in both flanks was monitored. The growth of GMTAP or B78H1Q9TAP tumors was recorded as a Kaplan-Meier plot. In both GMTAP and B78H1Q9TAP challenge groups, six mice also had detectable GMQ9TAP tumor growth. {blacksquare} and •, Animals in which GMQ9TAP tumor growth was detected. Numbers shown in the plot for B78H1Q9TAP tumor growth correspond to the animal indicated in B. B, Mice with GMQ9TAP tumor outgrowth do not develop protective immunity against B78H1Q9TAP. The day tumor was detected in flank receiving B78H1Q9TAP ({square}) or GMQ9TAP ({blacksquare}) was recorded for each animal shown in the Kaplan-Meier plot in A. C, Immunity generated against GMQ9TAP challenge protects against subsequent challenge with B78H1Q9TAP. Mice that had been challenged with GMQ9TAP tumor cells 4 wk earlier (immunized; n = 10) or had remained tumor-free 100 days after GMQ9TAP challenge and had received multiple boosting immunizations (boosted; n = 6) received an s.c. injection of 1 x 105 live B78H1Q9TAP cells in the opposite flank. Naive animals (n = 5) were included to monitor normal B78H1Q9TAP tumor outgrowth. D, Protection against secondary GMQ9TAP challenge does not require NK cells. Naive mice (primary challenge) or mice tumor-free 100 days after GMQ9TAP challenge (secondary challenge) were injected with 1 x 105 live GMQ9TAP cells in the absence (intact) or the presence of anti-asialo-GM1 NK cell-depleting Ab (anti-NK). Data shown are the percentage of mice that remained tumor-free 100 days after the challenge. Ten mice were used in each group. For survivor mice, no tumor growth was detected after secondary or additional boosting immunizations (>100 animals).

 
This hypothesis was tested in the second approach. B6 mice initially primed with live GMQ9TAP cells were given a second challenge 4 wk later in the opposite flank with B78H1Q9TAP cells. Partial protection was observed, with no tumor outgrowth detected in three of 10 preimmunized mice and delayed tumor take found in another four of 10 animals (Fig. 4C, immunized). Preimmunization with GMQ9TAP had no protective effect on outgrowth of vector-transfected B78H1, with tumor growth detectable in all mice by day 30 (data not shown). When similar experiments were performed in mice that had survived the GMQ9TAP challenge (tumor-free for >100 days) and had received several subsequent rounds of boosting immunizations, more dramatic protection against B78H1Q9TAP challenge was observed (Fig. 4C, boosted), with five of six mice remaining tumor-free. In addition, GMQ9TAP survivor animals were fully protected against subsequent challenges with GMQ9TAP, with no tumor growth observed in any of these animals after boosting immunizations (Fig. 4D). Furthermore, survivor mice were resistant to subsequent GMQ9TAP challenges even when depleted of NK cells in vivo via i.p. administration of anti-asialo-GM1 (Fig. 4D), thus demonstrating that NK cells are not required once protective immunity has been established.

In contrast, mice that were capable of clearing low doses of Q9-negative GMTAP tumor cells were unable to reject a subsequent challenge with B78H1Q9TAP cells. Mice immunized with sublethal doses of GMTAP tumor (three mice surviving the 3 x 103 dose and five mice surviving the 1 x 103 dose, from Fig. 1) were challenged in the opposite flank with 1 x 105 live B78H1Q9TAP cells. Outgrowth of poorly immunogenic B78H1Q9TAP cells in GMTAP-immunized animals displayed similar kinetics as outgrowth in naive animals, with tumors detectable in all mice by day 43 (data not shown).

Taken together, these experiments demonstrate that the protective immunity elicited in response to the initial challenge with GMQ9TAP is Q9-specific and adaptive, because the mediators of the rejection do not eliminate Q9-negative GMTAP cells, but gain specific activity against Q9-positive tumors as a consequence of previous exposure. Although NK cells constitute an important component of the immune response during the initial challenge with B78H1 melanoma variants (Fig. 2) (13), they are not required for rejection of secondary challenges (Fig. 4D). This result is consistent with the interpretation that a memory pool of Q9-restricted tumor-reactive CTL is amplified in immunized/boosted animals.

