Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen.

Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.     

Defining cytolytic T lymphocyte recognition of chemically modified self. I. Response to trinitrophenyl-H-2Kk

We used purified class I antigen incorporated into liposomes to examine the response of secondary cytolytic T lymphocytes (CTL) to chemically modified self. By generating the secondary response in the presence of T cell helper factor, the level of CTL response was limited by CTL recognition of added antigen rather than by helper cell generation of lymphokines. We found a strong secondary response against chemically modified self when spleen cells from trinitrophenyl (TNP)-primed C3H/HeJ mice were stimulated with a) TNP-modified liposomes containing H-2Kk, b) liposomes containing H-2Kk purified from TNP-modified RDM-4 (H-2k) cells, or c) liposomes containing the limited trypsin proteolysis product of H-2Kk that had been directly modified with TNP. In contrast, we were not able to generate a significant CTL response with unmodified H-2Kk incorporated into vesicles along with TNP-modified membrane components lacking H-2Kk. These results suggest that TNP-modified H-2Kk is a major antigenic site recognized by CTL from C3H/HeJ mice after priming against TNP-modified self.     

In vivo priming of mouse CTL precursors directed to product of a newly defined minor H-42 locus is under a novel control of class II MHC gene

In a previous study, we discovered a new mouse minor histocompatibility antigen encoded by a locus at 8.5 cM apart from the H-2 complex, and we have since named the locus H-42. One allele of H-42, which is named H-42a, had been elucidated, but the other alleles, which we tentatively named H-42b, have not been elucidated. In the present study, we explored MHC control on the anti-H-42a cytotoxic T lymphocyte (CTL) responsiveness in H-42b mice. In vivo immunization (i.v. injection) of H-42b mice with 5 to 30 X 10(6) spleen cells (SC) bearing allogeneic H-42a antigen but carrying H-2 complex (mouse MHC) matched with the H-42b mice failed to prime anti-H-42a CTL but induced stable and specific anti-H-42a CTL unresponsiveness, i.e., tolerance, in the H-42b recipient mice. In contrast, H-2 heterozygous H-42b F1 mice injected with SC bearing H-42a alloantigen on either of the parental H-2 haplotypes were effectively primed to generate anti-H-42a CTL. Exploration of the region or subregion in the H-2 complex of H-42a donor SC that should be compatible with H-42b recipient mice for the induction of their anti-H-42a CTL tolerance demonstrated that the compatibility at I region, most probably I-A subregion, but not at K, S, or D region, determined the induction of the tolerance. MHC class II compatible H-42a skin graft (SG) to H-42b mice, however, consistently primed the anti-H-42a CTL in the H-42b recipients. These results were discussed in several aspects, including uniqueness of MHC class II control on the CTL response to minor H-42a antigen, possibility of inactivation of responding anti-H-42a precursor CTL or helper T cells in H-42b mice by encountering the veto cells present in MHC class II-matched H-42a SC population, and significance of the present observations as a mechanism of CTL tolerance to self-components.     

The role of T cells in anti-herpes simplex virus immunity. I. Induction of antigen-specific cytotoxic T lymphocytes.

Mice infected with herpes simplex virus develop little or no cytotoxic T lymphocyte (CTL) response. However, in lymph nodes (LN's) draining a local site infected with HSV, antigen-specific CTL precursors are sensitized, which upon transfer to in vitro culture conditions develop within 72 hr into effective CTL. The in vivo blockade of CTL differentiation can be overcome by cyclophosphamide, suggesting that a cyclophosphamide-sensitive mechanism blocks the in vivo generation of HSV-immune CTL. The cytolytic activity of HSV-immune CTL is H-2 restricted and antigen specific. Thus CTL sensitized toward HSV type 1 discriminate between syngeneic targets infected with either the immunologic HSV variant type 1 or type 2 (and vice versa). H-2-matched target cells exposed for 30 min to infectious HSV are lysed within 60 min of contact with CTL. Since HSV replication is believed to require more than 4 to 5 hr, the data suggest that either the expression of HSV-dependent "early proteins" takes place within 30 to 90 min or cell membrane-integrated HSV virion represents the target antigen of CTL.     

