The shared tumor associated antigen cyclin-A2 is recognized by high-avidity T-cells.

Cyclin-A2, a key cell cycle regulator, has been shown to be overexpressed in various types of malignancies with little expression in normal tissue. Such tumor-associated genes potentially are useful targets for cancer immunotherapy. However, high-avidity cyclin-specific T cells are considered to be thymically deleted. We identified at least one nonameric HLA-A*0201 binding cyclin-A2 epitope by a reverse immunology approach. Using a highly efficient T-cell expansion system that is based on CD40-activated B (CD40-B) cells as sole antigen-presenting cells we successfully generated cyclin-A2 specific CTL from HLA-A*0201(+) donors. Interestingly, high-avidity cyclin-A2 specific CTL lines, which recognized peptide-pulsed and antigen expressing target cells, were indeed generated by stimulation with CD40-B cells when pulsed with low concentrations of peptide, whereas CD40-B cells pulsed at saturating concentrations could only induce low-avidity CTL, which recognized peptide-pulsed target cells only. One high-avidity CTL line was subcloned and CTL clones, whose peptide concentration required for half-maximal lysis were less than 1 nM, could lyse cyclin-A2 expressing tumor cells. Taken together, cyclin A2 is an attractive candidate for immune intervention in a significant number of cancer patients and high-avidity T cells can be readily generated using CD40-B cells as antigen-presenting cells.

Cyclin D1-specific cytotoxic T lymphocytes are present in the repertoire of cancer patients: implications for cancer immunotherapy.

PURPOSE: Cyclin D1, a key cell cycle regulator, is overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for cancer immunotherapy. Nevertheless, it had previously been suggested that efficient T cells recognizing cyclin D1-derived epitopes are absent from the repertoire because of thymic deletion. We attempted to induce autologous CTL from healthy donors and patients with cyclin D1-overexpressing tumors using a highly efficient T-cell expansion system based on CD40-activated B cells as antigen-presenting cells. EXPERIMENTAL DESIGN: Cyclin D1-derived, HLA-A*0201-restricted epitopes were predicted by multiple computer algorithms, screened in HLA-A2-binding assays, and used for T-cell stimulation. The generated CTL lines and clones were analyzed by IFN-gamma enzyme-linked immunosorbent spot assay or cytolysis assay. RESULTS: After screening, at least two naturally processed and presented HLA-A*0201-binding cyclin D1 epitopes were identified. CTL specific for these epitopes could be successfully generated from HLA-A2(+) donors. T cells efficiently recognized target cells pulsed with the cognate peptide and cyclin D1-expressing tumor cell lines in an HLA-A*0201-restricted manner. More importantly, HLA-A*0201-matched, primary cyclin D1(+) tumor cells were efficiently recognized by cyclin D1-specific CTL. These CTL could be generated from patients with mantle cell lymphoma and cyclin D1(+) colon cancer. CONCLUSIONS: These results underscore that cyclin D1 needs to be considered as a target for broad-based antitumor immunotherapy.

Lethal graft-versus-host disease in congenital neutropenia caused by p14 deficiency after allogeneic bone marrow transplantation from an HLA-identical sibling.

The molecular heterogeneity of severe congenital neutropenia (SCN) is increasingly recognized and may influence the risk-benefit assessment of therapeutic strategies. We report on a patient with p14 deficiency who succumbed to severe grade IV graft-versus-host disease (GvHD) after a human leukocyte antigen-identical bone marrow transplantion (BMT) from a sibling donor. Before BMT, in vitro generated p14-deficient dendritic cells showed a markedly elevated tumor necrosis factor (TNF-) alpha production upon toll-like receptor stimulation. We hypothesize that p14 deficiency predisposes to GvHD through increased TNF-alpha production. Adequate genetic testing is needed to prospectively assess potential risk factors for GvHD in defined SCN subgroups.

Intrathecal rituximab treatment for pediatric post-transplant lymphoproliferative disorder of the central nervous system.

Post-transplant lymphoproliferative disorder (PTLD) in the central nervous system (CNS) has a poor prognosis. New therapeutic approaches should be explored. We report our experience with intrathecal administration of rituximab in a 10-year-old kidney allograft recipient with PTLD in the CNS. After standard treatment had failed, we tried to treat the patient by administering rituximab directly into the cerebral ventricle through an Omaya reservoir, in addition to conventional intrathecal and systemic chemotherapy. This strategy resulted in a disappearance of clinical symptoms and a negative positron emission tomogram. Intrathecal administration of rituximab may be a feasible approach in children with PTLD in the CNS. However, its specific role in our patient remains uncertain.

CNS or bone marrow involvement as risk factors for poor survival in post-transplantation lymphoproliferative disorders in children after solid organ transplantation.

