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Tissue-resident memory CD8+ T (Trm) cells in the liver are critical for long-term protection against pre-erythrocytic Plasmodium infection. Such protection can usually be induced with three to five doses of i.v. administered radiation-attenuated sporozoites (RAS). To simplify and accelerate vaccination, we tested a DNA vaccine designed to induce potent T cell responses against the SYVPSAEQI epitope of Plasmodium yoelii circumsporozoite protein. In a heterologous “prime-and-trap” regimen, priming using gene gun–administered DNA and boosting with one dose of RAS attracted expanding Ag-specific CD8+ T cell populations to the liver, where they became Trm cells. Vaccinated in this manner, BALB/c mice were completely protected against challenge, an outcome not reliably achieved following one dose of RAS or following DNA-only vaccination. This study demonstrates that the combination of CD8+ T cell priming by DNA and boosting with liver-homing RAS enhances formation of a completely protective liver Trm cell response and suggests novel approaches for enhancing T cell–based pre-erythrocytic malaria vaccines.

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.

Dogs given nonmyeloablative conditioning and marrow grafts from two 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. Here, we used trichimeras to evaluate strategies of adoptive immunotherapy to solid tumors, using the kidney as a tumor surrogate. Three DLA-identical trichimeric recipients were established by simultaneously infusing marrow from two DLA-identical donor dogs into a DLA-identical recipient conditioned with 2 Gy total body irradiation and given a short course of postgrafting immunosuppression. After confirming stable hematopoietic engraftment, a kidney was transplanted from one of the two marrow donors into each respective trichimeric recipient. Peripheral blood lymphocytes (PBL) from each kidney donor were then used to sensitize the alternate marrow donor. Donor lymphocyte infusions (DLI) from the sensitized dogs were given to the trichimeric recipients, whereupon chimerism, graft-versus-host disease (GvHD), and kidney rejection were monitored. After DLI, we observed both prompt rejection of the transplanted marrow-donor kidney and disappearance of corresponding hematopoietic chimerism. Presumably, owing to shared minor histocompatibility antigens, host chimerism also disappeared and GvHD in skin, gut, and liver developed. The native kidneys, while showing lymphocytic infiltration, remained functionally normal. The current study demonstrated that under certain experimental conditions, the kidney, an organ ordinarily not involved in graft-versus-host reactions, can be targeted by sensitized donor lymphocytes.

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.

Biomarkers for early detection of epithelial ovarian cancer (EOC) are urgently needed. Patients can generate antibodies to tumor-associated antigens (TAAs). We tested multiplex detection of antibodies to candidate ovarian TAAs and statistical modeling for discrimination of sera of EOC patients and controls. Binding of serum antibody of women with EOC or healthy controls to candidate TAA-coated microspheres was assayed in parallel. A Bayesian model/variable selection approach using Markov Chain Monte Carlo computations was applied to these data, and serum CA125 values, to determine the best predictive model. The selected model was subjected to area under the receiver-operator curve (AUC) analysis. The best model generated an AUC of 0.86 [95% confidence interval (95% CI), 0.78-0.90] for discrimination between sera of EOC patients and healthy patients using antibody specific to p53, NY-CO-8, and HOXB7. Inclusion of CA125 in the model provided an AUC of 0.89 (95% CI, 0.84-0.92) compared with an AUC of 0.83 (95% CI, 0.81-0.85) using CA125 alone. However, using TAA responses alone, the model discriminated between independent sera of women with nonmalignant gynecologic conditions and those with advanced-stage or early-stage EOC with AUCs of 0.71 (95% CI, 0.67-0.76) and 0.70 (95% CI, 0.48-0.75), respectively. Serum antibody to p53 and HOXB7 is positively associated with EOC, whereas NY-CO-8-specific antibody shows negative association. Bayesian modeling of these TAA-specific serum antibody responses exhibits similar discrimination of patients with early-stage and advanced-stage EOC from women with nonmalignant gynecologic conditions and may be complementary to CA125. (Cancer Res 2006; 66(3): 1792-8)

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.

