Induction of tumor-reactive T helper responses by a posttranslational modified epitope from tumor protein p53

Posttranslational modifications regulate the function and stability of proteins, and the immune system is able to recognize some of these modifications. Therefore, the presence of posttranslational modifications increases the diversity of potential immune responses to a determinant antigen. The stimulation of tumor-specific CD4⁺ helper T lymphocytes (HTLs) is considered important for the production of anti-tumor antibodies by B cells and for the generation and persistence of CD8⁺ cytotoxic T lymphocytes, and in some instances, HTLs can directly reduce tumor cell growth. Identification of MHC class II-restricted peptide epitopes from tumor-associated antigens including those generated from posttranslational protein modifications should enable the improvement of peptide-based cancer immunotherapy. We describe here an MHC class II binding peptide from the tumor protein p53, which possesses an acetylated lysine at position 120 (p53_{110-124/AcK120}) that is effective in eliciting CD4⁺ T cell responses specific for the acetylated peptide. Most importantly, the acetylated peptide-reactive CD4 HTLs recognized the corresponding naturally processed posttranslational modified epitope presented by either dendritic cells loaded with tumor cell lysates or directly on tumors expressing p53 and the restricting MHC class II molecules. Treatment of tumor cells with a histone deacetylase inhibitor augmented their recognition by the p53_{110-124/AcK120}-reactive CD4⁺ T cells. These findings prove that the epitope p53_{110-124/AcK120} is immunogenic for anti-tumor responses and is likely to be useful for cancer immunotherapy.     

A functional recombinant single-chain T cell receptor fragment capable of selectively targeting antigen-presenting cells

Specificity in the immune system is dictated and regulated by specific recognition of peptide/major histocompatibility complexes (MHC) by the T cell receptor (TCR). Such peptide/MHC complexes are a desirable target for novel approaches in immunotherapy because of their highly restricted fine specificity. Recently a potent anti-human p53 CD8(+) cytotoxic T lymphocyte (CTL) response has been developed in HLA-A2 transgenic mice after immunization with peptides corresponding to HLA-A2 motifs from human p53. An alpha/beta T-cell receptor was cloned from such CTL which exhibited a moderately high affinity to the human p53(149-157) peptide. In this report, we investigated the possibility of using a recombinant tumor-specific TCR for antigen-specific elimination of cells that express the specific MHC-peptide complex. To this end, we constructed a functional single-chain Fv fragment from the cloned TCR and fused it to a very potent cytotoxic molecule, a truncated form of Pseudomonas exotoxin A (PE38). The p53 TCR scFv-P38 fusion protein was generated by in vitro refolding from bacterially-expressed inclusion bodies, and was found to be functional by its ability to bind antigen-presenting cells (APC) which express the specific p53-derived peptide. Moreover, we have shown that the p53-specific TCR scFv-PE38 molecule specifically kills APC in a peptide-dependent manner. These results represent the first time that a TCR-derived recombinant single-chain Fv fragment has been used as a targeting moiety to deliver a cytotoxic effector molecule to cells and has been able to mediate the efficient killing of the particular cell population that expresses the specific MHC/peptide complex. Similarly to antibody-based targeting approaches, TCR with tumor cell specificity represent attractive candidates for generating new, very specific targeting moieties for various modes of cancer immunotherapy.     

Generation of T cell help through a MHC class I-restricted TCR

CD4+ T cells that are activated by a MHC class II/peptide encounter can induce maturation of APCs and promote cytotoxic CD8+ T cell responses. Unfortunately, the number of well-defined tumor-specific CD4+ T cell epitopes that can be exploited for adoptive immunotherapy is limited. To determine whether Th cell responses can be generated by redirecting CD4+ T cells to MHC class I ligands, we have introduced MHC class I-restricted TCRs into postthymic murine CD4+ T cells and examined CD4+ T cell activation and helper function in vitro and in vivo. These experiments indicate that Ag-specific CD4+ T cell help can be induced by the engagement of MHC class I-restricted TCRs in peripheral CD4+ T cells but that it is highly dependent on the coreceptor function of the CD8beta-chain. The ability to generate Th cell immunity by infusion of MHC class I-restricted Th cells may prove useful for the induction of tumor-specific T cell immunity in cases where MHC class II-associated epitopes are lacking.     

