Brad Stone

Standard Treatments Induce Antigen-Specific Immune Responses in Prostate Cancer

Purpose: Prostate tumors express antigens that are recognized by the immune system in a significant proportion of patients; however, little is known about the effect of standard treatments on tumor-specific immunity. Radiation therapy induces expression of inflammatory and immune-stimulatory molecules, and neoadjuvant hormone therapy causes prominent T-cell infiltration of prostate tumors. We therefore hypothesized that radiation therapy and hormone therapy may initiate tumor-specific immune responses. Experimental Design: Pretreatment and posttreatment serum samples from 73 men with nonmetastatic prostate cancer and 50 cancer-free controls were evaluated by Western blotting and SEREX (serological identification of antigens by recombinant cDNA expression cloning) antigen arrays to examine whether autoantibody responses to tumor proteins arose during the course of standard treatment. Results: Western blotting revealed the development of treatment-associated autoantibody responses in patients undergoing neoadjuvant hormone therapy (7 of 24, 29.2%), external beam radiation therapy (4 of 29, 13.8%), and brachytherapy (5 of 20, 25%), compared with 0 of 14 patients undergoing radical prostatectomy and 2 of 36 (5.6%) controls. Responses were seen within 4 to 9 months of initiation of treatment and were equally prevalent across different disease risk groups. Similarly, in the murine Shionogi tumor model, hormone therapy induced tumor-associated autoantibody responses in 5 of 10 animals. In four patients, SEREX immunoscreening of a prostate cancer cDNA expression library identified several antigens recognized by treatment-associated autoantibodies, including PARP1, ZNF707 + PTMA, CEP78, SDCCAG1, and ODF2. Conclusion: We show for the first time that standard treatments induce antigen-specific immune responses in prostate cancer patients. Thus, immunologic mechanisms may contribute to clinical outcomes after hormone and radiation therapy, an effect that could potentially be exploited as a practical, personalized form of immunotherapy.

Immunoediting of Cancers May Lead to Epithelial to Mesenchymal Transition

Tumors evade both natural and pharmacologically induced (e.g., vaccines) immunity by a variety of mechanisms, including induction of tolerance and immunoediting. Immunoediting results in reshaping the immunogenicity of the tumor, which can be accompanied by loss of Ag expression and MHC molecules. In this study, we evaluated immunoediting in the neu-transgenic mouse model of breast cancer. A tumor cell line that retained expression of rat neu was generated from a spontaneous tumor of the neu-transgenic mouse and, when injected into the non-transgenic parental FVB/N mouse, resulted in the development of a strong immune response, initial rejection, and ultimately the emergence of neu Ag-loss variants. Morphologic and microarray data revealed that the immunoedited tumor cells underwent epithelial to mesenchymal transition accompanied by an up-regulation of invasion factors and increased invasiveness characteristic of mesenchymal tumor cells. These results suggest that immunoediting of tumor results in cellular reprogramming may be accompanied by alterations in tumor characteristics including increased invasive potential. Understanding the mechanisms by which tumors are immunoedited will likely lead to a better understanding of how tumors evade immune detection.

Sporadic ALS has compartment-specific aberrant exon splicing and altered cell–matrix adhesion biology

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive weakness from loss of motor neurons. The fundamental pathogenic mechanisms are unknown and recent evidence is implicating a significant role for abnormal exon splicing and RNA processing. Using new comprehensive genomic technologies, we studied exon splicing directly in 12 sporadic ALS and 10 control lumbar spinal cords acquired by a rapid autopsy system that processed nervous systems specifically for genomic studies. ALS patients had rostral onset and caudally advancing disease and abundant residual motor neurons in this region. We created two RNA pools, one from motor neurons collected by laser capture microdissection and one from the surrounding anterior horns. From each, we isolated RNA, amplified mRNA, profiled whole-genome exon splicing, and applied advanced bioinformatics. We employed rigorous quality control measures at all steps and validated findings by qPCR. In the motor neuron enriched mRNA pool, we found two distinct cohorts of mRNA signals, most of which were up-regulated: 148 differentially expressed genes (P ≤ 10−3) and 411 aberrantly spliced genes (P ≤ 10−5). The aberrantly spliced genes were highly enriched in cell adhesion (P ≤ 10−57), especially cell–matrix as opposed to cell–cell adhesion. Most of the enriching genes encode transmembrane or secreted as opposed to nuclear or cytoplasmic proteins. The differentially expressed genes were not biologically enriched. In the anterior horn enriched mRNA pool, we could not clearly identify mRNA signals or biological enrichment. These findings, perturbed and up-regulated cell–matrix adhesion, suggest possible mechanisms for the contiguously progressive nature of motor neuron degeneration. Data deposition: GeneChip raw data (CEL-files) have been deposited for public access in the Gene Expression Omnibus (GEO), www.ncbi.nlm.nih.gov/geo, accession number GSE18920.