Expansion of activated/memory CD8+ T cells after immunization with GMQ9TAP tumor

The in vitro cytotoxicity assays and in vivo challenge experiments are indicative of Q9-restricted melanoma-reactive CTL being raised in response to GMQ9TAP tumor cells. Such CTL would be expected to display an activated/memory phenotype. To assess the activation/memory status of CD8+ T cells from mice exposed to various Q9-negative and Q9-positive B78H1 melanoma derivatives, freshly isolated splenocytes (Fig. 5, A and B) and lymphocytes from draining ipsilateral inguinal lymph nodes (Fig. 5, C and D) were stained ex vivo and analyzed by flow cytometry for CD44 activation/memory marker expression levels. When CD8+ T cells were examined 5 days after inoculation with class I-negative B78H1 vector and GMTAP tumors and the poorly immunogenic B78H1Q9TAP variant, no statistically significant increase in the percentage of CD8+ T cells with high CD44 expression levels was observed compared with cells harvested from naive mice (Fig. 5, A and C). In contrast, a statistically significant increase in the percentage of activated CD8+CD44high T cells was seen in both the spleen (Fig. 5A) and draining lymph node (Fig. 5C) 5 days after a single challenge with the highly immunogenic GMQ9TAP tumor cells. Because a measurable change in the numbers of activated CD8+ T cells was detected only in response to GMQ9TAP, additional monitoring for memory CD8+ T cells in survivors of the tumor challenge was performed only in mice inoculated with this highly immunogenic Q9-positive B78H1 derivative. The percentage of CD8+ T cells with high CD44 expression levels remained elevated 100 days after GMQ9TAP challenge in the inguinal lymph node (Fig. 5D). There was a partial reduction in CD8+CD44high T cell numbers in the spleen from the same mice. The percentage of CD44high cells in the CD8+ T cell population became further elevated in survivor mice that received multiple rounds of boosting immunizations with GMQ9TAP tumor cells (Fig. 5, B and D). The persistence of an elevated CD8+CD44high T cell fraction in mice that had rejected the tumor implies that a memory pool of tumor-reactive CTL was established in these mice.



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  		FIGURE 5. Percentage of CD8+ T cells with activated/memory phenotype increases only after challenge with GMQ9TAP. Splenocytes (A and B) or lymphocytes harvested from ipsilateral inguinal lymph nodes (C and D) taken from B6 mice that had been challenged with various B78H1 melanoma derivatives were stained for CD3, CD8, and CD44 expression and analyzed by flow cytometry. Live cells determined by forward and side scatter were gated on CD3+CD8+ cells. The percentage of these CD8+ T cells that expressed high levels of CD44 was recorded. CD44 expression analysis was performed on cells harvested from mice 5 days after tumor challenge (A and C), or after various time points postchallenge with GMQ9TAP (B and D). Mice receiving boosting immunizations (B and D) were given at least three subsequent rounds of s.c. injections of 1 x 105 live GMQ9TAP cells, with at least 2 wk between each injection. Analysis was performed on cells harvested 5 days after the last boost. Data shown are the mean ± SE from five mice per group. Student’s t test was performed to determine whether tumor-challenged groups were statistically different from the unchallenged naive group. Groups showing statistically significant differences from the naive group are indicated by asterisks; numerals above the bars indicate p values: 1, p < 0.0005; 2, p < 0.005; 3, p < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CD8+ CTL have long been known as a vital component of the immune system’s tumor rejection response. Such CD8+ T cell-mediated immunity is dependent on Ag-specific recognition of tumor cells. For CD8+ T cells to recognize tumor cells, intracellular tumor-derived Ags must be presented on the surface of tumor cells in the context of MHC class Ia molecules. Indeed, since the first studies demonstrating that tumor cells presenting highly immunogenic Ags are rejected by CTL (29), numerous tumor Ags have been identified (14, 15, 16).

However, despite extensive evidence indicating that tumor cells are eliminated by the immune system, cancers can still manifest themselves in immunocompetent hosts. One of the primary strategies by which they are able to evade antitumor CTL is through the alteration of MHC class Ia expression (1, 4, 30). In many cases, restoration of MHC class Ia deficiencies abrogates the ability of tumor variants to escape CTL-mediated elimination (31, 32).