Lung cancer patients’ CD4<Superscript>+</Superscript> T cells are activated in vitro by MHC II cell-based vaccines despite the presence of myeloid-derived suppressor cells

BACKGROUND: Advanced non-small cell lung cancer (NSCLC) remains an incurable disease. Immunotherapies that activate patients' T cells against resident tumor cells are being developed; however, these approaches may not be effective in NSCLC patients due to tumor-induced immune suppression. A major cause of immune suppression is myeloid-derived suppressor cells (MDSC). Because of the strategic role of CD4(+) T lymphocytes in the activation of cytotoxic CD8(+) T cells and immune memory, we are developing cell-based vaccines that activate tumor-specific CD4(+) T cells in the presence of MDSC. The vaccines are NSCLC cell lines transfected with costimulatory (CD80) plus major histocompatibility complex class II (MHC II) genes that are syngeneic to the recipient. The absence of invariant chain promotes the presentation of endogenously synthesized tumor antigens, and the activation of MHC II-restricted, tumor-antigen-specific CD4(+) T cells. METHODS: Potential vaccine efficacy was tested in vitro by priming and boosting peripheral blood mononuclear cells from ten NSCLC patients who had varying levels of MDSC. CD4(+) T cell activation was quantified by measuring Type 1 and Type 2 cytokine release. RESULTS: The vaccines activated CD4(+) T cells from all ten patients, despite the presence of CD33(+)CD11b(+) MDSC. Activated CD4(+) T cells were specific for NSCLC and did not cross-react with tumor cells derived from non-lung tissue or normal lung fibroblasts. CONCLUSIONS: The NSCLC vaccines activate tumor-specific CD4(+) T cells in the presence of potent immune suppression, and may be useful for the treatment of patients with NSCLC.     

Lack of effector cell function and altered tetramer binding of tumor-infiltrating lymphocytes

Tumor-specific CD8 T cell responses to MCA102 fibrosarcoma cells expressing the cytotoxic T cell epitope gp33 from lymphocytic choriomeningitis virus were studied. MCA102(gp33) tumors grew progressively in C57BL/6 mice, despite induction of peripheral gp33-tetramer(+) T cells that were capable of mediating antiviral protection, specific cell rejection, and concomitant tumor immunity. MCA102(gp33) tumors were infiltrated with a high number ( approximately 20%) of CD11b(+)CD11c(-) macrophage-phenotype cells that were able to cross-present the gp33 epitope to T cells. Tumor-infiltrating CD8 T cells exhibited a highly activated phenotype but lacked effector cell function. Strikingly, a significant portion of tumor-infiltrating lymphocytes expressed TCRs specific for gp33 but bound MHC tetramers only after cell purification and a 24-h resting period in vitro. The phenomenon of "tetramer-negative T cells" was not restricted to tumor-infiltrating lymphocytes from MCA102(gp33) tumors, but was also observed when Ag-specific T cells derived from an environment with high Ag load were analyzed ex vivo. Thus, using a novel tumor model, allowing us to trace tumor-specific T cells at the single cell level in vivo, we demonstrate that the tumor microenvironment is able to alter the functional activity of T cells infiltrating the tumor mass.     

Identification and characterization of the immunodominant rat HER-2/neu MHC class I epitope presented by spontaneous mammary tumors from HER-2/neu-transgenic mice

The HER-2/neu (neu-N)-transgenic mice are a clinically relevant model of breast cancer. They are derived from the parental FVB/N mouse strain and are transgenic for the rat form of the proto-oncogene HER-2/neu (neu). In this study, we report the identification of a MHC class I peptide in the neu protein that is recognized by CD8(+) T cells derived from vaccinated FVB/N mice. This 10-mer was recognized by all tumor-specific FVB/N T cells generated regardless of the TCR Vbeta region expressed by the T cell or the method of vaccination used, establishing it as the immunodominant MHC class I epitope in neu. T cells specific for this epitope were able to cure FVB/N mice of transplanted neu-expressing tumor cells, demonstrating that this is a naturally processed peptide. Altered peptide analogs of the epitope were analyzed for immunogenicity. Vaccination with dendritic cells pulsed with a heteroclitic peptide provided FVB/N and neu-N mice with increased protection against tumor challenge as compared with mice immunized with dendritic cells loaded with either wild-type or irrelevant peptide. Discovery of this epitope allows for better characterization of the CD8(+) T cell responses in the neu-N mouse model in which neu-specific tolerance must be overcome to produce effective antitumor immunity.     

