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Chronic diseases that result in end-stage organ damage cause inflammation, which can reveal sequestered self-antigens (SAgs) in that organ and trigger autoimmunity. The thymus gland deletes self-reactive T-cells against ubiquitously expressed SAgs, while regulatory mechanisms in the periphery control immune responses to tissue-restricted SAgs. It is now established that T-cells reactive to SAgs present in certain organs (e.g., lungs, pancreas, and intestine) are incompletely eliminated, and the dysregulation of peripheral immuneregulation can generate immune responses to SAgs. Therefore, chronic diseases can activate self-reactive lymphocytes, inducing tissue-restricted autoimmunity. During organ transplantation, donor lymphocytes are tested against recipient serum (i.e., cross-matching) to detect antibodies (Abs) against donor human leukocyte antigens, which has been shown to reduce Ab-mediated hyperacute rejection. However, primary allograft dysfunction and rejection still occur frequently. Because donor lymphocytes do not express tissue-restricted SAgs, preexisting Abs against SAgs are undetectable during conventional cross-matching. Preexisting and de novo immune responses to tissue-restricted SAgs (i.e., autoimmunity) play a major role in rejection. In this review, we discuss the evidence that supports autoimmunity as a contributor to rejection. Testing for preexisting and de novo immune responses to tissue-restricted SAgs and treatment based on immune responses after organ transplantation may improve short- and long-term outcomes after transplantation.

Esophageal cancer (EC) is an aggressive cancer type that is increasing at a high rate in the US and worldwide. Extensive sequencing of EC specimens has shown that there are no consistent driver mutations that can impact treatment strategies. The goal of this study was to identify activated tyrosine kinase receptors (TKRs) in EC samples as potential targets in the treatment of EC. Activated tyrosine kinase receptors were detected using a dot-blot array for human TK receptors. Human esophageal cancer cell lines were transplanted into immunocompromised mice, and tumor xenografts were subjected to tyrosine kinase inhibitors based on the dot-blot array data. Using the OE33 esophageal cancer cell line, we identified activated EGF receptor (EGFR), as well as ErbB2 and ErbB3. Treatment of this cell line with erlotinib, a specific inhibitor of EGFR, did not impact the growth of this tumor cell line. Treating the OE33 cell line with afatinib, a pan-EGFR family inhibitor resulted in the growth inhibition of OE33, indicating that the ErbB2 and ErbB3 receptors were contributing to tumor cell proliferation. Afatinib treatment of mice growing OE33 tumors inhibited growth of the OE33 tumor cells. Activated tyrosine kinase receptors were readily detected in both cancer cell lines and human esophageal cancer samples. By identifying the activated receptors and then using the appropriate tyrosine kinase inhibitors, we can block tumor growth in vitro and in animal xenografts. We propose that identifying and targeting activated TKRs can be used as a personalized EC tumor treatment strategy.

Mammaglobin-A (Mam-A) is a 10 kDa secretory protein that is overexpressed in 80 % of primary and metastatic human breast cancers. Previous studies from our laboratory demonstrated that Mam-A cDNA vaccine can induce Mam-A-specific CD8 T cell responses and mediate regression of human breast cancer xenografts in NOD/SCID mice. In this article, we present our results on a phase I clinical trial of a Mam-A cDNA vaccination in breast cancer patients with stage-IV metastatic disease, including the impact of vaccination on the expression of the inducible co-stimulator molecule (ICOS) on CD4 T cells. Specimens from seven patients with stage-IV metastatic cancer were available for these analyses. Patients were vaccinated with a Mam-A cDNA vaccine on days 0, 28, and 56, and immune responses were assessed at serial time points following vaccination. At 6 months following the first vaccination, flow cytometric analysis demonstrated a significant increase in the frequency of CD4+ICOS hi T cells from 5 ± 2 % pre-vaccination to 23 ± 4 % ( p  < 0.001), with a concomitant decrease in the frequency of CD4+FoxP3+ T cells (regulatory T cells [Treg]) from 19 ± 6 to 10 ± 5 % ( p  < 0.05). ELISpot analysis of CD4+ICOS hi sorted T cells demonstrated that following vaccination the cytokines produced by Mam-A-specific T cells switched from IL-10 (78 ± 21 spm pre-vaccination to 32 ± 14 spm 5 months post-vaccine p  < 0.001) to IFN-γ (12 ± 6 spm pre-vaccination to 124 ± 31 spm 5 months post-vaccine p  < 0.001). The ratio of CD4+ICOS hi T cells to CD4+FoxP3+ T cells increased from 0.37 ± 0.12 before vaccination to 2.3 ± 0.72 ( p  = 0.021) following vaccination. Further, these activated CD4+ICOS hi T cells induced preferential lysis of human breast cancer cells expressing Mam-A protein. We conclude that Mam-A cDNA vaccination is associated with specific expansion and activation of CD4+ICOS hi T cells, with a concomitant decrease in Treg frequency. These encouraging results strongly suggest that Mam-A cDNA vaccination can induce antitumor immunity in breast cancer patients.