Functional investigations of the class Ib family members have lagged behind class Ia studies. Recently, however, research in this area has accelerated as it became clear that many of the nonpolymorphic class Ib proteins in human and mouse models perform specialized functions in the immune system and exert important antitumor effects via a variety of different, nonclassical pathways. For example, human {beta}2m-independent MHC class I chain-related A and B proteins guide {gamma}{delta} T cells in NKG2D-mediated killing of epithelial tumors (33). Similarly, murine functional homologues of MHC class I chain-related products, retinoic acid early inducible gene product-1{beta}, -{gamma}, and -{delta} and H60, can promote tumor rejection by NK and/or CD8+ T cells (34, 35), suggesting that the NKG2D ligands act directly to sensitize NK cells for killing (35) and/or perform costimulatory functions potentiating the cytotoxicity of Ag-specific CTL (34). Another effective antitumor pathway depends on the activation of NKT cells guided by the class Ib Ag CD1d, although the exact mechanism is not understood (36).

Surprisingly, some of the best-characterized class Ib proteins, such as Qa-2 products, have not been examined for their potential impact on tumor rejection. This oversight may have its roots in the mistaken assumption that Qa-2 Ags have restricted tissue distribution consistent with their undetectable expression on nearly all established tissue cultured tumor cell lines. Our recent observations suggesting that Qa-2 is widely expressed in normal tissues, but is selectively silenced in tumors (10), prompted us to focus on Qa-2 Ags as possible targets of antitumor immune surveillance.

The Qa-2 subfamily is comprised of several closely related genes and has frequently undergone contractions and expansions, as evidenced by strain-specific deletions/duplications of the two prototypical Qa-2 genes: Q8 and Q9 (5, 37). The best-characterized gene from this pair, Q9, closely resembles class Ia in {alpha}1, {alpha}2, and {alpha}3 domain primary amino acid sequence and three-dimensional structure (9). In normal cells, Q9 associates with a wide variety of nonameric self peptides that bind to the shallow Q9 groove via a unique anchor combination: histidine at position 7 and a hydrophobic amino acid at position 9 (11, 12). The canonical Q9 is attached to cell surfaces via a glycosylphosphatidylinositol moiety rather than a hydrophobic transmembrane segment used by class Ia MHC (38).

In this paper we report that ectopic expression of the canonical, membrane-bound Q9 promotes rejection of model syngeneic class Ia-negative melanoma cells via a CTL-mediated pathway. Several lines of evidence support this conclusion. First, CD8+ T cell-deficient mice, such as {beta}2m KO and CD8 KO, were unable to reject Q9-bearing tumors, whereas CD8+ strains supported generation of cytotoxic CD8+ T cells that specifically recognized Q9 on target melanoma cells. In vitro recognition of Q9-bearing targets was abolished by blocking with Ab against Q9 or CD8 proteins and was independent of GM-CSF expression in target or stimulator cells. Second, we demonstrated that protective immunity resulted in Q9-specific immunological memory. Mice surviving the original challenge with live, Q9-expressing, GM-CSF-transduced tumor cells were resistant to subsequent challenges with lethal doses of poorly immunogenic, non-GM-CSF-transduced, Q9-positive melanoma, even when depleted of NK cells. Thus, even though NK cells may be needed to reduce the initial tumor burden when the tumor is first injected into mice, they are not necessary for host survival during the secondary challenge. These observations suggest that antitumor CTL effector and memory cells are generated in immunized mice. Consistent with this prediction, we were able to detect an accumulation of CD8+ T cells with an activated phenotype (CD44high) in spleen and draining lymph node shortly after (5 days) tumor inoculation. Furthermore, the persistence of CD8+ T cells expressing high levels of CD44 activation/memory marker months after the initial tumor challenge indicated that a sizable memory pool of antitumor Q9-specific CTL was indeed established in immunized mice.

