Immunization with DNA coding for gp100 results in CD4 T-cell independent antitumor immunity.

BACKGROUND: Xenogeneic DNA immunization can exploit small differences in expressed protein sequence resulting in immune recognition of self-molecules. We hypothesized that immunizing mice with xenogeneic DNA coding for the human melanosomal membrane glycoprotein gp100 would overcome immune ignorance or tolerance and result in tumor immunity. We also investigated the immunologic mechanisms of the antitumor immunity. METHODS: C57BL/6 mice were immunized with DNA coding for human gp100, mouse gp100, or control vector by gene gun. After immunization, mice were challenged with a syngeneic melanoma expressing gp100, and tumor growth was analyzed. Mice deficient in major histocompatibility complex class I or class II molecules were similarly studied to assess the immunologic mechanism of the tumor protection. RESULTS: There was significant tumor protection after vaccination with xenogeneic human gp100 DNA. Class I, but not class II, major histocompatibility complex molecules were required for tumor immunity. In addition, mice immunized with human gp100 demonstrated autoimmunity manifested as coat color depigmentation. CONCLUSIONS: Immunization with xenogeneic DNA coding for the melanosomal glycoprotein gp100 results in tumor protection and autoimmune depigmentation. These results show that xenogeneic DNA vaccines can lead to cancer immunity without CD4(+) T-cell help with potential implications for rational vaccine design.

Age-related dysregulation in CD8 T cell homeostasis: kinetics of a diversity loss.

Relative diversity and representation of peripheral T cells bearing different TCR Vbeta families are remarkably tightly regulated between birth and advanced adulthood. By contrast, individual elderly humans and C3H.SW and B10.BR aged mice display drastic disruption in such regulation. It was suggested that the alterations in the murine aged T cell compartment were due to age-related clonal T cell expansions (TCE). Here, we studied the kinetics of homeostatic dysregulation of T cell populations in aged C57BL/6 (B6) mice. Using mAb staining, we show that the percentages of alphabeta+CD8+ or CD4+ T cells bearing different TCRVbeta elements remain virtually constant in mice up to 12 mo of age. In 22-mo-old mice, however, there is a dramatic disturbance of this pattern owing to the emergence of CD8+ TCE. Expanded T cells did not show any obvious bias in Vbeta usage and were derived in all cases examined thus far from a single clone. TCE appeared later in life, compared with B cell clonal expansions. However, and in contrast to those detected in humans, TCE were frequently unstable disappearing within 2-4 mo, with other TCE appearing within the same time frame. Additional studies carried on thymic T cells, thymectomized mice, and young T transferred cells into Rag1-/- mice suggest that the clonal expansions occur in the periphery and that their onset is accelerated by decreased thymic output and/or function(s).

MHC polymorphism can enrich the T cell repertoire of the species by shifts in intrathymic selection.

The murine class I molecule H-2Kb and its natural gene conversion variant, H-2Kbm8, which differs from H-2Kb solely at 4 aa at the bottom of the peptide-binding B pocket, are expressed in coisogenic mouse strains C57BL/6 (B6) and B6.C-H-2bm8 (bm8). These two strains provide an excellent opportunity to study the effects of Mhc class I polymorphism on the T cell repertoire. We recently discovered a gain in the antiviral CTL repertoire in bm8 mice as a consequence of the emergence of the Mhc class I allele H-2Kbm8. In this report we sought to determine the mechanism behind the generation of this increased CTL diversity. Our results demonstrate that repertoire diversification occurred by a gain in intrathymic positive selection. As previously shown, the emergence of the same Mhc allele also caused a loss in positive selection of T cell repertoire specific for another Ag, OVA-8. This indicates that a reciprocal loss-and-gain pattern of intrathymic selection exists between H-2Kb and H-2Kbm8. Therefore, in the thymus of an individual, a new Mhc allele can select new T cell specificities, while abandoning some T cell specificities selected by the wild-type allele. A byproduct of this repertoire shift is a net gain of T cell repertoire of the species, which is likely to improve its survival fitness.

Coupling and uncoupling of tumor immunity and autoimmunity.