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.

In the major histocompatibility complex (MHC)-matched hematopoietic cell transplantation (HCT) setting, minor histocompatibility antigens (miHAs) are implicated in curative graft-versus-tumor (GVT) responses for patients with hematologic malignancies, as well as the morbidity of graft rejection and graft-versus-host disease (GVHD). Minor histocompatibility antigens are MHC class I- and class II-presented endogenous peptides derived from nonsynonymous disparities within coding regions between the donor and recipient. These include unique Y chromosome disparities (H-Y) in female into male HCT. Genetic disparities that give rise to miHAs including H-Y are only antigenic when presented in the context of specific MHC molecules, a requirement termed HLA-restricted and dog leukocyte antigen (DLA)-restricted, in humans and dogs, respectively. Tissue-selective expression of miHAs suggests that it may be possible to augment and separate GVT responses from GVHD using a miHA vaccine.1 Although some miHAs are known in humans, formidable obstacles of efficacy, safety, and feasibility currently prevent the translation of our knowledge of miHAs into an established immunotherapy.2 We seek to establish a recombinant miHA vaccine in the canine model of allogeneic HCT to provide a large outbred animal model capable of addressing the challenges faced in implementing a miHA vaccine in human allogeneic HCT. With minimum-intensity conditioning, DLA-identical marrow infusion, and a short course of postgrafting immunosuppression, the canine model produces stable mixed donor-recipient hematopoietic chimeras.3 This mixed chimerism is a state of tolerance between donor and recipient cells and is not affected by “unsensitized” donor lymphocyte infusions (DLIs).4,5 However, if the donor is first sensitized to miHAs via recipient-derived skin implants, organ transplantation, or injections of allogeneic peripheral blood mononuclear cells (PBMCs), then a sensitized DLI breaks tolerance resulting in full donor chimerism that is often accompanied by GVHD.4-7 Thus, stable mixed chimerism provides a reproducible in vivo model to test donor T cell sensitization against recipient miHAs. A graphic reproduction of published results on chimerism analyses after unsensitized and miHA-sensitized DLI into DLA-identical mixed chimeras is provided in Figure ​Figure11 as a reference to interpret the results of this pilot study.4 FIGURE 1 Reproduction of published results following unsensitized DLI and miHA-sensitized DLI into eight stable mixed chimeric recipients.4 The chimerism results were shown as percent donor PBMC on the y axis, with weeks after the DLI shown on the x axis. Eight ... A major challenge facing the development of a recombinant miHA vaccine in the canine model is the lack of characterized miHAs. T cell cloning reagents used to characterize miHAs in humans are not yet available in the canine model. Instead, we postulated that making a vaccine encoding large sections of Y chromosome gene disparities may overcome the lack of peptide-level characterization of miHAs in the canine model and allow us to further develop this model through the use of female transplant donors and male transplant recipients. At the time of vaccine development, the canine genome had only 3 Y chromosome gene sequences available including ubiquitously transcribed tetratricopeptide repeat containing, Y-linked (UTY), selected mouse CDNA on Y (SMCY), and sex determining region Y (SRY). Attempts were made to clone the most disparate sections with respect to their X homologues of the large genes UTY and SMCY, as well as the entire small SRY gene. After 2 cloning attempts, 3 domains of canine SMCY encoding approximately 60% of the total disparities with the X-homologue SMCX were cloned, as well as the entire SRY gene. These clones were shuttled into DNA expression plasmids and replication-deficient human adenovirus type 5 (rAd5) vectors. Three female HCT donors then received 2 doses of the expression plasmids delivered by particle-mediated epidermal delivery (PMED) 4 weeks apart, and then 4 weeks later received an intramuscular boost of rAd5. Four weeks after the rAd5 boost injection, a DLI into their respective male mixed hematopoietic chimeric recipients resulted in a significant increase in donor chimerism in 1 of 3 hosts, representing the first functional miHA response to a recombinant miHA vaccine in a large animal model.8 In our initial vaccine series, we observed relatively weak antigen-specific T cell responses at the time of the first DLI. We hypothesized that “inadequate donor sensitization” may explain why the observed increase in donor chimerism did not reach full donor chimerism and explain the lack of change in donor chimerism in the other 2 recipients. Because 2 of the original donor-recipient transplant pairs were still available, including the pair whose recipient demonstrated an increase in donor chimerism after the first DLI, we were able to address this hypothesis with 4 additional PMED boost injections in the 2 female donors followed by a second DLI into their respective male mixed hematopoietic chimeric recipients.