Because Q9 Ags are structurally similar to class Ia and bind endogenous self-derived peptides (11, 12), it is probable that CTL-mediated elimination of melanoma involves recognition of a tumor Ag(s) presented in the Q9 groove. We have observed that the TAP pathway, which is needed for peptide loading of Q9, is essential for generation of an effective CTL response against Q9-positive tumors, because the TAP2-negative, Q9-transfected cells grew in the syngeneic hosts with kinetics similar to those of the parental B78H1 cells. The identity of the tumor Ag(s) that may be presented to Q9-restricted CTL is currently unknown, nor is it known whether it represents a shared or B78H1 melanoma-specific peptide.

CD4+ T cells have been reported in many models to be required for optimal antitumor CTL responses (39, 40). In our system we failed to detect any adverse effects of CD4 deficiency on rejection of Q9-positive melanoma in CD4 KO mice. Thus, it appears that Q9-restricted CTL are independent of CD4+ T cell help in the tumor take assay. Similar independence from CD4+ T cell help was observed in CTL responses induced by cross-priming, where the frequency of CTL precursors was high (41). Alternatively, it is also possible that the dependence is only partial and could not be observed in the experimental model of CD4 KO mice, which supports the development of CD4–CD8– CD4 lineage T cells (27) and class II MHC-educated CTL (28).

The prominent role of CD8+ T cells in elimination of tumors in animal models and in cancer patients is well documented (1, 16). Although the bulk of the research in this area reinforced the idea that CTL antitumor effects occur via recognition of tumor Ag in the context of the classical class I MHC, more recent studies led to the suggestion that the less polymorphic nonclassical class I loci are also likely to be involved. Griffiths et al. (42) reported that Qa-1 molecules, the murine homologues of human HLA-E that bind leader peptides of class I H chains and promote allogeneic antitumor responses, may also contribute to the rejection of melanoma cells in syngeneic hosts. In the African clawed frog, Xenopus laevis, CD8+ T cells were shown to reject syngeneic class Ia-negative tumor (43). Because the frog tumor cells express class Ib transcripts (44), it was proposed that the antitumor cytotoxic response operates via class Ib Ags. In the human system, studies of CD8+ TCR {alpha}{beta} T cells reactive with prostate and colon cancer cells lines led to a surprising conclusion that these T cells recognize a shared tumor Ag that was processed through the classical, proteasome-dependent, MHC class I pathway, but was presented by a nonclassical, as yet to be defined, MHC-like molecule (45). This interpretation was based on the findings that tumor Ag presentation was diminished by inhibitors of endogenous/cytosolic class I assembly, required {beta}2m expression, and was not inhibited by mAb directed against MHC class Ia or class II.

Finally, it is of interest to consider HLA-C as a potential candidate for MHC unrestricted class I presentation of shared tumor-associated Ags. The HLA-C locus combines features of the classical and nonclassical class I molecules. The expression of HLA-C at the cell surface is reduced to 1/10th the levels of HLA-A and -B, and its importance in Ag presentation of the rapidly evolving microbial pathogens has been questioned (46, 47). In comparison with HLA-A and -B, HLA-C alleles are more closely related to each other, with less variation in the region encoding the peptide-binding groove and more at other nucleotide positions (46, 47). Nevertheless, several independent studies identified HLA-C as a restriction element for diverse tumor-associated Ags (48, 49, 50).

In the present study we identified the nonclassical class Ib MHC molecule Q9 as a target of a potent antitumor CTL response in mice. Future studies are warranted to establish whether a functional homologue of this MHC molecule exists in other animal models or humans and if its nonpolymorphic structure and unique peptide binding properties are selectively advantageous for induction of effective responses against cancer-associated neoantigens.


   Acknowledgments
 
We thank Drs. James Forman and Thorbald van Hall for helpful discussions and expert advice during these studies.


