So far, only 6 other mHAgs with hematopoietic system—specific tissue distribution have been described; 5 are encoded by autosomal genes, and 1 is encoded on the Y chromosome Table 1 ; reviewed in ref. Despite inclusion of these mHAgs, the potential number of patients that could be treated remains low due to the phenotypic frequencies of the mHAgs and the HLA restriction molecule.
Various biochemical and molecular approaches have been used to characterize mHAgs.
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The strength of this approach is that the identified peptide is by definition present on the cell surface 14 , The recent determination of the complete human genome sequence has facilitated identification of the gene encoding the relevant peptide. The drawback of the classical approach is the need for highly specialized equipment and personnel. Alternatively, cDNA library screening has successfully been executed for the identification of antigenic minor histocompatibility peptides.
Although this technique can be applied for identifying autosomal mHAgs 16 , 17 , it is particularly powerful for identifying H-Y epitopes, for which there are only a limited number of candidate genes 18 — Another possibility for the chemical identification of human mHAgs was put forward by Gubarev et al.
The families studied consist of 3 generations, and all individuals have been typed for 3,—10, genetic markers 23 , This approach led to the localization of mHAgs on chromosomes 22 22 and 11 At that time, these mHAg loci could not be further refined, leaving the biochemical structure of the epitopes unsolved. The first indications that this approach could indeed lead to the molecular identification of minor histocompatibility peptides were provided by a retrospective study on the HA-8 antigen Combining the genetic linkage data with HLA-binding prediction tools on nonsynonymous single nucleotide polymorphism—containing DNA sequences yielded an epitope that matched the eluted one.
The genetic linkage analyses combined with the T cell reactivities specific for mHAgs in question resulted in 46 candidate genes. Further identification was facilitated by the fact that ACC-1— and ACC-2—specific T cell clones only recognize cells of the hematopoietic system. The only gene that was reported by databases to match the expression pattern was BCL2A1.
In this issue of the JCI , de Rijke et al. To circumvent the problem that tissue distribution data in the various databases might be incomplete or incorrect, real-time PCR analysis of candidate genes was performed. This additional selection procedure appeared to be crucial for identifying the correct gene. The results clearly show that molecular identification via genetic linkage analyses can successfully be executed for mHAgs with a limited tissue distribution.
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Genetic linkage identification of minor histocompatibility epitopes with a broad expression pattern, such as the GVHD-associated mHAgs, might turn out to be more difficult. J Immunol ; : — Selective changes in expression of HLA class I polymorphic determinants in human solid tumors. Antigenic heterogeneity of skin tumors of nonmelanocyte origin: analysis with monoclonal antibodies to tumor-associated antigens and to histocompatibility antigens. J Natl Cancer Inst ; 71 : — Immunohistochemical analysis of malignant melanomas and nevocellular nevi with monoclonal antibodies to distinct monomorphic determinants of HLA antigens.
Cancer Res ; 44 : — Loss of HLA haplotype and B locus down-regulation in melanoma cell lines. Int J Cancer ; 91 : — Quantitative correlation between HLA class I allele expression and recognition of melanoma cells by antigen-specific cytotoxic T lymphocytes. Cancer Res ; 55 : — Cancer Res ; 65 : — Down-regulation of HLA class I antigen-processing molecules in malignant melanoma: association with disease progression. Am J Pathol ; : — Level of HLA antigens in locoregional metastases and clinical course of the disease in patients with melanoma. Cancer Res ; 48 : — J Clin Invest ; 87 : — J Clin Invest ; 91 : — Loss of functional beta2-microglobulin in metastatic melanomas from five patients receiving immunotherapy.
J Natl Cancer Inst ; 88 : — J Clin Invest ; : — Immune selection of hot-spot b2-microglobulin gene mutations, HLA-A2 allospecificity loss, and antigen-processing machinery component down-regulation in melanoma cells derived from recurrent metastases following immunotherapy. Cellular and humoral immunity to different types of human neoplasms. Nature ; : — Tumour-specific antibodies in human malignant melanoma and their relationship to the extent of the disease. Br Med J ; 3 : — Treating cancer with genetically engineered T cells. Trends Biotechnol ; 29 : — Cationic lipid-based gene delivery systems: pharmaceutical perspectives.
Pharm Res ; 14 : — Direct gene transfer with DNA-liposome complexes in melanoma: expression, biologic activity, and lack of toxicity in humans. Safety and short-term toxicity of a novel cationic lipid formulation for human gene therapy. Hum Gene Ther ; 4 : — Lipid-DNA complexes induce potent activation of innate immune responses and antitumor activity when administered intravenously.
Plasmid DNA activates murine macrophages to induce inflammatory cytokines in a CpG motif-independent manner by complex formation with cationic liposomes. Biochem Biophys Res Commun ; : — Immunostimulation mechanism of LPD nanoparticle as a vaccine carrier. Mol Pharm ; 2 : 22— Vaccination with liposome-DNA complexes elicits enhanced antitumor immunity. Cancer Gene Ther ; 13 : — Cationic lipid:bacterial DNA complexes elicit adaptive cellular immunity in murine intraperitoneal tumor models.
Cancer Res ; 60 : — Tumor treatment with complexes of cationic lipid and noncoding plasmid DNA results in the induction of cytotoxic T cells and systemic tumor elimination. Mol Ther ; 6 : — Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science ; : — A toll-like receptor recognizes bacterial DNA. Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol ; 20 : — J Leukoc Biol ; 77 : 71— Lipofection indirectly increases expression of endogenous major histocompatibility complex class I molecules on tumor cells. Cancer Gene Ther ; 5 : — Melanoma Res ; 7 Suppl 2 : S67—S Characterization of the major histocompatibility complex class I deficiencies in B16 melanoma cells.