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).

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.

Although hematopoietic cell transplantation (HCT) is generally accomplished using a single donor, multiple donors have been used to enhance the speed of engraftment, particularly in the case of umbilical cord blood grafts. Here we posed the question in the canine HCT model whether stable dual-donor chimerism could be established using 2 DLA-identical donors. We identified 8 DLA-identical littermate triplets in which the marrow recipients received 2 Gy total body irradiation followed by marrow infusions from 2 donors and postgrafting immunosuppression. All 8 dogs showed initial “trichimerism,” which was sustained in 5 dogs, while 2 dogs rejected one of the allografts and remained mixed chimeras, and 1 dog rejected both allografts. Immune function in one trichimeric dog, as tested by mixed leukocyte culture response and antibody response to sheep red blood cells, was found to be normal. Five dogs received kidney grafts from one of their respective marrow donors at least 6 months after HCT without immunosuppressive drugs, and grafts in 4 dogs are surviving without rejection. In summary, following nonmyeloablative conditioning, simultaneous administration of marrow grafts from 2 DLA-identical littermates could result in sustained trichimerism, and immunologic tolerance could include a kidney graft from one of the marrow donors.

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.

Abstract 4123 Adoptive transfer of tumor antigen-sensitized lymphocytes has been a long-studied approach for the treatment of solid malignancies. Transfer of lymphocytes sensitized against minor histocompatibility (H) antigens, disparate between the marrow donor and recipient, may be a similar approach to enable improved host versus tumor responses in the treatment of hematological malignancies. We have developed a pre-clinical canine transplant model that establishes a state of stable mixed donor-recipient hematopoietic chimerism after a non-myeloablative conditioning regimen, dog leukocyte antigen (DLA)-identical sibling marrow graft, and a short course of post-grafting immunosuppression. Residual recipient hematopoiesis represents a surrogate for relapse from the underlying hematologic malignancy. The only way to eliminate the residual recipient hematopoiesis is by a donor lymphocyte infusion (DLI) from a donor sensitized by minor H antigens though either an infusion of cryopreserved recipient peripheral blood mononuclear cells, a kidney graft, or a skin graft. A DLI from an unsensitized donor does not alter chimerism in the recipient. This model lends itself to testing of vaccines to minor H antigens. We hypothesized that vaccinating donor dogs with autologous antigen presenting cells (APC) expressing recipient minor H antigens followed by DLI will result in conversion to full donor hematopoiesis in the mixed chimerism recipient, providing the experimental evidence in a large animal model of the potential therapeutic benefit for a minor H antigen vaccine to treat relapsed disease. To test our hypothesis, we sensitized two donor dogs with autologous APC's transfected with poly(A)-selected mRNA from their respective recipients followed by DLI into these mixed chimeric recipients. We performed subcutaneous injections into the donor of 5 to 12 x 10⋀6 CD40-ligand stimulated donor B-cells electroporated with mRNA isolated from PHA blasts from the chimeric recipient every three weeks for a total of three vaccinations. This was followed by a DLI infusion into the chimeric recipient. There was no change in chimerism in the recipient dogs, and we were unable to demonstrate an Elispot response in the donor dogs to the transfected recipient mRNA. Due to the difficulty in isolating sufficient quantities of mRNA and a lack of an ELISpot response, we changed the source of antigen. Utilizing the canine genome sequence database we identified non-synonymous disparities between the × and Y chromosome genes of SMCY, UTY, and SRY. We created ‘minigenes’ encoding these disparities flanked by a T7 promoter and a 3' beta globin untranslated region with a poly(A) tail. These minigenes encoded 80% of the Y chromosome-associated disparities between canine SMCY, UTY, and their × chromosome homologs. The entire SRY gene was included as there is no × homolog for SRY. Minigenes were transcribed in vitro with a cap analogue to produce fully translatable mRNA. These Y chromosome-specific minigenes produced a strongly positive ELISpot in a female dog that had been sensitized with cells from a DLA-identical male sibling. We then performed three consecutive intravenous vaccinations at least one week apart with a combination of 25 to 40 × 106̂ CD34-derived donor dendritic cells and CD40 ligand-stimulated B cells transfected with these Y chromosome-specific minigenes. Following the third vaccination, we demonstrated a weak positive ELISpot reaction to the Y chromosome-specific minigenes. However, a DLI into a mixed chimeric recipient failed to convert the recipient to full donor hematopoiesis and did not elicit any graft versus host disease. These studies suggest that in vivo sensitization of the marrow donor against recipient minor H antigens presented by autologous dendritic cells will require additional immune-modulation to accomplish improved graft versus tumor effects in the treatment of hematological diseases. Disclosures: No relevant conflicts of interest to declare.