PURPOSE: To identify prognostic factors of survival in pediatric post-transplantation lymphoproliferative disorder (PTLD) after solid organ transplantation. PATIENTS AND METHODS: A multicenter, retrospective case analysis of 55 pediatric solid organ graft recipients (kidney, liver, heart/lung) developing PTLD were reported to the German Pediatric-PTLD registry. Patient charts were analyzed for tumor characteristics (histology, immunophenotypes, cytogenetics, Epstein-Barr virus [EBV] detection), stage, treatment, and outcome. Probability of overall and event-free survival was analyzed in defined subgroups using univariate and Cox regression analyses. RESULTS: PTLD was diagnosed at a median time of 29 months after organ transplantation, with a significantly shorter lag time in liver (0.83 years) versus heart or renal graft recipients (3.33 and 3.10 years, respectively; P = .001). The 5-year overall and event-free survival was 68% and 59%, respectively, with 59% of patients surviving 10 years. Stage IV disease with bone marrow and/or CNS involvement was associated independently with poor survival (P = .0005). No differences in outcome were observed between early- and late-onset PTLD, monomorphic or polymorphic PTLD, and EBV-positive or EBV-negative PTLD, respectively. Patients with Burkitt or Burkitt-like PTLD and c-myc translocations had short survival (< 1 year). CONCLUSION: Stage IV disease is an independent risk factor for poor survival in pediatric PTLD patients. Prospective multicenter trials are needed to delineate additional risk factors and to assess treatment approaches for pediatric PTLD.

CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.

CD40-activated B cells (CD40-B cells) have previously been introduced as an alternative source of antigen-presenting cells for immunotherapy. CD40-B cells can prime naive and expand memory T cells, and they can be generated in large numbers from very small amounts of peripheral blood derived from healthy individuals or cancer patients alike. Administration of CD40-B cells as a cellular adjuvant would require these cells to migrate toward secondary lymphoid organs and attract T cells in situ, processes guided by specific chemokines and chemokine receptors. Here, we demonstrate that primary, human CD40-B cells express a pattern of adhesion molecules and chemokine receptors necessary for homing to secondary lymphoid organs and have the capacity to migrate to cognate ligands. Furthermore, we show that CD40-B cells express important T-cell attractants and induce strong T-cell chemotaxis. These findings further support the use of CD40-B cells as cellular adjuvant for cancer immunotherapy.

Identification of a new HLA-A*0201-restricted cryptic epitope from CYP1B1.

Cytochrome P450 1B1 (CYP1B1) was recently shown to be a candidate tumor antigen broadly expressed in solid and hematologic malignancies. Nevertheless, use of such self-antigens as targets for immune intervention can be limited because of loss of high-avidity T cells during negative selection in the thymus. Recent data suggest that targeting of cryptic epitopes may represent a way to circumvent such self-tolerance and induce efficient antitumor CTL responses. Here, we present the identification and characterization of a novel, cryptic HLA-A*0201-binding peptide from CYP1B1. The nanomer CYP246 was identified by epitope deduction using algorithms to predict HLA-A*0201-binding peptides. CYP246 is characterized by strong initial HLA-A*0201 binding but a short MHC/peptide binding half-life. Expansion of high-avidity CTL was readily possible using autologous CD40-activated B cells from normal donors and cancer patients as antigen-presenting cells, suggesting that an intact T-cell repertoire can be expanded for this epitope. Lysis of CYP1B1-expressing, HLA-A*0201+ tumor cell lines and primary tumor cells confirmed that sufficient levels of CYP246 are presented by tumor cells for effector CTL killing. These findings indicate that CYP246 is a candidate cryptic epitope for immune interventions in which tumor CYP1B1 is targeted.

Autoantibodies frequently detected in patients with aplastic anemia.

Although accumulating evidence strongly suggests that aplastic anemia (AA) is a T cell-mediated autoimmune disease, no target antigens have yet been described for AA. In autoimmune diseases, target autoantigens frequently induce not only cellular T-cell responses but also humoral B-cell responses. We hypothesized that the presence of antigen-specific autoantibodies could be used as a "surrogate marker" for the identification of target T-cell autoantigens in AA patients. We screened a human fetal liver library for serologic reactivity against hematopoietic stem/progenitor cell antigens and isolated 32 genes. In 7 of 18 AA patients, an immunoglobulin G (IgG) antibody response was detected to one of the genes, kinectin, which is expressed in all hematopoietic cell lineages tested including CD34+ cells. No response to kinectin was detected in healthy volunteers, multiply transfused non-AA patients, or patients with other autoimmune diseases. Epitope mapping of IgG autoantibodies against kinectin revealed that the responses to several of the epitopes were shared by different AA patients. Moreover, CD8+ cytotoxic T cells raised against kinectin-derived peptides suppressed the colony formation of granulocyte macrophage colony-forming units (CFU-GMs) in an HLA class I-restricted fashion. These results suggest that kinectin may be a candidate autoantigen that is involved in the pathophysiology of AA.

