Maria P. Bettinotti

Heterogeneous expression of melanoma-associated antigens and HLA-A2 in metastatic melanoma in vivo.

MART‐1/MelanA and Pmel17/gp100 are melanoma‐associated antigens (MAAs) that can be recognized by tumor‐infiltrating lymphocytes (TILs) capable of mediating successful adoptive therapy in vivo. Analysis of melanoma cell lines in vitro has demonstrated that heterogeneous antigen expression in the context of class I MHC is a significant co‐factor in determining the recognition of melanoma targets by cytotoxic lymphocytes (CTLs). In this study, 217 specimens from 103 patients with metastatic melanoma were examined for the expression of MART‐1/MelanA (monoclonal antibody \[MAb\] M27C10) and Pmel17/gp100 (HMB45 MAb) by immuno‐histochemistry. Marked heterogeneity in the expression of both MAAs was confirmed by analysis of the percentage of positively staining tumor cells or the average intensity of tumor staining. We also noted heterogeneity of expression among multiple lesions taken from different anatomic sites within a patient. A dissociation was noted in the detection of MART‐1 and gp100 in some lesions, with gp100 being undetectable in 24% of the lesions and MART‐1 being undetectable in 11%. In several cases, loss of one MAA was not associated with loss of the other MAA, suggesting that MART‐1 can represent a useful additional marker for the diagnosis of melanoma in gp100 (HMB45)‐negative lesions. Of the 217 specimens, 155 were obtained from HLA‐A*0201 patients, of which 6% were negative for HLA‐A2, 8% were negative for MART‐1/MelanA and 21% were negative for Pmel17/gp100. The potential significance of our findings is illustrated by a case study in which a patient with melanoma experienced rapid tumor progression in association with loss of either MAA or HLA expression in several lesions. Int. J. Cancer 75:517‐524, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Natural variation of the expression of HLA and endogenous antigen modulates CTL recognition in an In vitro melanoma model

Increasing attention has been devoted to elucidating the mechanism of lost or decreased expression of MHC or melanoma‐associated antigens (MAAs), which may lead to tumor escape from immune recognition. Loss of expression of HLA class I or MAA has, as an undisputed consequence, loss of recognition by HLA class I–restricted cytotoxic T cells (CTLs). However, the relevance of down‐regulation remains in question in terms of frequency of occurrence. Moreover the functional significance of epitope down‐regulation, defining the relationship between MHC/epitope density and CTL interactions, is a matter of controversy, particularly with regard to whether the noted variability of expression of MHC/epitope occurs within a range likely to affect target recognition by CTLs. In this study, bulk metastatic melanoma cell lines originated from 25 HLA‐A*0201 patients were analyzed for expression of HLA‐A2 and MAAs. HLA‐A2 expression was heterogeneous and correlated with lysis by CTLs. Sensitivity to lysis was also independently affected by the amount of ligand available for binding at concentrations of 0.001 to 1 mM. Natural expression of MAA was variable, independent from the expression of HLA‐A*0201, and a significant co‐factor determining recognition of melanoma targets. Thus, the naturally occurring variation in the expression of MAA and/or HLA documented by our in vitro results modulates recognition of melanoma targets and may (i) partially explain CTL–target interactions in vitro and (ii) elucidate potential mechanisms for progressive escape of tumor cells from immune recognition in vivo. Int. J. Cancer: 80, 781–790 (1999).

A Tumor-Infiltrating Lymphocyte from a Melanoma Metastasis with Decreased Expression of Melanoma Differentiation Antigens Recognizes MAGE-12