Self-antigens, in the form of differentiation antigens, are commonly recognized by the immune system on melanoma and other cancers. We have shown previously that active immunization of mice against the melanocyte differentiation antigen, a tyrosinase-related protein (TRP) gp75(TRP-1) (the brown locus protein) expressed by melanomas, could induce tumor immunity and autoimmunity manifested as depigmentation. In this system, tumor immunity and autoimmunity were mediated by autoantibodies. Here, we characterize immunity against another tyrosinase family glycoprotein TRP-2 (the slaty locus protein), using the same mouse model and method of immunization. As observed previously for gp75(TRP-1), immunity was induced by DNA immunization against a xenogeneic form of TRP-2, but not against the syngeneic gene, and depended on CD4(+) cells. Immunization against TRP-2 induced autoantibodies and autoreactive cytotoxic T cells. In contrast to immunization against gp75(TRP-1), both tumor immunity and autoimmunity required CD8(+) T cells, but not antibodies. Only autoimmunity required perforin, whereas tumor immunity proceeded in the absence of perforin. Thus, immunity induced against two closely related autoantigens that are highly conserved throughout vertebrate evolution involved qualitatively different mechanisms, i.e., antibody versus CD8(+) T cell. However, both pathways led to tumor immunity and identical phenotypic manifestations of autoimmunity.

The final maturation of at least some single-positive CD4(hi) thymocytes does not require T cell receptor-major histocompatibility complex contact.

The majority ( approximately 70%) of postselection CD4(+) single-positive (SP) thymocytes are CD8(lo)CD4(hi). These cells express very low levels of CD8, undetectable by flow cytofluorimetric (FCM) analysis, but sufficiently high to allow purification by panning. Unlike the fully mature CD8(-)CD4(hi) thymocytes, which account for the remaining approximately 30% of the SP CD4(+) thymocytes, CD8(lo)CD4(hi) cells are functionally immature and short-lived unless they receive an unidentified maturation signal from the thymus. In this study, we tested the hypothesis that this signal is provided by a T cell receptor (TCR)-major histocompatibility complex (MHC) class II interaction. Using intrathymic transfer, we show that the immature CD8(lo)CD4(hi) cells could complete their intrathymic maturation and populate the peripheral lymphoid organs in the absence of MHC class II (and class I) molecules. Furthermore, in mice devoid of class II (and class I) molecules, the progeny of CD8(lo)CD4(hi) cells was long-lived and functionally reactive to allogeneic class II molecules, although their numbers in the spleen and the mesenteric lymph node were approximately 40-50% lower than those in class II(+) mice 5 mo after transfer. Control experiments demonstrated that the surviving cells did not originate from the contaminating mature thymocytes. These results demonstrate that the final maturation, proliferation, and peripheral survival (up to 5 mo) of at least some postselection CD4(+) SP cells do not require the TCR-MHC class II interaction. They also indicate that the TCR-MHC class II interaction(s) required for the intrathymic development of long-lived CD4(+) SP cells occurs before the CD4(hi) SP stage of development.

Cellular basis of B cell clonal populations in old mice.

Previous studies from this laboratory have shown that >85% of old mice have stable B cell clonal populations detectable by Ig heavy chain complementary-determining region 3 mRNA size analysis and confirmed by sequence analysis. B cells from the same clone are frequently detected in several lymphoid compartments of the same mouse. We now report the phenotype of all ten stable B cell clonal populations detected in five 20-month-old C57BL/6 mice. These clonal B cells appear to develop in the periphery and nine of the ten B cell clonal populations expressed the CD5 cell surface marker. Stable B cell expansions may be dominated by cells at two stages of differentiation. Some B cell populations were detected with DNA as well as RNA and represent large clonal populations of B cells, detectable in several lymphoid compartments. These populations are found predominantly in B cell populations expressing CD45R/B220 and the mRNA coding for the membrane-bound form of the mu Ig heavy chain, which suggests a predominance of B lymphocytes in these populations. In other cases, smaller clonal populations were detected only in splenic RNA samples. These clonal populations were found predominantly among CD45R/B220- B cells and did not express the membrane-bound form of the micro Ig heavy chain. We offer the hypothesis that the B cell clonal populations present in old mice may be precursors of the two types of B cell neoplasms which are dominated by CD5+ B cells (B cell chronic lymphocytic leukemia) or plasma cells (multiple myeloma).

Cellular requirements for the monoclonal antibody-mediated eradication of an established solid tumor.