Peptide-loaded dendritic cells prime and activate MHC-class I-restricted T cells more efficiently than protein-loaded cross-presenting DC

Undifferentiated and differentiated dendritic cells (uDC and dDC, respectively), derived from the bone marrow, were studied in vitro and in vivo. Ovalbumin (OVA) and two OVA-derived peptides binding to H-2K(b) and I-A(b), respectively, were used. Two IL-2 secreting T cell hybridomas specific for the OVA-derived epitopes were used in the in vitro read-out. The ability to cross-present the H-2K(b) binding OVA(257-264)-peptide (SIINFEKL) was restricted to dDC, which express CD11c(+), CD86(+), and MHC-II(+). In vitro, the antigenicity of SIINFEKL-loaded DC declined at a slower rate than that of OVA-pulsed DC. Moreover, SIINFEKL-loaded DC were up to 50 times more efficient than DC-pulsed with OVA-protein for generation of an H-2K(b)-restricted response. Immunization of mice with SIINFEKL-loaded DC resulted in a much stronger H-2K(b)-restricted response than immunization with OVA-pulsed DC. These data might have important implications for the choice of antigen source in the design of DC-based vaccines.     

Depletion of macrophages expressing I-J antigen results in efficient generation of alloreactive cytotoxic T lymphocytes

The role of suppressor macrophages (S-M phi) produced during generation of cytotoxic T lymphocytes (CTL) stimulated with allogeneic lymphocytes was investigated. Splenic CTL from C3H/He mice (H-2k) were generated by in vivo immunization and subsequent in vitro stimulation by splenic lymphocytes from C57B1/6 mice (H-2b) in mixed lymphocyte reaction (MLR). In addition to in vitro standard 51Cr release assay, the CTL activity was mainly measured in vivo using the Winn assay against EL-4 thymoma cells in B6C3F1 mice (H-2b/k). In mice injected with CTL plus EL-4 cells survival rate was 20% compared with no survival of mice treated with normal spleen cells plus EL-4 cells. The antitumor activity of the CTL was significantly increased when immunized mice were treated with a 5 mg/kg ip dose of indomethacin at the time of immunization (80% survival). Macrophages were depleted from spleen cells of immunized mice by plastic adherence or carbonyl-iron treatment, replaced with an equivalent number of M phi from normal mice, and then introduced into a 5-day MLR. When the antitumor activity of the cells isolated from this MLR was measured in the Winn assay, 90-100% survival in EL-4-bearing mice was observed. In contrast, none of the mice inoculated with EL-4 alone and 20% of the mice that received CTL obtained after alloimmunization followed by MLR in addition to EL-4 survived. These results of CTL activity were confirmed by in vitro cytotoxicity tests. When the M phi isolated from spleens of immunized mice were analyzed for I-Jk antigen expression, a 2.5-fold increase was detected, compared with splenic M phi obtained from normal C3H/He mice. In contrast, Ia and I-Ak antigen expression was equivalent in M phi isolated from normal or immunized C3H/He mice. When immune spleen cells were treated with anti-I-Jk antiserum followed by complement and then, subjected to the MLR, the antitumor activity of CTL was significantly enhanced (80% survival). However, treatment of these cells with anti-I-Ak antiserum and complement did not alter CTL activity. These data suggest that the increase of S-M phi expressing I-Jk+ antigen to be induced during alloimmunization results in suppression of allospecific CTL-generation in MLR.     