Serologic analysis of ovarian tumor antigens reveals a bias toward antigens encoded on 17q

We utilized SEREX immunoscreening to identify a set of novel tumor antigens that are associated with human serous ovarian cancer and may prove useful for the early detection and treatment of this disease. Extensive screening with a panel of sera from 25 late‐stage ovarian cancer patients against 3 independent cDNA libraries identified a set of 9 antigens that were immunogenic in more than 1 patient and not in a panel of 20–45 normal female serum donors. These antigens include p53, NY‐ESO‐1, UBQLN1, HOXB6, TOP2A, putative helicase‐RUVBL (RUVBL), HMBA‐inducible (HEXIM1), DDX5 and HDCMA. Ten of 25 ovarian cancer patients (40%) expressed serum IgG to at least 1 of these antigens, while 14% (4/25) had antibodies to 2 or more antigens. Unexpectedly, 4 antigens identified in this screen, DDX5, HEXIM1, TOP2A and HOXB6, are encoded within a region of 17q that also includes the genes for HER2/neu, Homeobox‐B7 and BRCA1. Real‐time RT‐PCR analysis showed that mRNA for HER2/neu and 3 SEREX‐defined antigens, TOP2A, HOXB6 and DDX5, was more abundant in ovarian tumors than most normal tissues, including normal and benign ovarian tissues, suggesting that elevated expression of genes encoded within this region of chromosome 17 is a common event in ovarian tumors. Thus, these abnormal expression patterns combined with the endogenous immune response suggests that these antigens represent potential targets for immunotherapy.

A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations

Development of an antimalarial subunit vaccine inducing protective cytotoxic T lymphocyte (CTL)-mediated immunity could pave the way for malaria eradication. Experimental immunization with sporozoites induces this type of protective response, but the extremely large number of proteins expressed by Plasmodium parasites has so far prohibited the identification of sufficient discrete T-cell antigens to develop subunit vaccines that produce sterile immunity. Here, using mice singly immunized with Plasmodium yoelii sporozoites and high-throughput screening, we identified a unique CTL response against the parasite ribosomal L3 protein. Unlike CTL responses to the circumsporozoite protein (CSP), the population of L3-specific CTLs was not expanded by multiple sporozoite immunizations. CSP is abundant in the sporozoite itself, whereas L3 expression does not increase until the liver stage. The response induced by a single immunization with sporozoites reduces the parasite load in the liver so greatly during subsequent immunizations that L3-specific responses are only generated during the primary exposure. Functional L3-specific CTLs can, however, be expanded by heterologous prime-boost regimens. Thus, although repeat sporozoite immunization expands responses to preformed antigens like CSP that are present in the sporozoite itself, this immunization strategy may not expand CTLs targeting parasite proteins that are synthesized later. Heterologous strategies may be needed to increase CTL responses across the entire spectrum of Plasmodium liver-stage proteins.

Discovery of T Cell Antigens by High-Throughput Screening of Synthetic Minigene Libraries

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.

Adoptive immunotherapy against allogeneic kidney grafts in dogs with stable hematopoietic trichimerism.