Following subcutaneous implantation, the murine lymphoma E.G7 [a variant of EL-4, transfected with the chicken ovalbumin (OVA) gene] up-regulates the CD4 molecule. We previously showed that the administration of an anti-CD4 monoclonal antibody (mAb) to EG.7-bearing mice leads to a rapid and complete regression of large established tumors. This tumor regression was shown to require both CD8+ cells and functional Fcgamma receptors (FcgammaR), as it failed to occur in mice depleted of CD8 cells, or mice genetically deficient in FcgammaI/III (gamma-/-mice). Using adoptive transfer, we now show that the FcgammaR+ cells required for this regression are the CD11b+ (phagocytic) cells. Furthermore, experiments using peptide tolerization demonstrated that the critical CD8 CTL population in this model is tumor specific. Analysis of tumors at various stages of regression revealed a massive CD11b+FcgammaR+ and a marginal CD8 infiltration. In the presence of the CTL determinant OVA-8 on tumor cells and of the antitumor mAb, this CD8 infiltration became remarkable, and correlated with tumor regression. These results identify the specific cellular effectors essential for the mAb-mediated tumor regression, and suggest that FcgammaR-activated macrophages induced an expansion of tumor-eliminating CTL in situ.

Heteroclitic immunization induces tumor immunity.

In tumor transplantation models in mice, cytotoxic T lymphocytes (CTLs) are typically the primary effector cells. CTLs recognize major histocompatibility complex (MHC) class I-associated peptides expressed by tumors, leading to tumor rejection. Peptides presented by cancer cells can originate from viral proteins, normal self-proteins regulated during differentiation, or altered proteins derived from genetic alterations. However, many tumor peptides recognized by CTLs are poor immunogens, unable to induce activation and differentiation of effector CTLs. We used MHC binding motifs and the knowledge of class I:peptide:TCR structure to design heteroclitic CTL vaccines that exploit the expression of poorly immunogenic tumor peptides. The in vivo potency of this approach was demonstrated using viral and self-(differentiation) antigens as models. First, a synthetic variant of a viral antigen was expressed as a tumor antigen, and heteroclitic immunization with peptides and DNA was used to protect against tumor challenge and elicit regression of 3-d tumors. Second, a peptide from a relevant self-antigen of the tyrosinase family expressed by melanoma cells was used to design a heteroclitic peptide vaccine that successfully induced tumor protection. These results establish the in vivo applicability of heteroclitic immunization against tumors, including immunity to poorly immunogenic self-proteins.

Maturation of B cell precursors is impaired in thymic-deprived nude and old mice.

We have previously reported that bone marrow B cell precursors from thymic-deprived nude and old mice express less recombination-activating gene-1 (RAG-1) mRNA than they do in young euthymic mice. We now report that both nude and old mice have decreased bone marrow pre-B cells and that fewer pre-B cells express RAG protein. This combination of events appears to be the basis for the lower level of bone marrow RAG mRNA in thymic-deprived mice. A link between thymic function and B cell development was suggested by the similar kinetics of thymic involution and of declining bone marrow RAG-1 gene expression during aging. Support for this hypothesis was obtained by demonstrating that injection of supernatant medium from activated CD8+ but not CD4+ young T cells from mice increases RAG mRNA, RAG protein, and the number of bone marrow pre-B cells in nude and old mice. Furthermore, in vivo CD8+ T cells also regulate bone marrow RAG gene expression. Thus, mice deficient in CD8+ T cells expressed levels of RAG-1 mRNA in their bone marrow that were only 10% of those observed in normal or CD4+ T cell-deficient mice. IL-16 was detected in the supernatant medium from activated T cell cultures, and injection of nanogram quantities of recombinant IL-16 (rIL-16) into nude or old mice increased the levels of RAG mRNA in bone marrow B cell precursors and the number of bone marrow pre-B cells. We conclude that the impaired development of B cells within the bone marrow of thymic-deprived nude and old mice can be reversed, at least in part, by the administration of rIL-16.

Clonal expansions of B lymphocytes in old mice.