Mammaglobin-A (MAM-A) is a secretory protein that is overexpressed in 80 % of human breast cancers. Its near-universal expression in breast cancer as well as its exquisite tissue specificity makes it an attractive target for a breast cancer prevention vaccine, and we recently initiated a phase 1 clinical trial of a MAM-A DNA vaccine. Previously, we have identified multiple MAM-A CD8 T cell epitopes using a reverse immunology candidate epitope approach based on predicted binding, but to date no attempt has been made to identify epitopes using an unbiased approach. In this study, we used human T cells primed in vitro with autologous dendritic cells expressing MAM-A to systematically identify MAM-A CD8 T cell epitopes. Using this unbiased approach, we identified three novel HLA-A2-restricted MAM-A epitopes. CD8 T cells specific for these epitopes are able to recognize and lyse human breast cancer cells in a MAM-A-specific, HLA-A2-dependent fashion. HLA-A2 + /MAM-A + breast cancer patients have an increased prevalence of CD8 T cells specific for these novel MAM-A epitopes, and vaccination with a MAM-A DNA vaccine significantly increases the number of these CD8 T cells. The identification and translational validation of novel MAM-A epitopes has important implications for the ongoing clinical development of vaccine strategies targeting MAM-A. The novel MAM-A epitopes represent attractive targets for epitope-based vaccination strategies, and can also be used to monitor immune responses. Taken together these studies provide additional support for MAM-A as an important therapeutic target for the prevention and treatment of breast cancer.

Bone marrow transplantation (BMT) benefits nonmalignant diseases but is limited by regimen-related toxicity, graft-versus-host disease (GVHD), donor availability, and graft rejection (GR). To overcome some of these barriers, we developed a new conditioning strategy for these patients. In total, 16 patients received Campath-1H (33/48 mg; days -21 to -19), fludarabine (150 mg/m(2); days -8 to -4), melphalan (140/70 mg/m(2); day -3), and transplant using related/unrelated stem cells. GVHD prophylaxis included cyclosporine/methylprednisolone for cord cells. Other recipients also received methotrexate. Risk factors for GR included multiple transfusions (6), low stem cell numbers (1), and immunologic/metabolic disorders (3). Donor engraftment was present in 14/16 recipients. Neutrophils (ANC>0.5 x 10(9)/l) and platelets (>50 x 10(9)/l) engrafted at a median of 13 and 24 days. Two patients died of Pseudomonas sepsis prior to engraftment, one of CMV disease, and another of intracranial hemorrhage. With median follow-up of 281 days (78-907), 12/16 are stable/improved, or cured. Acute GVHD was absent (n=10) or mild and transient (grade1-2 skin) (n=4). There was no chronic GVHD. Toxicities were predominantly early infections within 100 days, and correlated with lymphopenia (CD4+ T and B cells). Stable engraftment and low incidence of significant GVHD, irrespective of age or stem cell source, make this reduced-intensity regimen attractive for nonmalignant disorders.

Mammaglobin-A (MGBA), a 10-kD protein, is over expressed in 80% of primary and metastatic human breast cancers. Breast cancer patients demonstrate high frequencies of CD8 + cytotoxic T lymphocytes (CTL) specific to MGBA. Defining CD8 + CTL responses to HLA class I-restricted MGBA-derived epitopes assumes significance in the context of our ongoing efforts to clinically translate vaccine strategies targeting MGBA for prevention and/or treatment of human breast cancers. In this study, we define the CD8 + CTL response to MGBA-derived candidate epitopes presented in the context of HLA-B7, which has a frequency of 17.7% in Caucasian and 15.5% in African American populations. We identified seven MGBA-derived candidate epitopes with high predicted binding scores for HLA-B7 using a computer algorithm. Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-B7 indicated that MGBA B7.3 (VSKTEYKEL), B7.6 (KLLMVLMLA), B7.7 (NPQVSKTEY), and B7.1 (YAGSGCPLL) have the highest HLA-B7 binding affinities. Further, two CD8 + CTL cell lines generated in vitro against T2.B7 cells individually loaded with MGBA-derived candidate epitopes showed significant cytotoxic activity against MGBA B7.1, B7.3, B7.6, and B7.7. In addition, the same CD8 + CTL lines lysed the HLA-B7 + /MGBA + human breast cancer cell line DU-4475 but had no significant cytotoxicity against HLA-B7 − or MGBA − breast cancer cell lines. Cold-target inhibition studies strongly suggest that MGBA B7.3 is an immunodominant epitope. In summary, our results define HLA-B7-restriced, MGBA-derived, CD8 + CTL epitopes with all of the necessary features for developing novel vaccine strategies against HLA-B7 expressing breast cancer patients.