Cancer Res ; 61 : — Expression and modulation of a retrovirus-associated antigen by murine melanoma cells. J Exp Med ; : — A cell surface molecule with a function in human T cell activation. Eur J Immunol ; 16 : — Nature ; : 76— Immunologic correlates of the abscopal effect in a patient with melanoma. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer.
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Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. Di Rosa F, Santoni A. These peptide differences between donor and patient that may account for the curative GvL are often the result of non-synonymous coding single nucleotide polymorphisms cSNPs that yield a protein with a single amino acid difference between the donor and patient  , .
Donor T cells obtained from a donor who is homozygous for a certain allele in a given HLA epitope would be predicted to be intolerant towards an HLA epitope derived from the alternate allele. In such cases, donor T cells would be predicted to mount an immune response upon exposure to the alternate allele epitope if it is expressed by the patient.
Traditional mHA discovery methods begin with the establishment of a donor-derived T cell clone obtained from the patient post-SCT that reacts to an unknown antigen expressed on an antigen presenting cell usually, but not always, obtained from the recipient  ,  ,  ,  ,  , . These cloning approaches, by themselves, are unable to provide immediate information regarding the minor antigen's HLA-restriction, chemical structure, tissue restriction or allele frequency — characteristics that are important considerations if mHA therapeutics are to be developed clinically.
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Determination of these properties requires further experimentation that can only be performed after the initial cloning  , . Because each SCT has thousands of potential mHA mismatches: some unique to that particular donor patient pair and others common in multiple transplant pairs, each with varying levels of immunogenicity, the individual characterization of T cell clones is insufficient to screen all of the potential mHA in the human population. If therapeutic decision-making that is based on minor histocompatibility antigen analysis is to become part of standard clinical practice, methods must be developed to efficiently evaluate multiple candidate mHAs and determine which ones have the properties e.
With the advent of robust high-throughput genomic platforms, cSNP analysis of patient populations is being performed in many diverse fields, and should be applicable to mHA discovery  ,  , . To test our hypothesis, we applied this general approach to a SCT patient cohort with the aim of identifying a common, immunodominant mHA.
Additionally, T4A possesses several properties that make it a good target mHA: 1 T4A mHA mismatch occurs at the theoretical maximum frequency for any given SCT population and 2 T4A expression is highly restricted to hematopoietic tissue and leukemia. Written informed consent for blood sample collection was obtained from all patients and donors through an M. Research was conducted according to the principles in the Declaration of Helsinki. The addition of these patients increased our test cohort from 36 to 57 patients.
This analysis was performed at the University of Texas at Houston School of Medicine microarray core facility. Candidate peptides were synthesized by the M. Anderson peptide core facility using traditional f-moc chemistry, and peptides for the iTopia Epitope Discovery System were synthesized by Bioynthesis Lewisville, TX. All peptides were purified by HPLC, and molecular weights were confirmed by mass spectrometry. After incubation, T2 cells were washed in sterile phosphate buffered saline PBS twice and incubated with unconjugated BB7.
Tetramers were made, in our laboratory, for all of the peptides that bound HLA-A2 using the T2 binding assay according to published protocols . Monomers were then reacted with phycoerythrin PE conjugated streptavidin and subsequently purified on kDa membranes to yield PE-conjugated tetramers. Single stain controls were used for compensation purposes.
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Samples were washed once after incubation and analyzed immediately on a Cyan flow cytometer. Normal donor buffy coats that served as negative controls were purified by ficol-hypaque purification and tested for HLA-A2 expression using BB7. Survival differences were compared using the log-rank test.
Louis, MO. The next day, the membrane was washed for 30 min in TBS The gel was then transferred to a PME membrane using an electric gel blotter at 15 mV for 50 min and blocked with 2. The membranes we then developed as described above. Table 1 shows the clinical characteristics of the 57 patient cohort. Neither of these mismatches occurred in the HLA-A locus. Figure 1 shows a schematic of how candidate cSNP alleles were identified as potential mediators of clinically significant immune responses.
The 57 patients were clinically classified according to remission status and GvHD as described in Materials and Methods and represented in Figure 1A. For each cSNP a disparity involving a homozygous donor and genetically nonidentical i. All other genetic combinations were scored as genetically predictive of no immune response gIR-, unshaded boxes. A strong association i. The 40 cSNPs that yielded the lowest P-value were further characterized in this pilot study.
All 57 patients were classified by GvHD and remission status 1A. The same assignments can be made for the recipient R. No gIR is predicted if a donor is heterozygous at a given locus or if the donor and recipient share the same genotype gIR-, unshaded boxes. Because of the large number of cSNPs tested the problem of false-positive results from multiple testing is significant in our method.
From the 40 cSNPs 16 were removed at this step because they could not be mapped to a known protein sequence with the given allelic variation. From the 15 identified cSNPs, we ordered 23 peptides with 17 subsequently being made at high purity Table 2. Two of the 23 ordered peptides, P6 and P20 Table 2 , did not meet our loose binding criteria but were selected to further assess the binding algorithms' performance characteristics.
Six of the 23 peptides that were ordered could not be synthesized efficiently because of their hydrophobicity and the resulting difficulty with purification.
Figure 2F shows a representative binding assay of one of the peptides that did not bind HLAA according to our assay. Unpulsed T2 cells are shown as shaded traces. Test peptide-pulsed T2 cells are shown as traces with dashed lines, and PR1-pulsed T2 cells are shown as solid black lines.
A representative non-binding peptide is shown 2F.
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