The effect of age on the diversity of the murine Ig heavy chain repertoire has been studied in unimmunized C57BL/6 mice. We examined the heterogeneity of complementarity-determining region 3 (CDR3) sizes of Ig mRNA of the IgM and IgG isotypes using two VH families, VHJ558 and VHQ52, which together account for approximately 65% of the Ab repertoire. The broad and bell-shaped profiles representing the diversity of the VHJ558 family in the spleen of 2- to 6-mo-old C57BL/6 mice becomes significantly less diverse after 12 mo of age and by 18 mo of age, single CDR3 sizes that dominate the profiles can be observed in the spleens of > 85% of the mice. Readable sequences have been obtained from 40 dominant mRNA CDR3 size species indicating that they represent clonal populations of B lineage. There are no significant homologies among these sequences. Clones of B lymphocytes that express a dominant CDR3 mRNA species can also be found in the bone marrow, the mesenteric lymph nodes, and the thymus of C57BL/6 mice > 18 mo of age. Some clones of B cells can be detected in only one lymphoid compartment; others are found in two or more compartments. The splenic B cell clones in C57BL/6 mice > 18 mo of age are stable for at least 2 mo. The CDR3 mRNA species that dominate the splenic repertoire of Ig mRNA-expressing cells in vivo do not dominate the repertoire of splenic B cells activated in vitro by bacterial LPS, suggesting that they represent a modest population of B cells expressing high levels of Ig mRNA.

Synergy between an antibody and CD8+ cells in eliminating an established tumor.

We investigated the effector mechanisms operating during the rejection of a transplantable solid lymphoma E.G7 (H-2b) which expresses the gene encoding chicken ovalbumin (OVA). Anti-OVA cytotoxic T lymphocytes (CTL) completely and specifically protected animals from the onset of, but could not eradicate established, E.G7 tumors. The growth of the same lymphoma was also effectively prevented by the antibody GK1.5, whose target molecule, CD4, was expressed on E.G7 cells in vivo. Furthermore, GK1.5 was able to eradicate established solid E.G7 tumors. GK1.5-mediated tumor elimination was due to its antitumor activity, and not to the elimination of regulatory CD4+ cells, based on unimpaired tumor growth in the absence of GK1.5 in animals that genetically lack CD4 T cells. In vitro, GK1.5 did not kill tumor cells: complement activation or apoptosis induction were not evident. In vivo, GK1.5-mediated tumor regression did not depend on natural killer cells, but it absolutely required CD8+ cells and intact Fcgamma receptor. We conclude that, in the E.G7 model, the collaboration of antibody and CTL immunity was crucial for the successful immunotherapy of established tumors. The mechanism of this collaboration is discussed.

Priming for T-cell-mediated rejection of established tumors by cutaneous DNA immunization.

DNA immunization has been shown to elicit both antibody and CTL responses against antigens expressed by infectious organisms. Because CTL responses have been implicated in rejection of cancer, we investigated whether DNA immunization by particle bombardment using a gene gun could induce CTL responses that were capable of rejecting tumors in mice. DNA immunization by particle bombardment using genes encoding beta-galactosidase and ovalbumin primed mice to generate CTLs in two genetic backgrounds (DBA/2 and C57BL/6 strains, respectively). DNA immunization was more potent in inducing CTLs than immunization with an optimized regimen of ovalbumin peptide plus immune adjuvant. Immunity induced by DNA immunization protected mice against s.c. challenge with tumors expressing the beta-galactosidase antigen. Tumors were rejected even when DNA immunization was started 3 or 7 days after tumor challenge as tumors were becoming established. Tumor rejection required CD8(+) T cells, confirming a role for CTLs in vivo. These studies show that DNA immunization by particle bombardment can efficiently induce CTL responses that are capable of rejecting even established tumors.

The critical role of a solvent-exposed residue of an MHC class I-restricted peptide in MHC-peptide binding.