Twenty separate tumor infiltrating lymphocyte (TIL) bulk cultures and a tumor cell line were originated simultaneously from a fine needle aspiration biopsy of a metastasis in a patient with melanoma (F001) previously immunized with the HLA-A*0201-associated gp100:209–217(210 M) peptide. None of the TIL recognized gp100. However, 12 recognized autologous (F001-MEL) and allogeneic melanoma cells expressing the HLA haplotype A*0201, B*0702, Cw*0702. Further characterization of F001-MEL demonstrated loss of gp100/PMel17, severely decreased expression of other melanoma differentiation Ags and retained expression of tumor-specific Ags. Transfection of HLA class I alleles into B*0702/Cw*0702-negative melanoma cell lines identified HLA-Cw*0702 as the restriction element for F001-TIL. A cDNA library from F001-MEL was used to transfect IFN-α-stimulated 293 human embryonal kidney (293-HEK) cells expressing HLA-Cw*0702. A 100-gene pool was identified that induced recognition of 293-HEK cells by F001-TIL. Subsequent cloning of the pool identified a cDNA sequence homologous, except for one amino acid (aa 187 D→A), to MAGE-12. Among 25 peptide sequences from MAGE-12 with the HLA-Cw*0702 binding motif, MAGE-12:170–178 (VRIGHLYIL) induced IFN-γ release by F001-TIL when pulsed on F001-EBV-B cells at concentrations as low as 10 pg/ml. Peptide sequences from MAGE-1, 2, 3, 4a, and 6 aligned to MAGE-12:170–178 were not recognized by F001-TIL. In summary a TIL recognizing a MAGE protein was developed from an HLA-A*0201 expressing tumor with strongly reduced expression of melanoma differentiation Ags. Persisting tumor-specific Ag expression maintained tumor immune competence suggesting that tumor-specific Ags/melanoma differentiation Ags may complement each other in the context of melanoma Ag-specific vaccination.

Unmasking cryptic epitopes after loss of immunodominant tumor antigen expression through epitope spreading

The basis of intra‐tumoral and systemic T cell reactivity toward cancer remains unclear. In particular the role that peripheral stimuli, whether endogenous or exogenous, play in shaping acquired immune response toward cancer remains poorly understood. In this study we document the surfacing of systemic immune reactivity toward a cryptic epitope from the MAGE‐12 gene (MAGE‐12:170–178), after temporary regression of a single melanoma metastasis, in response to gp100/PMel17‐specific vaccination. This emergence was unlikely related to unusually high expression of MAGE‐12 by the tumor, by the influence of analog epitopes to MAGE‐12:170–178. Because MAGE‐12 was unlikely to be expressed at sites other than the tumor, the demonstration of MAGE‐12:170–178 reactivity in post‐ but not pre‐vaccination circulating lymphocytes suggests that the systemically observed immune response was influenced by events induced by the vaccine at tumor site or draining lymph nodal areas. Possibly, as suggested by pre‐clinical models, immunologic ignorance is the default response toward cancer in humans unless unusual stimulatory conditions occur in peripheral tissues. Surfacing of MAGE‐12 specificity occurred in association with loss of gp100/PMel 17 targeted by the vaccine. This finding suggests that vaccinations might have effects beyond their intrinsic specificity and may trigger broader immune responses through epitope spreading by inducing changes within the tumor microenvironment. This may have important practical implication for the development of immunization strategies. Published 2001 Wiley‐Liss, Inc.

CD177: A member of the Ly-6 gene superfamily involved with neutrophil proliferation and polycythemia vera

Genes in the Leukocyte Antigen 6 (Ly-6) superfamily encode glycosyl-phosphatidylinositol (GPI) anchored glycoproteins (gp) with conserved domains of 70 to 100 amino acids and 8 to 10 cysteine residues. Murine Ly-6 genes encode important lymphocyte and hematopoietic stem cell antigens. Recently, a new member of the human Ly-6 gene superfamily has been described, CD177. CD177 is polymorphic and has at least two alleles, PRV-1 and NB1. CD177 was first described as PRV-1, a gene that is overexpressed in neutrophils from approximately 95% of patients with polycythemia vera and from about half of patients with essential thrombocythemia. CD177 encodes NB1 gp, a 58–64 kD GPI gp that is expressed by neutrophils and neutrophil precursors. NB1 gp carries Human Neutrophil Antigen (HNA)-2a. Investigators working to identify the gene encoding NB1 gp called the CD177 allele they described NB1. NB1 gp is unusual in that neutrophils from some healthy people lack the NB1 gp completely and in most people NB1 gp is expressed by a subpopulation of neutrophils. The function of NB1 gp and the role of CD177 in the pathogenesis and clinical course of polycythemia vera and essential thrombocythemia are not yet known. However, measuring neutrophil CD177 mRNA levels has become an important marker for diagnosing the myeloproliferative disorders polycythemia vera and essential thrombocythemia.