Determination of structural principles underlying three different modes of lymphocytic choriomeningitis virus escape from CTL recognition

Lymphocytic choriomeningitis virus infection of H-2(b) mice generates a strong CD8(+) CTL response mainly directed toward three immunodominant epitopes, one of which, gp33, is presented by both H-2D(b) and H-2K(b) MHC class I molecules. This CTL response acts as a selective agent for the emergence of viral escape variants. These variants generate altered peptide ligands (APLs) that, when presented by class I MHC molecules, antagonize CTL recognition and ultimately allow the virus to evade the cellular immune response. The emergence of APLs of the gp33 epitope is particularly advantageous for LCMV, as it allows viral escape in the context of both H-2D(b) and H-2K(b) MHC class I molecules. We have determined crystal structures of three different APLs of gp33 in complex with both H-2D(b) and H-2K(b). Comparison between these APL/MHC structures and those of the index gp33 peptide/MHC reveals the structural basis for three different strategies used by LCMV viral escape mutations: 1) conformational changes in peptide and MHC residues that are potential TCR contacts, 2) impairment of APL binding to the MHC peptide binding cleft, and 3) introduction of subtle changes at the TCR/pMHC interface, such as the removal of a single hydroxyl group.     

Cytotoxic T lymphocyte responses to minor H-43 alloantigens in H-43a and H-43b mice. Both anti-H-43b and anti-H-43a CTL activities are generated exclusively in the context of the same H-2Kb restriction element

We have previously demonstrated that anti-H-43a CTL response of H-43b responder mice was exclusively restricted by self H-2Kb (Kb) but not by the other nine self MHC class I alleles from independent origins, i.e., Kbml,d,k,s and Db,d,k,q,s. In the present study, we verified that Kf,q,r and Df,r alleles could also not serve as restricting class I elements in the CTL response to H-43a alloantigen. Another notable observation made in the earlier study was the fact that, in H-43 incompatibility of the alternative combination, H-43a mice were incapable of generating CTL activity against H-43b alloantigen. However, by means of employing new in vivo immunization procedures, we discovered that some but not all genetically identical H-43a responder mice could mount anti-H-43b CTL response restricted by self Kb. Again, no anti-H-43b CTL activity could be generated in the context of self Kk, Kj, Db or Dk molecules. Although the number of class I alleles we examined is still limited, these results indicate that antigenic fragments derived from the processed H-43a and H-43b alloantigens possess an indistinguishable epitope (agretope), and that such agretope either interacts only with the privileged Kb molecules or allows to bestow the immunogenic conformation of allodeterminants on the fragments solely in the context of the restricting Kb element.     

Overcoming T cell tolerance to the hepatitis B virus surface antigen in hepatitis B virus-transgenic mice

The sequence of the hepatitis B virus (HBV) major envelope (Env) protein (ayw subtype) was scanned for the presence of H-2(d,b) motifs. Following binding and immunogenicity testing, two new H-2(d)-restricted epitopes (Env.362 and Env.364) were identified. These epitopes induced CTLs capable of recognizing naturally processed HBV-Env, but were apparently generated with lower efficiency than the previously defined dominant Env.28 epitope. Next, HBV-transgenic mice that express all of the HBV proteins and produce fully infectious particles were immunized with a mixture of lipopeptides encompassing the Env.28, Env.362, and Env.364 epitopes. Significant CTL responses were obtained, but they had no effect on viral replication in the liver, nor did they induce an inflammatory liver disease. However, in adoptive transfer experiments, CTL lines generated from the HBV-transgenic mice following immunization were able to inhibit viral replication in vivo without causing hepatitis. This is in contrast to CTL lines derived from nontransgenic mice that displayed both antiviral and cytopathic effects, presumably because they displayed higher avidity for the viral epitopes than the transgenic CTLs. These results suggest that T cell tolerance to HBV can be broken with appropriate immunization but the magnitude and characteristics of the resultant T cell response are significantly different from the response in HBV-naive individuals since their antiviral potential is stronger than their cytotoxic potential. This has obvious implications for immunotherapy of chronic HBV infection.     