   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 by National Institutes of Health Grant 2R01AI19624 and National Institute of Allergy and Infectious Diseases Grant T32A1005284. Back

2 Address correspondence and reprint requests to Dr. Iwona Stroynowski, Center for Immunology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9093. E-mail address: iwona.stroynowski{at}utsouthwestern.edu Back

3 Abbreviations used in this paper: KO, knockout; {beta}2m, {beta}2-microglobulin. Back

Received for publication May 24, 2004. Accepted for publication July 21, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Chang, C.-C., M. Campoli, S. Ferrone. 2003. HLA class I defects in malignant lesions: what have we learned?. Keio J. Med. 4:220.
  2. Sette, A., R. Chesnut, J. Fikes. 2001. HLA expression in cancer: implications for T cell-based immunotherapy. Immunogenetics 53:255.[Medline]
  3. Houghton, A. N., J. S. Gold, N. E. Blachere. 2001. Immunity against cancer: lessons learned from melanoma. Curr. Opin. Immunol. 13:134.[Medline]
  4. Rees, R. C., S. Mian. 1999. Selective MHC expression in tumours modulates adaptive and innate antitumor responses. Cancer Immunol. Immunother. 48:374.[Medline]
  5. Kumanovics, A., T. Takada, K. F. Lindahl. 2003. Genomic organization of the mammalian MHC. Annu. Rev. Immunol. 21:629.[Medline]
  6. Stroynowski, I., K. Fischer Lindahl. 1994. Antigen presentation by non-classical class I molecules. Curr. Opin. Immunol. 6:38.[Medline]
  7. Stroynowski, I., J. Forman. 1995. Novel molecules related to MHC antigens. Curr. Opin. Immunol. 7:97.[Medline]
  8. O’Callaghan, C. A., J. I. Bell. 1998. Structure and function of the human MHC class Ib molecules HLA-E, HLA-F and HLA-G. Immunol. Rev. 163:129.[Medline]
  9. He, X.-L., P. Tabaczewski, J. Ho, I. Stroynowski, K. C. Garcia. 2001. Promiscuous antigen presentation by the nonclassical MHC Ib Qa-2 is enabled by a shallow, hydrophobic groove and self-stabilized peptide conformation. Structure 9:1213.[Medline]
  10. Ungchusri, T., E. Y. Chiang, G. Brown, M. Chen, P. Tabaczewski, L. Timares, I. Stroynowski. 2001. Widespread expression of the nonclassical class I Qa-2 antigens in hemopoietic and nonhemopoietic cells. Immunogenetics 53:455.[Medline]
  11. Tabaczewski, P., E. Chiang, M. Henson, I. Stroynowski. 1997. Alternative peptide binding motifs of Qa-2 class Ib molecules define rules for binding of self and nonself peptides. J. Immunol. 159:2771.[Abstract]
  12. Joyce, S., P. Tabaczewski, R. H. Angeletti, S. G. Nathenson, I. Stroynowski. 1994. A nonpolymorphic major histocompatiblity complex class Ib molecule binds a large array of diverse self-peptides. J. Exp. Med. 93:161.
  13. Chiang, E. Y., M. Henson, I. Stroynowski. 2003. Correction of defects responsible for impaired Qa-2 class Ib MHC expression on melanoma cells protects mice from tumor growth. J. Immunol. 170:4515.[Abstract/Free Full Text]
  14. Weber, J.. 2002. Peptide vaccines for cancer. Cancer Invest. 20:208.[Medline]
  15. Renkvist, N., C. Castelli, P. F. Robbins, G. Parmiani. 2001. A listing of human tumor antigens recognized by T cells. Cancer Immunol. Immunother. 50:3.[Medline]
  16. Van der Bruggen, P., Y. Zhang, P. Chaux, V. Stroobant, C. Panichelli, E. S. Schultz, J. Chapiro, B. J. Van den Eynde, F. Brasseur, T. Boon. 2002. Tumor-specific shared antigenic peptides recognized by human T cells. Immunol. Rev. 188:51.[Medline]
  17. Cresswell, P., N. Bangia, T. Dick, G. Diedrich. 1999. The nature of the MHC class I peptide loading complex. Immunol. Rev. 172:21.[Medline]