Introduction Mammaglobin-A(mam-A) is expressed in over 80% of human breast tumors. We recently reported that mam-A DNA vaccination resulted in breast cancer immunity in a preclinical model. Here we investigated whether mam-A HLA-class-I tetramers could be used to monitor and define the role of CD8 + cytotoxic T-lymphocytes(CTL) in mediating breast cancer immunity following mam-A DNA vaccination. Study Design Mam-A DNA vaccination was performed in HLA-A2 + huCD8 + transgenic mice. HLA-A2 tetramers carrying the immunodominant mamA2.1 peptide were used to monitor CD8 + CTL. Human breast cancer colonies were developed in immunodeficient SCID-beige mice. ELISPOT was used to correlate frequency of mamA2.1 tetramer + CD8 + T cells and IFN-γ production [spots per million cells (spm)] in human subjects. Results Vaccination of HLA-A2 + huCD8 + mice with mam-A DNA vaccine, but not empty vector, led to the expansion of mamA2.1 tetramer + CD8 + T-cells in peripheral blood (<0.5% pre-vaccination compared to >2.0% post-vaccination). CD8 + T cells from vaccinated mice specifically lysed UACC-812(HLA-A2 + /mam-A + , 25% lysis) but not MDA-MB-415(HLA-A2 − /mam-A + ) or MCF-7(HLA-A2 + /mam-A − ) breast cancer cells. Adoptive transfer of purified CD8 + T cells from vaccinated mice into immunodeficient SCID-beige mice with established human breast cancer colonies led to tetramer + CD8 + T-cell infiltration with regression of UACC-812 but not MCF-7 tumors. HLA-A2 + breast cancer patients revealed increased frequency of mamA2.1 tetramer + CD8 + T-cells compared to normal controls (2.86 ± 0.8% vs. 0.71 ± 0.1%, P  = 0.01) that correlated with the IFN-γ response to mamA2.1 peptide (48.1 ± 20.9 vs. 2.9 ± 0.8 spm, P  = 0.03). Conclusions CD8 + T-cells are crucial in mediating breast cancer immunity following mam-A DNA vaccination. Mam-A HLA-class-I tetramers can be effectively used to monitor development of CD8 + T-cells following mam-A vaccination.

Mammaglobin-A (Mam-A) is a 10 kDa secretory protein that is overexpressed in 80 % of primary and metastatic human breast cancers. Previous studies from our laboratory demonstrated that Mam-A cDNA vaccine can induce Mam-A-specific CD8 T cell responses and mediate regression of human breast cancer xenografts in NOD/ SCID mice. In this article, we present our results on a phase I clinical trial of a Mam-A cDNA vaccination in breast cancer patients with stage-IV metastatic disease, including the impact of vaccination on the expression of the inducible co-stimulator molecule (ICOS) on CD4 T cells. Specimens from seven patients with stage-IV metastatic cancer were available for these analyses. Patients were vaccinated with a Mam-A cDNA vaccine on days 0, 28, and 56, and immune responses were assessed at serial time points following vaccination. At 6 months following the first vaccination, flow cytometric analysis demonstrated a significant increase in the frequency of CD4+ [ICOS.sup.hi] T cells from 5 ± 2 % pre-vaccination to 23 ± 4% (p < 0.001), with a concomitant decrease in the frequency of CD4+ FoxP3 + T cells (regulatory T cells [Treg]) from 19 ± 6 to 10 ± 5% (p < 0.05). ELISpot analysis of CD4+ [ICOS.sup.hi] sorted T cells demonstrated that following vaccination the cytokines produced by Mam-A-specific T cells switched from IL-10 (78 ± 21 spm pre-vaccination to 32 ± 14 spm 5 months post-vaccine p < 0.001) to IFN-γ (12 ± 6 spm pre-vaccination to 124 ± 31 spm 5 months post-vaccine p < 0.001). The ratio of CD4+[ICOS.sup.hi] T cells to CD4+FoxP3 + T cells increased from 0.37 ± 0.12 before vaccination to 2.3 ± 0.72 (p = 0.021) following vaccination. Further, these activated CD4+[ICOS.sup.hi] T cells induced preferential lysis of human breast cancer cells expressing Mam-A protein. We conclude that Mam-A cDNA vaccination is associated with specific expansion and activation of CD4+[ICOS.sup.hi] T cells, with a concomitant decrease in Treg frequency. These encouraging results strongly suggest that Mam-A cDNA vaccination can induce antitumor immunity in breast cancer patients. Keywords DNA vaccine * Mammaglobin-A * Breast cancer * T cells * ICOS