Cytotoxic T-Lymphocyte Epitope Immunodominance in the Control of Choroid Plexus Tumors in Simian Virus 40 Large T Antigen Transgenic Mice

The simian virus 40 (SV40) large tumor antigen (Tag) is a virus-encoded oncoprotein which is the target of a strong cytotoxic T-lymphocyte (CTL) response. Three immunodominant H-2(b)-restricted epitopes, designated epitopes I, II/III, and IV, have been defined. We investigated whether induction of CTLs directed against these Tag epitopes might control Tag-induced tumors in SV11(+) (H-2(b)) mice. SV11(+) mice develop spontaneous tumors of the choroid plexus due to expression of SV40 Tag as a transgene. We demonstrate that SV11(+) mice are functionally tolerant to the immunodominant Tag CTL epitopes. CTLs specific for the H-2Kb-restricted Tag epitope IV were induced in SV11(+) mice following adoptive transfer with unprimed C57BL/6 spleen cells and immunization with recombinant vaccinia viruses expressing either full-length Tag or the H-2Kb-restricted epitope IV as a minigene. In addition, irradiation of SV11(+) mice prior to adoptive transfer with unprimed C57BL/6 spleen cells led to the priming of epitope IV-specific CTLs by the endogenous Tag. Induction of epitope IV-specific CTLs in SV11(+) mice by either approach correlated with increased life span and control of the choroid plexus tumor progression, indicating that CTLs specific for the immunodominant Tag epitope IV control the progressive growth of spontaneous tumors induced by this DNA virus oncogene in transgenic mice.     

Capsid-specific cytotoxic T lymphocytes recognize three distinct H-2D(b)-restricted regions of the BeAn strain of Theiler's virus and exhibit different cytokine profiles

The role of virus-specific cytotoxic T lymphocytes (CTL) in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a viral model for multiple sclerosis, is not yet clear. To investigate the specificity and function of CTL generated in response to TMEV infection, we generated a panel of overlapping 20-mer peptides encompassing the entire capsid and leader protein region of the BeAn strain of TMEV. Binding of these peptides to H-2K(b) and H-2D(b) class I molecules of resistant mice was assessed using RMA-S cells. Several peptides displayed significant binding to H-2K(b), H-2D(b), or both. However, infiltrating cytotoxic T cells in the central nervous system of virus-infected mice preferentially lysed target cells pulsed with VP2(111-130/121-140) or VP2(121-130), a previously defined CTL epitope shared by the DA strain of TMEV and other closely related cardioviruses. In addition, at a high effector-to-target cell ratio, two additional peptides (VP2(161-180) and VP3(101-120)) sensitized target cells for cytolysis by infiltrating T cells or splenic T cells from virus-infected mice. The minimal epitopes within these peptides were defined as VP2(165-173) and VP3(110-120). Based on cytokine profiles, CTL specific for these subdominant epitopes are Tc2, in contrast to CTL for the immunodominant epitope, which are of the Tc1 type. Interestingly, CTL function towards both of these subdominant epitopes is restricted by the H-2D molecule, despite the fact that these epitopes bind both H-2K and H-2D molecules. This skewing toward an H-2D(b)-restricted response may confer resistance to TMEV-induced demyelinating disease, which is known to be associated with the H-2D genetic locus.     

Quantitation of CD8(+) T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large T antigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombinants expressing full-length T antigen or epitope minigenes

The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2(b)) mice is directed against three H2-D(b)-restricted epitopes, I, II/III, and V, and one H2-K(b)-restricted epitope, IV. Epitopes I, II/III, and IV are immunodominant, while epitope V is immunorecessive. We investigated whether this hierarchical response was established in vivo or was due to differential expansion in vitro by using direct enumeration of CD8(+) T lymphocytes with Tag epitope/major histocompatibility complex class I tetramers and intracellular gamma interferon staining. The results demonstrate that epitope IV-specific CD8(+) T cells dominated the Tag-specific response in vivo following immunization with full-length Tag while CD8(+) T cells specific for epitopes I and II/III were detected at less than one-third of this level. The immunorecessive nature of epitope V was apparent in vivo, since epitope V-specific CD8(+) T cells were undetectable following immunization with full-length Tag. In contrast, high levels of epitope V-specific CD8(+) T lymphocytes were recruited in vivo following immunization and boosting with a Tag variant in which epitopes I, II/III, and IV had been inactivated. In addition, analysis of the T-cell receptor beta (TCRbeta) repertoire of Tag epitope-specific CD8(+) cells revealed that multiple TCRbeta variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCRbeta10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8(+) T-cell responses is established in vivo.     