  18. Chiang, E. Y., M. Henson, I. Stroynowski. 2002. The nonclassical major histocompatibility complex molecule Qa-2 protects tumor cells from natural killer cell- and lymphokine-activated killer cell-mediated cytolysis. J. Immunol. 168:2200.[Abstract/Free Full Text]
  19. Ozato, K., D. H. Sachs. 1981. Monoclonal antibodies to mouse MHC antigens. III. Hybridoma antibodies reacting to antigens of the H-2b haplotype reveal genetic control of isotype expression. J. Immunol. 126:317.[Abstract]
  20. Hasenkrug, K. J., J. M. Cory, J. H. Stimpfling. 1987. Monoclonal antibodies defining mouse tissue antigens encoded by the H-2 region. Immunogenetics 25:136.[Medline]
  21. Jones, B., C. A. Janeway. 1981. Cooperative interaction of B lymphocytes with antigen-specific helper T lymphocytes is MHC restricted. Nature 292:547.[Medline]
  22. Tabaczewski, P., I. Stroynowski. 1994. Expression of secreted and glycosylphosphatidylinositol-bound Qa-2 molecules is dependent on functional TAP-2 peptide transporter. J. Immunol. 152:5268.[Abstract]
  23. Bosma, M., W. Schuler, G. Bosma. 1988. The scid mouse mutant. Curr. Top. Microbiol. Immunol. 137:197.[Medline]
  24. Bannai, M., H. Oya, T. Kawamura, T. Naito, T. Shimizu, H. Kawamura, C. Miyaji, H. Watanabe, K. Hatakeyama, T. Abo. 2000. Disparate effect of beige mutation on cytotoxic function between natural killer and natural killer T cells. Immunology 100:165.[Medline]
  25. Fung-Leung, W. P., M. W. Schilham, A. Rahemtulla, T. M. Kundig, M. Vollenweider, J. Potter, W. van Ewijk, T. Mak. 1991. CD8 is needed for development of cytotoxic T cells but not helper T cells. Cell 65:443.[Medline]
  26. Koller, B. H., P. Marrack, J. W. Kappler, O. Smithies. 1990. Normal development of mice deficient in {beta}2M, MHC class I proteins, and CD8+ T cells. Science 248:1227.[Abstract/Free Full Text]
  27. Rahemtulla, A., W. P. Fung-Leung, M. W. Schilham, T. M. Kundig, S. R. Sambhara, A. Narendran, A. Arabian, A. Wakeham, C. J. Paige, R. M. Zinkernagel, et al 1991. Normal development and function of CD8+ cells but markedly decreased helper cell activity in mice lacking CD4. Nature 353:180.[Medline]
  28. Tyznik, A. J., J. C. Sun, M. J. Bevan. 2004. The CD8 population in CD4-deficient mice is heavily contaminated with MHC class II-restricted T cells. J. Exp. Med. 199:559.[Abstract/Free Full Text]
  29. Sibille, C., P. Chomez, C. Wildmann, A. van Pel, E. de Plaen, J. Maryanski, V. de Bergeyck, T. Boon. 1990. Structure of the gene of tum– transplantation antigen P198: a point mutation generates a new antigenic peptide. J. Exp. Med. 172:35.[Abstract/Free Full Text]
  30. Seliger, B., M. J. Maeurer, S. Ferrone. 2000. Antigen-processing machinery breakdown and tumor growth. Immunol. Today 21:455.[Medline]
  31. Tanaka, K., K. J. Isselbacher, G. Khoury, G. Jay. 1985. Reversal of oncogenesis by the expression of a major histocompatibility complex class I gene. Science 228:26.[Abstract/Free Full Text]
  32. Wallich, R., N. Bulbuc, G. J. Hammerling, S. Katzav, S. Segal, M. Feldman. 1985. Abrogation of metastatic properties of tumour cells by de novo expression of H-2K antigens following H-2 gene transfection. Nature 315:301.[Medline]
  33. Groh, V., R. Rhinehart, H. Secrist, S. Bauer, K. H. Grabstein, T. Spies. 1999. Broad tumor-associated expression and recognition by tumor-derived {gamma}{delta} T cells of MICA and MICB. Proc. Natl. Acad. Sci. USA 96:6879.[Abstract/Free Full Text]
  34. Diefenbach, A., E. R. Jensen, A. M. Jamieson, D. H. Raulet. 2001. Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity. Nature 413:165.[Medline]