Functional analysis of birch pollen allergen Bet v 1-specific regulatory T cells

Allergen-specific immunotherapy using peptides is an efficient treatment for allergic diseases. Recent studies suggest that the induction of CD4+ regulatory T (Treg) cells might be associated with the suppression of allergic responses in patients after allergen-specific immunotherapy. Our aim was to identify MHC class II promiscuous T cell epitopes for the birch pollen allergen Bet v 1 capable of stimulating Treg cells with the purpose of inhibiting allergic responses. Ag-reactive CD4+ T cell clones were generated from patients with birch pollen allergy and healthy volunteers by in vitro vaccination of PBMC using Bet v 1 synthetic peptides. Several CD4+ T cell clones were induced by using 2 synthetic peptides (Bet v 1(141-156) and Bet v 1(51-68)). Peptide-reactive CD4+ T cells recognized recombinant Bet v 1 protein, indicating that these peptides are produced by the MHC class II Ag processing pathway. Peptide Bet v 1(141-156) appears to be a highly MHC promiscuous epitope since T cell responses restricted by numerous MHC class II molecules (DR4, DR9, DR11, DR15, and DR53) were observed. Two of these clones functioned as typical Treg cells (expressed CD25, GITR, and Foxp3 and suppressed the proliferation and IL-2 secretion of other CD4+ T cells). Notably, the suppressive activity of these Treg cells required cell-cell contact and was not mediated through soluble IL-10 or TGF-beta. The identified promiscuous MHC class II epitope capable of inducing suppressive Treg responses may have important implication for the development of peptide-based Ag-specific immunotherapy to birch pollen allergy.     

Immunization with a mimotope of GD2 ganglioside induces CD8+ T cells that recognize cell adhesion molecules on tumor cells

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.     

CD8+ T cell recognition of polymorphic wild-type sequence p5365–73 peptides in squamous cell carcinoma of the head and neck

The TP53 tumor suppressor gene contains a well-studied polymorphism that encodes either proline (P) or arginine (R) at codon 72, and over half of the world’s population is homozygous for R at this codon. The wild-type sequence (wt) p53 peptide, p53_65–73, has been identified as a CD8+ T cell-defined tumor antigen for use in broadly applicable cancer vaccines. However, depending on the TP53 codon 72 polymorphism of the recipient, the induced responses to the peptides incorporating R (p53_72R) or P (p53_72P) can be “self” or “non-self.” Thus, we sought to determine which wt p53_65–73 peptide should be used in wt p53-based cancer vaccines. Despite similar predicted HLA-A2-binding affinities, the p53_72P peptide was more efficient than the p53_72R peptide in HLA-A2 stabilization assays. In vitro stimulation (IVS) of CD8+ T cells obtained from healthy HLA-A2⁺ donors with these two peptides led to the generation of CD8+ T cell effectors in one-third of the samples tested, at a frequency similar to the responsiveness to other wt p53 peptides. Interestingly, regardless of their p53 codon 72 genotype, CD8+ T cells stimulated with either p53_72P or p53_72R peptide were cross-reactive against T2 cells pulsed with either peptide, as well as HLA-A2⁺ head and neck cancer (HNC) cell lines presenting p53_72P and/or p53_72R peptides for T cell recognition. Therefore, the cross-reactivity of CD8+ T cells for the polymorphic wt p53_65–73 peptides, irrespective of their p53 codon 72 polymorphism, suggests that employing either peptide in wt p53-based vaccines can result in efficient targeting of this epitope.     

Immunoglobulin framework-derived peptides function as cytotoxic T-cell epitopes commonly expressed in B-cell malignancies

Although the idiotypic structures of immunoglobulin from malignant B cells were the first tumor-specific determinants recognized, and clinical vaccination trials have demonstrated induction of tumor-specific immunity, the function of immunoglobulin-specific CD8+ cytotoxic T lymphocytes in tumor rejection remains elusive. Here, we combined bioinformatics and a T cell-expansion system to identify human immunoglobulin-derived peptides capable of inducing cytotoxic T-lymphocyte responses. Immunogenic peptides were derived from framework regions of the variable regions of the immunoglobulin that were shared among patients. Human-leukocyte-antigen-matched and autologous cytotoxic T lymphocytes specific for these peptides killed primary malignant B cells, demonstrating that malignant B cells are capable of processing and presenting such peptides. Targeting shared peptides to induce T-cell responses might further improve current vaccination strategies in B-cell malignancies.     