Molecularly engineered vaccine which expresses an immunodominant T-cell epitope induces cytotoxic T lymphocytes that confer protection from lethal virus infection

Identification of a single viral T-cell epitope, associated with greater than 95% of the virus-specific cytotoxic T-lymphocyte (CTL) activity in BALB/c (H-2d) mice (J. L. Whitton, A. Tishon, H. Lewicki, J. Gebhard, T. Cook, M. Salvato, E. Joly, and M. B. A. Oldstone, J. Virol. 63:4303-4310, 1989), permitted us to design a CTL vaccine and test its ability to protect against a lethal virus challenge. Here we show that a single immunization with a recombinant vaccinia virus-lymphocytic choriomeningitis virus (LCMV) vaccine (VVNPaa1-201) expressing the immunodominant epitope completely protected H-2d mice from lethal infection with LCMV but did not protect H-2b mice. Furthermore, we show that the success or failure of immunization was determined entirely by the host class I major histocompatibility glycoproteins. The difference in outcome between mice of these two haplotypes was consistent with the presence or absence in the immunizing sequences of an epitope for CTL recognition and is correlated with the induction of LCMV-specific H-2-restricted CTL in H-2d mice. Protection is not conferred by a humoral immune response, since LCMV-specific antibodies were not detectable in sera from VVNPaa1-201-immunized mice. In addition, passive transfer of sera from vaccinated mice did not confer protection upon naive recipients challenged with LCMV. Hence, the molecular dissection of viral proteins can uncover immunodominant CTL epitope(s) that can be engineered into vaccines that elicit CTL. A single CTL epitope can protect against a lethal virus infection, but the efficacy of the vaccine varies in a major histocompatibility complex-dependent manner.     

In vivo augmentation of tumor-specific CTL responses by class I/peptide antigen complexes on microspheres (large multivalent immunogen)

Tumor membrane Ag immobilized on cell size microspheres (large multivalent immunogen (LMI)) was previously shown to augment tumor-specific CTL activity and reduce tumor growth, and a clinical trial examining this approach is in progress. In the current study, LMI treatment has been examined using adoptive transfer of TCR-transgenic CD8 T cells to visualize Ag-specific cells during the response. OT-I T cells specific for H-2K(b)/OVA(257-264) were transferred into mice that were then challenged with LMI made by immobilizing H-2K(b)/OVA(257-264) on microspheres (K(b)/OVA(257-264)-LMI) alone, or along with i.p. challenge with OVA-expressing E.G7 tumor. K(b)/OVA(257-264)-LMI caused significant reduction of tumor growth when administered to E.G7-bearing mice. When administered alone, the K(b)/OVA(257-264)-LMI caused only weak clonal expansion of OT-I cells in the spleen and lymph nodes, although most of the OT-I cells up-regulated expression of CD44 and VLA-4. In contrast, K(b)/OVA(257-264)-LMI administration to E.G7-bearing mice stimulated no detectable expansion of OT-I cells in the spleen and lymph nodes but caused a rapid increase in the number of OT-I cells in the peritoneal cavity, the site of the growing tumor. These results demonstrate the potential for using class I/tumor peptide complexes for immunotherapy. In addition, they suggest a model for the mechanism of CTL augmentation in which recognition of the LMI Ag results in altered trafficking of the tumor-specific CD8 T cells so that they reach the site of a growing tumor more rapidly and in greater numbers, where they may further expand and acquire effector function.     

Full length antigen priming enhances the CTL epitope-based DNA vaccine efficacy

Although CD8⁺ cytotoxic T lymphocyte (CTL) epitope-based DNA vaccination is valuable experience on vaccine research but many attempts are still continued to achieve acceptable protective response. To study the role of full length antigen in CTL epitope immunization, we evaluated cellular immunity of diverse patterns of complete Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) and the immunodominant CTL epitope (498–505) DNA injection in C57BL/6 mice. Optimal immune response was observed in the group immunized with the full length of gB in the first injection and CTL epitope in the second and third vaccination as assessed by lymphocyte proliferation assay (MTT), cytokine assay (ELISA) and CTL assay. B cell and spatially CD4⁺ T cell epitopes in full length protein might be important for appropriate priming of CTL immune response. These findings may have important implication for the improvement of CTL epitope based DNA vaccine against HSV and other pathogens.     