The immunodominant ovalbumin257-264 (OVA-8, SIINFEKL) and herpes simplex virus gB496-503 (HSV-8, SSIEFARL) peptides share 50% amino acid identity (residues P1, P3, P5 and P8) and bind with comparable efficacy to the murine MHC-encoded class I molecule H-2Kb. However, these two peptides bind differently to H-2Kbm8, a natural H-2Kb variant with a substitution in four amino acids on the floor of the peptide-binding site; HSV-8 binds with high and OVA-8 with a relatively low efficacy. To investigate which of the non-homologous peptide residues were responsible for this differential binding, we used substituted peptide variants and the class I thermodynamic stabilization assay. Variation at the solvent-exposed peptide residues P6 and P7 did not appreciably influence binding. By contrast, variation at the buried P2 and, surprisingly, at the solvent-exposed P4 residue was found to be important. Transplantation of the HSV-8 P2 or P4 residues onto the OVA-8 backbone created variant peptides O2S (P2I-->S) and O4E (P4N-->E) that bound considerably better to H-2Kbm8 than OVA-8. Furthermore, the double-substituted peptide, O2S4E, bound even better, revealing a cooperative effect of the two residues. The reciprocally substituted peptides H2I and H4N, generated by grafting the OVA-8 P2 and P4 residues onto the HSV-8 backbone respectively, bound to H-2Kbm8 slightly worse than HSV-8 but the double-substituted peptide H2I4N bound as poorly as OVA-8. Effects exerted by the P4 residue, which is solvent accessible and therefore available for the TCR contact, demonstrated that exposed peptide residues can, in certain situations, influence not only the TCR contact but also MHC-peptide binding.

T cell receptor (TCR) recognition of MHC class I variants: intermolecular second-site reversion provides evidence for peptide/MHC conformational variation.

We investigated mechanistic differences in antigen presentation between murine MHC class I variants H-2K(b) and H-2K(bm)8. H-2K(bm)8 differs from H-2K(b) by four residues at the floor of the peptide-binding site, affecting its B pocket which interacts with the second (P2) residue of the peptide. The rest of the molecule, including the T cell receptor (TCR)-contacting residues, is identical to H-2K(b). Due to this variation, CTLs that recognize the ovalbumin 257-264 and HSV gB 498-505 peptides on H-2K(b) cannot recognize them on H-2K(bm)8. This could be due to impaired peptide binding or an altered peptide: K(bm)8 conformation. Peptide binding studies ruled out the first explanation. Molecular modeling indicated that the most obvious consequence of amino acid variation between peptide/H-2K(b) and peptide/H-2K(bm)8 complexes would be a loss of the conserved hydrogen bond network in the B pocket of the latter. This could cause conformational variation of bound peptides. Intermolecular second-site reversion was used to test this hypothesis: P2-substituted OVA and HSV peptides, engineered to restore the hydrogen bond network of the B pocket, were the only ones which restored CTL recognition. These results provide a molecular understanding of peptide/MHC conformational variation.

CD4-independent in vivo priming of murine CTL by optimal MHC class I-restricted peptides derived from intracellular pathogens.

CTL combat intracellular pathogens by killing infected cells. The molecular targets of their attack are foreign peptides bound to self MHC encoded class I molecules. Immunization of mice with peptides containing CTL determinants was shown to elicit CD4-dependent CTL. Here, we have achieved in vivo CTL priming with naturally processed 8-10 amino acid long class I-restricted peptides emulsified in an adjuvant. A potent, reproducible and physiologically relevant response was obtained using peptides from an intracellular bacterium and five viruses (including HIV) in two murine MHC haplotypes. This method is suitable for multiple vaccination, since a 'cocktail' of peptides derived from three pathogens elicited effector CTL against each pathogen. Most importantly, peptide-induced CD8+CD4- CTL were CD4(+)-independent. These results have implications for CTL induction in situations where CD4 T cells are depleted or compromised, as is the case in HIV infection.

The majority of postselection CD4+ single-positive thymocytes requires the thymus to produce long-lived, functional T cells.

We have previously isolated, and characterized in vitro, two subsets of CD4hi T cell receptor (TCR)hi single positive (SP) thymocytes: CD8- and CD8lo. In this report, we have analyzed phenotypic, functional, and developmental characteristics of these "late" CD4hi SP thymocyte subsets. The TCRhi phenotype and the elimination of T cells expressing TCR V beta segments reactive with endogenous mouse mammary tumor virus (MMTV) products suggested that both subsets had undergone positive and negative selection. CD8-4hi thymocytes were functional, as judged by their ability to: (a) induce lethal graft versus host disease (GVHD); (b) survive and expand in peripheral lymphoid organs; and (c) proliferate, rather than undergo apoptosis, in response to in vitro TCR cross-linking. By contrast, CD8lo4hi cells could not induce GVHD, were unable to expand (and perhaps even survive) in peripheral organs and underwent apoptosis upon TCR cross-linking. However, when reintroduced into the thymus, these cells matured into functional, long-lived CD8-4hi lymphocytes. These results document an obligatory requirement for the thymic microenvironment in the final maturation of the majority of CD4hi SP postselection thymocytes, and demonstrate the existence of a previously unrecognized control point in T cell development.