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.     

CD4-directed peptide vaccination augments an antitumor response, but efficacy is limited by the number of CD8+ T cell precursors

Peptide vaccination is an immunotherapeutic strategy being pursued as a method of enhancing Ag-specific antitumor responses. To date, most studies have focused on the use of MHC class I-restricted peptides, and have not shown a correlation between Ag-specific CD8(+) T cell expansion and the generation of protective immune responses. We investigated the effects of CD4-directed peptide vaccination on the ability of CD8(+) T cells to mount protective antitumor responses in the DUC18/CMS5 tumor model system. To accomplish this, we extended the amino acid sequence of the known MHC class I-restricted DUC18 rejection epitope from CMS5 to allow binding to MHC class II molecules. Immunization with this peptide (tumor-derived extracellular signal-regulated kinase-II (tERK-II)) induced Ag-specific CD4(+) T cell effector function, but did not directly prime CD8(+) T cells. Approximately 31% of BALB/c mice immunized with tERK-II were protected from subsequent tumor challenge in a CD40-dependent manner. Priming of endogenous CD8(+) T cells in immunized mice was detected only after CMS5 challenge. Heightened CD4(+) Th cell function in response to tERK II vaccination allowed a 12-fold reduction in the number of adoptively transferred CD8(+) DUC18 T cells needed to protect recipients against tumor challenge as compared with previous studies using unimmunized mice. Furthermore, tERK-II immunization led to a more rapid and transient expansion of transferred DUC18 T cells than was seen in unimmunized mice. These findings illustrate that CD4-directed peptide vaccination augments antitumor immunity, but that the number of tumor-specific precursor CD8(+) T cells will ultimately dictate the success of immunotherapy.     

CD4+ T cell responses to self- and mutated p53 determinants during tumorigenesis in mice

We analyzed CD4+ T helper responses to wild-type (wt) and mutated (mut) p53 protein in normal and tumor-bearing mice. In normal mice, we observed that although some self-p53 determinants induced negative selection of p53-reactive CD4+ T cells, other p53 determinants (cryptic) were immunogenic. Next, BALB/c mice were inoculated with J774 syngeneic tumor cell line expressing mut p53. BALB/c tumor-bearing mice mounted potent CD4+ T cell responses to two formerly cryptic peptides on self-p53. This response was characterized by massive production of IL-5, a Th2-type lymphokine. Interestingly, we found that T cell response was induced by different p53 peptides depending upon the stage of cancer. Mut p53 gene was shown to contain a single mutation resulting in the substitution of a tyrosine by a histidine at position 231 of the protein. Two peptides corresponding to wt and mutated sequences of this region were synthesized. Both peptides bound to the MHC class II-presenting molecule (Ed) with similar affinities. However, only mut p53.225-239 induced T cell responses in normal BALB/c mice, a result strongly suggesting that high-affinity wt p53.225-239 autoreactive T cells had been eliminated in these mice. Surprisingly, CD4+ T cell responses to both mut and wt p53.225-239 peptides were recorded in J774 tumor-bearing mice, a phenomenon attributed to the recruitment of low-avidity p53.225-239 self-reactive T cells.     

Cell type-specific proteasomal processing of HIV-1 Gag-p24 results in an altered epitope repertoire

Proteasomes are critical for the processing of antigens for presentation through the major histocompatibility complex (MHC) class I pathway. HIV-1 Gag protein is a component of several experimental HIV-1 vaccines. Therefore, understanding the processing of HIV-1 Gag protein and the resulting epitope repertoire is essential. Purified proteasomes from mature dendritic cells (DC) and activated CD4(+) T cells from the same volunteer were used to cleave full-length Gag-p24 protein, and the resulting peptide fragments were identified by mass spectrometry. Distinct proteasomal degradation patterns and peptide fragments were unique to either mature DC or activated CD4(+) T cells. Almost half of the peptides generated were cell type specific. Two additional differences were observed in the peptides identified from the two cell types. These were in the HLA-B35-Px epitope and the HLA-B27-KK10 epitope. These epitopes have been linked to HIV-1 disease progression. Our results suggest that the source of generation of precursor MHC class I epitopes may be a critical factor for the induction of relevant epitope-specific cytotoxic T cells.     