The early expression of glycoprotein B from herpes simplex virus can be detected by antigen-specific CD8+ T cells

The immune response to cutaneous herpes simplex virus type 1 (HSV-1) infection begins with remarkable rapidity. Activation of specific cytotoxic T lymphocytes (CTL) begins within hours of infection, even though the response within the draining lymph nodes peaks nearly 5 days later. HSV gene products are classified into three main groups, alpha, beta, and gamma, based on their kinetics and requirements for expression. In C57BL/6 mice, the immunodominant epitope from HSV is derived from glycoprotein B (gB(498-505)). While gB is considered a gamma or "late" gene product, previous reports have indicated that some level of gene expression may occur soon after infection. Using brefeldin A as a specific inhibitor of viral antigen presentation to major histocompatibility complex class I-restricted CTL, we have formally addressed the timing of gB peptide expression in an immunologically relevant manner following infection. Presentation of gB peptide detected by T-cell activation was first observed within 2 h of infection. Comparison with another viral epitope expressed early during infection, HSV-1 ribonucleotide reductase, demonstrated that gB is presented with the same kinetics as this classical early-gene product. Moreover, this rapidity of gB expression was further illustrated via rapid priming of na?ve transgenic CD8(+) T cells in vivo after HSV-1 infection of mice. These results establish that gB is expressed rapidly following HSV-1 infection, at levels capable of effectively stimulating CD8(+) T cells.     

H-2D(b-/-) mice are susceptible to persistent infection by Theiler's virus

H-2(b) mice are resistant to persistent infection of the central nervous system by Theiler's virus. They clear the infection 7 to 10 days after intracranial inoculation. Resistance maps to the H-2D gene and not to the H-2K gene and is associated with a potent antiviral cytotoxic T-lymphocyte (CTL) response. We used H-2(b) mice in which the H-2D or the H-2K gene had been inactivated to dissect the respective roles of these genes in resistance. We report that H-2D(-/-) but not H-2K(-/-) mice were susceptible to persistent infection. Furthermore, whereas H-2K(-/-) mice mounted a vigorous virus-specific CTL response, similar to that of control C57BL/6 mice, the CTL response of H-2D(-/-) mice was nil or minimal. Using target cells transfected with the H-2D(b) or the H-2K(b) gene, we showed that the H-2K-restricted CTL response against the virus was minimal in H-2D(-/-) mice. These results demonstrate that the H-2D(b) and H-2K(b) genes play nonredundant roles in the resistance to this persistent infection.     

In vivo expansion of the residual tumor antigen-specific CD8+ T lymphocytes that survive negative selection in simian virus 40 T-antigen-transgenic mice

Mice that express the viral oncoprotein simian virus 40 (SV40) large T antigen (T-Ag) as a transgene provide useful models for the assessment of the state of the host immune response in the face of spontaneous tumor progression. Line SV11 (H2(b)) mice develop rapidly progressing choroid plexus tumors due to expression of full-length T-Ag from the SV40 promoter. In addition, T-Ag expression in the thymus of SV11 mice results in the deletion of CD8(+) T cells specific for the three H2(b)-restricted immunodominant epitopes of T-Ag. Whether CD8(+) T cells specific for the immunorecessive H2-D(b)-restricted epitope V of T-Ag survive negative selection in SV11 mice has not been determined. Immunization of SV11 mice with rVV-ES-V, a recombinant vaccinia virus expressing epitope V as a minigene, resulted in the induction of weak, but reproducible, epitope V-specific cytotoxic T-lymphocyte (CTL) responses. This weak lytic response corresponded with a decreased frequency of epitope V-specific CTL that could be recruited in SV11 mice. In addition, CTL lines derived from rVV-ES-V-immunized SV11 mice had reduced avidities compared to that seen with CTL derived from healthy mice. Despite this initial weak response, significant numbers of epitope V-specific CD8(+) T cells were detected in SV11 mice ex vivo following a priming-boosting approach and these cells demonstrated high avidity for epitope V. The results suggest that low numbers of tumor-reactive CD8(+) T cells with high avidity for epitope V survive negative selection in SV11 mice but can be expanded by specific boosting approaches in the tumor bearing host.     