  35. Cerwenka, A., J. L. Baron, L. L. Lanier. 2001. Ectopic expression of retinoic acid early inducible gene (RAE-1) permits natural killer cell-mediated rejection of a MHC class I-bearing tumor in vivo. Proc. Natl. Acad. Sci. USA 98:11521.[Abstract/Free Full Text]
  36. Kitamura, H., K. Iwakabe, T. Yahata, S. Nishimura, A. Ohta, Y. Ohmi, M. Sato, K. Takeda, K. Okumura, L. Van Kaer, et al 1999. The natural killer T (NKT) cell ligand {alpha}-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells. J. Exp. Med. 189:1121.[Abstract/Free Full Text]
  37. Devlin, J. J., E. H. Weiss, M. Paulson, R. Flavell. 1985. Duplicated gene pairs and alleles of class I genes in the Qa-2 region of the murine major histocompatibility complex: a comparison. EMBO J. 4:3203.[Medline]
  38. Stroynowski, I., M. Soloski, M. G. Low, L. Hood. 1987. A single gene encodes soluble and membrane-bound forms of the major histocompatibility Qa-2 antigen: anchoring of the product by a phospholipid tail. Cell 50:759.[Medline]
  39. 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.[Medline]
  40. Gao, F. G., V. Khammanivong, W. J. Liu, G. R. Leggatt, I. H. Frazar, G. J. P. Fernando. 2002. Antigen-specific CD4+ T-cell help is required to activate a memory CD8+ T cell to a fully functional tumor killer cell. Cancer Res. 62:6438.[Abstract/Free Full Text]
  41. Mintern, J. D., G. M. Davey, G. T. Belz, F. R. Carbone, W. R. Heath. 2002. Cutting edge: precursor frequency affects the helper dependence of cytotoxic T cells. J. Immunol. 168:977.[Abstract/Free Full Text]
  42. Griffiths, E., H. Ong, M. J. Soloski, M. F. Bachmann, P. S. Ohashi, D. E. Speiser. 1998. Tumor defense by murine cytotoxic T cells specific for peptide bound to nonclassical MHC class I. Cancer Res. 58:4682.[Abstract/Free Full Text]
  43. Rau, L., N. Cohen, J. Robert. 2001. MHC-restricted and -unrestricted CD8 T cells: an evolutionary perspective. Transplantation 72:1830.[Medline]
  44. Robert, J., C. Guiet, L. Du Pasquier. 1994. Lymphoid tumors of Xenopus laevis with different capacities for growth in larvae and adults. Dev. Immunol. 3:297.[Medline]
  45. Housseau, F., R. K. Bright, T. Simonis, M. I. Nishimura, S. L. Topalian. 1999. Recognition of a shared human prostrate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells. J. Immunol. 163:6330.[Abstract/Free Full Text]
  46. Zemmour, J., P. Parham. 1992. Distinctive polymorphism at the HLA-C locus: implications for the expression of HLA-C. J. Exp. Med. 176:937.[Abstract/Free Full Text]
  47. Adams, E. J., S. Cooper, G. Thomson, P. Parham. 2000. Common chimpanzees have greater diversity than humans at two of the three highly polymorphic MHC class I genes. Immunogenetics 51:410.[Medline]
  48. van der Bruggen, P., J. P. Szikora, P. Boel, C. Wildmann, M. Somville, M. Sensi, T. Boon. 1994. Autologous cytolytic T lymphocytes recognize a MAGE-1 nonapeptide on melanomas expressing HLA-Cw*1601. Eur. J. Immunol. 24:2134.[Medline]
  49. Chaux, P., R. Luiten, N. Demotte, V. Vantomme, V. Stroobant, C. Traversari, V. Russo, E. Schultz, G. R. Cornelis, T. Boon, P. van der Bruggen. 1999. Identification of five MAGE-A1 epitopes recognized by cytolytic T lymphocytes obtained by in vitro stimulation with dendritic cells transduced with MAGE-A1. J. Immunol. 163:2928.[Abstract/Free Full Text]
  50. Panelli, M. C., M. P. Bettinotti, K. Lally, G. A. Ohnmacht, Y. Li, P. Robbins, A. Riker, S. A. Rosenberg, F. M. Marincola. 2000. A tumor-infiltrating lymphocyte from a melanoma metastasis with decreased expression of melanoma differentiation antigens recognizes MAGE-12. J. Immunol. 164:4382.[Abstract/Free Full Text]



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