Identification of an SSX-2 epitope presented by dendritic cells to circulating autologous CD4+ T cells

Accumulating evidence supports the requirement for both tumor-specific CD8(+) and CD4(+) T cell responses for efficient tumor rejection to occur. Because of its expression in different tumor types, the cancer/testis Ag encoded by the synovial sarcoma X breakpoint 2 (SSX-2) gene is among the most relevant candidates for the development of generic cancer vaccines. The immunogenicity of SSX-2 has been previously corroborated by detection of specific humoral and CD8(+) T cell responses in cancer patients. In this study we report identification of the first CD4(+) T cell epitope encoded by SSX-2. The identified epitope mapped to the 19-34 region of the protein and was recognized by CD4(+) T cells from an Ag-expressing melanoma patient in association with HLA-DPB1*0101. The absence of detectable response in healthy donors and other patients suggests that SSX-2-specific CD4(+) T cells in the responder patient had been previously expanded in vivo in response to the autologous tumor. The epitope did not appear to be presented on the surface of tumor cells at levels sufficient to allow direct recognition. In contrast, it was efficiently presented by autologous dendritic cells, supporting the concept that processing by professional APC is the main pathway through which the CD4(+) T cell immunoresponse to tumor Ags occurs in vivo.     

Antigen processing for presentation to CD4+ T cells.

The immune system surveys the organism for the presence of foreign or abnormal structures. An important role in the immune response is assumed by T lymphocytes that recognize foreign antigen while tolerating self-proteins. T lymphocytes can recognize only peptide fragments that are presented to them by molecules of the major histocompatibility complex (MHC). Antigen processing for presentation to T cells involves distinct cellular compartments where peptides and MHC molecules interact. Whereas class I MHC molecules (recognized by CD8+ cytotoxic T cells) acquire peptides in an early biosynthetic compartment, class II molecules (recognized by CD4+ helper T cells) acquire peptides most efficiently in an endocytic compartment. It has emerged recently that the class II processing compartment can be fed not only from the outside with exogenous antigen but also from endogenous sources, including membrane-associated and cytosolic proteins. The potential sources of proteins that can trigger a helper T cell response during viral infections and that can induce self-tolerance are thus much wider than previously anticipated.     

The wild-type sequence (wt) p53(25-35) peptide induces HLA-DR7 and HLA-DR11-restricted CD4+ Th cells capable of enhancing the ex vivo expansion and function of anti-wt p53(264-272) peptide CD8+ T cells

Tumor peptide-based vaccines are more effective when they include tumor-specific Th cell-defined as well as CTL-defined peptides. Presently, two overlapping wild-type sequences (wt) p53 helper peptides, p53(108-122) and p53(110-124), have been identified as HLA-DR1- and/or HLA-DR4-restricted epitopes. These HLA-DR alleles are expressed by approximately 35% of subjects with cancer. To identify Th cell-defined wt p53 peptides suitable for use on the remaining subject population, a dendritic cell (DC)-based coculture system was developed. CD4+ T cells isolated from PBMC obtained from HLA-DR4- normal donors were stimulated ex vivo with autologous DC transfected with wt p53 or mutant p53 cDNA. Reactivity of T cells was tested in ELISPOT IFN-gamma assays against DC pulsed individually with a panel of algorithm-predicted, multiple HLA-DR-binding wt p53 peptides. The wt p53(25-35) peptide was identified as capable of inducing and being recognized by CD4+ T cells in association, at a minimum, with HLA-DR7 and -DR11 molecules, each of which is expressed by approximately 15% of the population. In addition, the presence of anti-p53(25-35) CD4+ Th cells was shown to enhance the in vitro generation/expansion of HLA-A2-restricted, anti-wt p53(264-272) CD8+ T cells, which from one donor were initially "nonresponsive" to the wt p53(264-272) peptide. The wt p53(25-35) peptide has attributes of a naturally presented Th cell-defined peptide, which could be incorporated into antitumor vaccines applicable to a broader population of subjects for whom a wt p53 helper peptide is presently unavailable, as well as used for monitoring anti-p53 Th cell activity in cancer subjects receiving p53-based immunotherapy.     