Dogs given nonmyeloablative conditioning and marrow grafts from 2 dog leukocyte antigen (DLA)-identical littermate donors developed stable trichimerism and stably accepted a subsequent kidney graft from one of the marrow donors without the need for immunosuppression. In this study, we used trichimeras to evaluate strategies for adoptive immunotherapy to solid tumors, using the kidney as a tumor surrogate. Three DLA-identical trichimeric recipients were established by simultaneously infusing marrow from 2 DLA-identical donor dogs into a DLA-identical recipient conditioned with 2 Gy of total body irradiation (TBI) and given a short course of postgraft immunosuppression. After stable hematopoietic engraftment was confirmed, a kidney was transplanted from 1 of the 2 marrow donors into each respective trichimeric recipient. Peripheral blood lymphocytes from each kidney donor were then used to sensitize the alternate marrow donor. The trichimeric recipients were given donor lymphocyte infusions (DLIs) from the sensitized dogs and monitored for chimerism, graft-versus-host disease (GVHD), and kidney rejection. After DLI, we observed both prompt rejection of the transplanted marrow and donor kidney and disappearance of corresponding hematopoietic chimerism. Presumably due to shared minor histocompatibility antigens, host chimerism also disappeared, and GVHD in skin, gut, and liver developed. The native kidneys, although exhibiting lymphocytic infiltration, remained functionally normal. This study demonstrates that under certain experimental conditions, the kidney—an organ ordinarily not involved in graft-versus-host reactions—can be targeted by sensitized donor lymphocytes.

Absence of SPARC leads to impaired lens circulation

SPARC is a matricellular glycoprotein involved in regulation of extracellular matrix, growth factors, adhesion, and migration. SPARC-null mice have altered basement membranes and develop posterior sub-capsular cataracts with cell swelling and equatorial vacuoles. Exchange of fluid, nutrients, and waste products in the avascular lens is driven by a unique circulating ion current. In the absence of SPARC, increased circulation of fluid, ions, and small molecules led to increased fluorescein distribution in vivo, loss of resting membrane polarization, and altered distribution of small molecules. Microarray analysis of SPARC-null lenses showed changes in gene expression of ion channels and receptors, matrix and adhesion genes, cytoskeleton, immune response genes, and cell signaling molecules. Our results confirm the hypothesis that the regulation of SPARC on cell-capsular matrix interactions can increase the circulation of fluid and ions in the lens, and the phenotype in the SPARC-null mouse lens is the result of multiple intersecting functional pathways.

Development of a minor histocompatibility antigen vaccine regimen in the canine model of hematopoietic cell transplantation

Background Minor histocompatibility antigen (miHA) vaccines have the potential to augment graft-versus-tumor effects without graft-versus-host disease (GVHD). We used mixed hematopoietic chimerism in the canine model of major histocompatibility complex–matched allogeneic hematopoietic cell transplantation as a platform to develop a miHA vaccination regimen. Methods We engineered DNA plasmids and replication-deficient human adenovirus type 5 constructs encoding large sections of canine SMCY and the entire canine SRY gene. Results Priming with replication-deficient human adenovirus type 5 constructs and boosting with ex vivo plasmid-transfected dendritic cells and cutaneous delivery of plasmids with a particle-mediated epidermal delivery device (PMED) in 2 female dogs induced antigen-specific T-cell responses. Similar responses were observed after a prime-boost vaccine regimen in three female hematopoietic cell transplantation donors. Subsequent donor lymphocyte infusion resulted in a significant change of chimerism in 1 of 3 male recipients without any signs of graft-versus-host disease. The change in chimerism in the recipient occurred in association with the development of CD4+ and CD8+ T-cell responses to the same peptide pools detected in the donor. Conclusions These studies describe the first in vivo response to miHA vaccination in a large, outbred animal model without using recipient cells to sensitize the donor. This model provides a platform for ongoing experiments designed to define optimal miHA targets and develop protocols to directly vaccinate the recipient.

Identification of novel HLA class II target epitopes for generation of donor-specific T regulatory cells

Therapies capable of generating host T regulatory cells (T(R)) responsive to donor-specific HLA-class II minor histocompatibility antigens have the potential to promote tolerance of a transplanted organ. Our group has developed a novel approach for the identification of potentially therapeutic T(R) target antigens. We perform parallel non-synonymous SNP genotyping of HLA-identical subject pairs to identify peptide variations expressed by only one of the two subjects. Variant peptide pairs are then evaluated for binding a shared HLA-class II allele. Minor peptides predicted to bind HLA-class II with greater affinity than the common variant peptide are tested for HLA class II binding and in vitro induction of suppressive CD4+ T cells. Using this approach we have identified multiple pairs of variant peptides capable of differential binding and induction of suppressive CD4+ T cells. These data demonstrate the feasibility of identifying potentially therapeutic HLA class II minor antigens for generation of donor-specific T(R).