Traditional methods for identifying minor histocompatibility antigens (mHags) are technically challenging and biased against discovery of mHags not expressed in the peripheral blood. In this work, we propose a rapid, unbiased, genetic approach for identification minor antigens resulting from disparities in coding non-synonymous SNPs (“C SNPS”). This approach is capable of testing for responses to candidate minor antigens expressed in virtually any tissue, including those expressed exclusively in tissues targeted by GVHD. The first step in our approach begins with comparison of donor and recipient C SNP genotypes generated using C SNP microarrays. These arrays interrogate approximately 80% of human C SNPs predicted to occur in greater than 5% of the population. Comparison of C SNP genotypes directly identifies protein-altering alleles present in the recipient but not the donor (hereafter referred to as “recipient-restricted” alleles), thereby identifying a transplant-specific set of candidate minor antigens. The second step utilizes conventional HLA-class I epitope prediction performed on all linear peptides that include amino-acid residues defined by a recipient-restricted allele. This two step filtering process identifies a small, “testable” number candidate minor peptide epitopes for an individual expressed HLA-class I allele. Candidate epitopes can then be synthesized, pooled and tested at diagnosis of GVHD using a commercial Granzyme B ELISPOT assay. As proof-of-principal for a direct genotyping approach, we have analyzed C SNP genotypes, performed epitope prediction and generated T-cell lines specific for candidate minor antigens using DNA and PBL from a pair of disease-free HLA-identical siblings. Analysis of 10,000 C SNPs shows that approximately 2,000 C SNP alleles are restricted to one sibling within the pair. BIMAS epitope prediction of short unique peptide sequences determined by each sibling-restricted allele identifies approximately 100 candidate minor epitopes predicted to bind HLA-A*0201. These candidate minor epitopes were ranked using expression microarrays performed on EBV-transformed LCL derived from each sibling, with candidates derived from highly expressed genes ranked above those from genes with lower expression levels. A pool of 12 candidate minor epitopes that were both unique to sibling “A” and derived from genes highly expressed in LCL were synthesized and used to generate CD8+ T-cell lines from sibling “B”. Stimulation utilized autologous (sibling “B”-derived) mature dendritic cells loaded with candidate minor epitopes. After several rounds of in-vitro stimulation, each T-cell line was tested for responses to EBV-LCL from sibling “B” and sibling “A” using a Granzyme B ELISPOT kit. Five out of sixteen lines responded to LCL from sibling A while no line responded to autologous LCL. Thus we show that this approach frequently generates CD8+ T-cell lines specific for sibling-derived target cells, suggesting that this approach efficiently identifies genuine, novel, endogenously processed and presented minor epitopes. Deconvolution of the peptide pool suggests that at least two out of the twelve candidate minor epitopes are naturally processed and presented.