Identification of a ras oncogene peptide that contains both CD4(+) and CD8(+) T cell epitopes in a nested configuration and elicits both T cell subset responses by peptide or DNA immunization

Mutations in ras proto-oncogenes are commonly found in a diversity of malignancies and may encode unique, non-self epitopes for T cell-mediated antitumor activity. In a BALB/c (H-2(d)) murine model, we have identified a single peptide sequence derived from the ras oncogenes that contained both CD8(+) and CD4(+) T cell epitopes in a nested configuration. This peptide reflected ras sequence 4-16, and contained the substitution of Gly to Val at position 12 ?i.e., 4-16(Val12)?. Mice immunized with this 13-mer peptide induced a strong antigen (Ag)-specific CD4(+) proliferative response in vitro. In contrast, mice inoculated with the wild-type ras sequence failed to generate a peptide-specific T cell response. Additionally, mice immunized with the ras 4-16(Val12) peptide concomitantly displayed an Ag-specific CD8(+) cytotoxic T lymphocyte (CTL) response, as determined by lysis of syngeneic tumor target cells incubated with the nominal 9-mer nested epitope peptide ?i.e., 4-12(Val12)?, as well as lysis of tumor target cells expressing the corresponding ras codon 12 mutation. Analysis of the Valpha- and Vbeta-chains of the T cell receptor (TCR) expressed by these CTL revealed usage of the Valpha1 and Vbeta9 subunits, consistent with the TCR phenotype of anti-ras Val12 CTL lines produced by in vivo immunization with the nominal peptide epitope alone. Moreover, immunization with the nested epitope peptide, as compared to immunization with either the 9-mer CTL peptide alone or an admixture of the 9-mer CTL peptide with an overlapping 13-mer CD4(+) T cell helper peptide ?i.e., 5-17(Val12)? lacking the class I N-terminus anchor site, enhanced the production of the CD8(+) T cell response. Finally, immunization with plasmid DNA encoding the ras 4-16(Val12) sequence led to the induction of both Ag-specific proliferative and cytotoxic responses. Overall, these results suggested that a single peptide immunogen containing nested mutant ras-specific CD4(+) and CD8(+) T cell epitopes: (1) can be processed in vivo to induce both subset-specific T lymphocyte responses; and (2) leads to the generation of a quantitatively enhanced CD8(+) CTL response, likely due to the intimate coexistence of CD4(+) help, which may have implications in peptide- or DNA-based immunotherapies.     

In vivo functional efficacy of tumor-specific T cells expanded using HLA-Ig based artificial antigen presenting cells (aAPC)

Adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability in the final T cell product and in the limited in vivo function and survival of ex vivo expanded antigen-specific cytotoxic T cells (CTL). This has stimulated interest in development of standardized artificial antigen presenting cells (aAPC) to reliably expand antigen specific CTL. However, for successful immunotherapy the aAPC ex vivo generated CTL must have anti-tumor activity in vivo. Here, we demonstrate that HLA-Ig based aAPC stimulated tumor-specific CTL from human peripheral blood T lymphocytes showed robust expansion and functional activity in a human/SCID mouse melanoma model. HLA-Ig based aAPC expanded CTL were detected in the peripheral blood up to 15 days after transfer. Non-invasive bioluminescence imaging of tumor bearing mice demonstrated antigen dependent localization of transferred CTL to the tumor site. Moreover, adoptive transfer of HLA-Ig based aAPC generated CTL inhibited the tumor growth both in prevention and treatment modes of therapy and was comparable to that achieved by dendritic cell expanded CTL. Thus, our data demonstrate potential therapeutic in vivo activity of HLA-Ig based aAPC expanded CTL to control tumor growth.