Bone marrow declines as a site of B-cell precursor differentiation with age: relationship to thymus involution.

The rearrangement of immunoglobulin genes in B-lymphocyte precursors requires the expression of the recombination activating genes (Rag), which leads to the generation of a highly diverse B-cell repertoire. We can use the level of Rag-1 mRNA in the bone marrow as an index of its capacity to support the maturation of B lymphocytes as all detectable bone marrow Rag-1 mRNA is expressed by B-cell precursors. In mouse bone marrow, Rag-1 mRNA increases during the first 2 months of life to reach its maximal level at 2 months of age. This level is maintained until 5 months of age and thereafter declines to a minimum level by 10 months of age. Thus, bone marrow Rag-1 mRNA is highest at the time when thymic function is maximal in euthymic mice. An association between thymic activity and bone marrow Rag-1 gene expression was supported by showing a low level of bone marrow Rag-1 mRNA in athymic nude mice at an age when this gene is maximally expressed in euthymic mice. Another characteristic of B cells in nude mice is their preferential rearrangement of diversity region (D)-proximal heavy-chain variable region (VH) genes. We demonstrated that injection of syngeneic splenic T cells into nude mice not only stimulates an increase in Rag-1 mRNA in their bone marrow B-cell precursors but also restores their random use of VH genes. Most interestingly, injection of supernatant medium from phytohemagglutinin-activated splenic T-cell cultures from young euthymic mice also induces both Rag-1 mRNA in bone marrow B-cell precursors and random use of VH genes. These findings suggest that thymic function can regulate both Rag-1 gene expression and VH gene use by bone marrow B-cell precursors.

A positively selecting thymic epithelial cell line lacks costimulatory activity.

The participation of costimulatory molecule interactions in positive selection of T lymphocytes was addressed by assessing the ability of a positively selecting thymic epithelial cell (TEC) line, 427.1, to stimulate allospecific CTL responses. Stimulation of H-2s spleen cells with the H-2b expressing 427.1 line does not result in the generation of cells capable of lysing H-2b target cell lines. The level of expression of MHC class I molecules by 427.1 is lower than that found in other stimulatory TEC lines. However, this finding does not account for the nonstimulatory phenotype. Up-regulation of MHC class I did not result in stimulation, and fusion of 427.1 cells with stimulatory TEC resulted in a line with low MHC class I molecule expression and stimulatory phenotype. The TEC line 427.1 does not express the costimulatory molecule B7/BB1, and transfection of the B7/BB1-encoding DNA results in expression of the molecule and conversion into a stimulatory phenotype, demonstrating directly that the non-stimulatory phenotype is a result of lack of costimulation. However, B7/BB1 expression does not improve the ability of 427.1 TECs to induce positive selection. Intrathymic injection of the B7/BB1 transfected, compared with mock transfected 427.1 cells, rescued fewer CD8+ mature thymocytes in beta 2-microglobulin negative mice. Therefore, unlike the peripheral T cell responses to Ag, positive selection of T cells in the thymus may not depend on the costimulation provided by the presenting cell.

Positive selection of the T-cell repertoire is affected by mutations in the peptide-binding site of MHC class I molecules.

H-2Kb mutant molecules (H-2Kbm) and the H-2Kb-restricted response to OVA and VSV N peptides were used to investigate the influence of polymorphism of structurally defined regions of the MHC class I molecules on intrathymic positive selection of the T-cell repertoire. We show that the positive selection of the T-cell repertoire in the thymus requires the self-peptide to be present in the MHC antigen-binding site. A correlation between the ability of four MHC molecules to present antigenic peptide and to positively select T cells specific for it was noted. The self-peptides involved in positive selection may therefore mimic the foreign peptide during intrathymic selection. A structural correlate of this mimicry may be a similar or identical binding requirement for the antigen-binding pocket(s)/residues of the MHC peptide-binding site.