The shared tumor-associated antigen cytochrome P450 1B1 is recognized by specific cytotoxic T cells.

Cytochrome P450 1B1 (CYP1B1), a drug-metabolizing extrahepatic enzyme, was recently shown to be overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for anticancer therapy, particularly cancer immunotherapeutics. We identified HLA-A*0201-binding peptides and a naturally processed and presented T-cell epitope capable of inducing CYP1B1-specific cytotoxic T lymphocytes (CTLs) in HLA-A2 transgenic mice. Furthermore, the induction of CYP1B1-specific T cells was demonstrated in healthy donors and cancer patients. These T cells efficiently lysed target cells pulsed with the cognate peptide. More important, HLA-A2-matched tumor cell lines and primary malignant cells were also recognized by CYP1B1-specific CTLs. These findings form the basis of a phase 1 clinical trial exploring a DNA-based vector encoding CYP1B1 for widely applicable cancer immunotherapy conducted at the Dana-Farber Cancer Institute.

Viral antigen-specific CD8+ T-cell responses are impaired in multiple myeloma.

Multiple myeloma (MM) is associated with defects of humoral and cellular immunity, however, little is known about the frequency and function of antigen-specific CD8+ T cells. Such information might be critical for the development of immunotherapy for MM patients. As a model, we assessed the frequency and proliferation of CD8+ T cells specific for HLA-A*0201-restricted immunodominant epitopes from influenza A (Inf A) and Epstein-Barr virus (EBV). Experiments in identical twins demonstrated reduced numbers of antigen-specific T cells after ex-vivo antigenic challenge in the MM twin when compared with the healthy twin. Similarly, the proliferation and frequency of EBV- and Inf A-specific T cells was also significantly reduced in a cohort of 24 previously untreated or conventionally treated MM patients when compared with 19 healthy individuals. In contrast, MM patients studied after receiving an autologous stem cell transplantation showed strikingly higher frequencies of EBV-specific T cells with potential to proliferate ex vivo, suggesting that EBV-specific T cells are readily expandable under these circumstances. These data identify an impaired response of CD8+ T cells in MM patients, which might in part explain the relatively limited success of anti-MM immunisations. Prospective studies will determine whether such immune assessment strategies may identify patients more likely to benefit from cancer immunotherapy.

Melanoma inhibitor of apoptosis protein (ML-IAP) is a target for immune-mediated tumor destruction.

The identification of antigens associated with tumor destruction is a major goal of cancer immunology. Vaccination with irradiated tumor cells engineered to secrete granulocyte-macrophage colony stimulating factor generates potent, specific, and long-lasting antitumor immunity through improved tumor antigen presentation by dendritic cells and macrophages. A phase I clinical trial of this immunization strategy in patients with disseminated melanoma revealed the consistent induction in distant metastases of dense T and B cell infiltrates that effectuated substantial tumor necrosis and fibrosis. To delineate the target antigens of this vaccine-stimulated tumor destruction, we screened a melanoma cDNA expression library with postimmunization sera from a long-term responding patient (K030). High-titer IgG antibodies recognized melanoma inhibitor of apoptosis protein (ML-IAP), a caspase antagonist containing a single baculoviral IAP repeat and a COOH-terminal RING domain. Although K030 harbored antibodies to ML-IAP at the time of study entry, multiple courses of vaccination over 4 years increased antibody titers and elicited isotype switching. Moreover, lymphocyte infiltrates in necrotic metastases included CD4+ and CD8+ T cells specific for ML-IAP, as revealed by proliferation, tetramer, enzyme-linked immunospot, and cytotoxicity analysis. Whereas melanoma cells in densely infiltrated lesions showed strong ML-IAP expression by immunohistochemistry, lethal disease progression was associated with the loss of ML-IAP staining and the absence of lymphocyte infiltrates. These findings demonstrate that ML-IAP can serve as a target for immune-mediated tumor destruction, but that antigen-loss variants can accomplish immune escape.

Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.

CD40 engagement is the major signal that induces B cells to efficiently present antigen to T cells. We previously demonstrated that human peripheral blood-derived CD40-activated B cells (CD40-B cells) function as antigen-presenting cells (APCs). Here, we have established a culture system to generate these APCs under clinically applicable conditions using guanylic acid-grade soluble trimeric CD40 ligand. To monitor APC function and antigen loading for these cells, simple and efficient quality control assays have been developed. Using this approach, we demonstrate that CD40-B cells from healthy donors and cancer patients are fully functional and equally expanded in long-term cultures. These B cells boost robust memory T-cell responses, but more importantly, they also prime naive T-cell responses against neoantigens ex vivo. CD40-B cells overcome current obstacles, such as the difficulty of isolation, generation, and long-term expansion observed with other APCs. Therefore, they are an excellent source of professional APCs for immune assessment, antigen discovery, and antigen-specific immunotherapy.