CD4+/CD25+ regulatory cells inhibit activation of tumor-primed CD4+ T cells with IFN-gamma-dependent antiangiogenic activity, as well as long-lasting tumor immunity elicited by peptide vaccination

CD25(+) regulatory T (T reg) cells suppress the activation/proliferation of other CD4(+) or CD8(+) T cells in vitro. Also, down-regulation of CD25(+) T reg cells enhance antitumor immune responses. In this study, we show that depletion of CD25(+) T reg cells allows the host to induce both CD4(+) and CD8(+) antitumoral responses following tumor challenge. Simultaneous depletion of CD25(+) and CD8(+) cells, as well as adoptive transfer experiments, revealed that tumor-specific CD4(+) T cells, which emerged in the absence of CD25(+) T reg cells, were able to reject CT26 colon cancer cells, a MHC class II-negative tumor. The antitumoral effect mediated by CD4(+) T cells was dependent on IFN-gamma production, which exerted a potent antiangiogenic activity. The capacity of the host to mount this antitumor response is lost once the number of CD25(+) T reg cells is restored over time. However, CD25(+) T reg cell depletion before immunization with AH1 (a cytotoxic T cell determinant from CT26 tumor cells) permits the induction of a long-lasting antitumoral immune response, not observed if immunization is conducted in the presence of regulatory cells. A study of the effect of different levels of depletion of CD25(+) T reg cells before immunization with the peptide AH1 alone, or in combination with a Th determinant, unraveled that Th cells play an important role in overcoming the suppressive effect of CD25(+) T reg on the induction of long-lasting cellular immune responses.     

Binding of MAR-DNA elements by mutant p53: Possible implications for its oncogenic functions

The tumor suppressor p53 is a multifunctional protein whose main duty is to preserve the integrety of the genome. This function of wild-type p53 as “guardian of the genome” is achieved at different levels, as a cell cycle checkpoint protein, halting the cell cycle upon DNA damage, and via a direct involvement in processes of DNA repair. Alternatively, p53 can induce apoptosis. Mutations in the p53 gene occur in about 50% of all human tumors and eliminate the tumor suppressor functions of p53. However, many mutant p53 proteins have not simply lost tumor suppressor functions but have gained oncogenic properties which contribute to the progression of tumor cells to a more malignant phenotype. The molecular basis for this gain of function of mutant p53 is still unknown. However, mutant (mut) p53 specifically binds to nuclear matrix attachment region (MAR) DNA elements. MAR elements constitute important higher order regulatory elements of chromatin structure and function. By binding to these elements, mut p53 could modulate important cellular processes, like gene expression, replication, and recombination, resulting in phenotypic alterations of the tumor cells. Mut p53 thus could be the first representative of a new class of oncogenes, which exert their functions via long-range alterations or perturbation of chromatin structure and function. © 1996 Wiley-Liss, Inc.     

Minor structural changes in a mutated human melanoma antigen correspond to dramatically enhanced stimulation of a CD4+ tumor-infiltrating lymphocyte line

While most immunotherapies for cancer have focused on eliciting specific CD8+ cytotoxic T lymphocyte killing of tumor cells, a mounting body of evidence suggests that stimulation of anti-tumor CD4+ T cell help may be required for highly effective therapy. Several MHC class II-restricted tumor antigens that specifically activate such CD4+ helper T lymphocytes have now been identified, including one from a melanoma tumor that is caused by a single base-pair mutation in the glycolytic enzyme triosephosphate isomerase. This mutation results in the conversion of a threonine residue to isoleucine within the antigenic epitope, concomitant with a greater than five log-fold increase in stimulation of a CD4+ tumor-infiltrating lymphocyte line. Here, we present the crystal structures of HLA-DR1 in complex with both wild-type and mutant TPI peptide antigens, the first structures of tumor peptide antigen/MHC class II complexes recognized by CD4+ T cells to be reported. These structures show that very minor changes in the binding surface for T cell receptor correspond to the dramatic differences in T cell stimulation. Defining the structural basis by which CD4+ T cell help is invoked in an anti-tumor immune response will likely aid the design of more effective cancer immunotherapies.