CMV pp65 and IE-1 T cell epitopes recognized by healthy subjects

BackgroundAdoptive immune and vaccine therapies have been used to prevent cytomegalovirus (CMV) disease in recipients of hematopoietic progenitor cell transplants, but the nature of T cell responses to CMV have not been completely characterized.MethodsPeptide pools and individual peptides derived from the immune-dominant CMV proteins pp65 and IE-1 and antigen-specific, cytokine flow cytometry were used to characterize the prevalence and frequency of CD4+ and CD8+ memory T cells in 20 healthy CMV-seropositive subjects.ResultsCD8+ T cell responses to pp65 were detected in 35% of subjects and to IE-1 in 40% of subjects. CD4+ T cell responses to pp65 were detected in 50% of subjects, but none were detected to IE-1. Several new IE-1 HLA class I epitopes were identified, including 4 restricted to HLA-C antigens. One region of IE-1 spanning amino acids 300 to 327 was rich in class I epitopes. The HLA class I restrictions of IE-1 peptides were more promiscuous than those of pp65 peptides.ConclusionSince naturally occurring CD4+ and CD8+ T cell responses to pp65 were detectable in many subjects, but only CD8+ T cell responses to IE-1 were detected, pp65 may be better than IE-1 for use in vaccine and adoptive immune therapies.

HLA-DRB5*0101 and -DRB1*1501 expression in the multiple sclerosis-associated HLA-DR15 haplotype.

The HLA region, and particularly the DR15 haplotype (containing the two DRB* genes DRB1*1501 and DRB5*0101 and the tightly linked DQ alleles DQA*0102 and DQB1*0602, which together form the DQw6 molecule) in Caucasians, shows the strongest genetic association with multiple sclerosis (MS). In the DR15 haplotype, two beta-chains HLA-DRB1*1501 and -DRB5*0101 are co-expressed resulting in two different surface HLA-DR alphabeta heterodimers, DR2b and DR2a. Most previous studies focused on DRB1*1501, however, both DR2a and DR2b may contribute to MS pathogenesis via antigen presentation to myelin-specific T lymphocytes. We therefore analyzed the expression of the two DR15 genes in various antigen presenting cells (APCs), central nervous system and thymic tissues. Transcript levels were higher for DRB5*0101 in all cell types and tissues. Both HLA-DR heterodimers were expressed at significant levels on the cell surface, where they showed a differential expression pattern in different APCs. They were similarly regulated after stimulation with interferon-gamma and interleukin-4. Finally, immunohistochemistry experiments indicated that both molecules were expressed in thymic tissue. Our results encourage future research to investigate the potential functional relevance of both genes for the pathogenesis of MS.

Clinical and immunological evaluation of patients with metastatic melanoma undergoing immunization with the HLA-Cw*0702-associated epitope MAGE-A12:170-178.

Patients with metastatic melanoma who expressed HLA‐Cw*0702 and whose tumors had demonstrable MAGE‐A12 expression were immunized with the peptide MAGE‐A12:170–178 administered subcutaneously in incomplete Freunds adjuvant (IFA). The peptide was administered either every week or every 3 weeks for 4 cycles. Patients were evaluated for toxicity and for immunologic and clinical response to peptide immunization. Pre‐treatment fine needle aspirates were obtained to document MAGE‐A12 expression for enrollment. MAGE‐A12 mRNA was identified in 62% of specimens. Nine patients were selected for vaccination based on MAGE‐A12 expression and the presence of HLA‐Cw*0702. The immune response was monitored both by tetrameric HLA‐Cw*0702/MAGE‐A12:170–178 complexes and by analysis of interferon‐γ mRNA transcription using a quantitative real‐time polymerase chain reaction assay after peptide‐specific stimulation. The samples consisted of circulating lymphocytes analyzed ex vivo or after 10 to 14 days of in vitro sensitization. One of 9 patients sustained an ongoing partial clinical response. No convincing evidence of enhancement of the systemic immune response against MAGE‐A12:170–178 could be documented. Because of the modest immunological and clinical results, the present protocol has been discontinued as new routes of administration are being considered.

Identification of immune dominant cytomegalovirus epitopes using quantitative real-time polymerase chain reactions to measure interferon-γ production by peptide-stimulated peripheral blood mononuclear cells

The identification of HLA restricted immune dominant cytotoxic T cell (CTL) epitopes limits immune therapy. Cytomegalovirus (CMV) disease remains a significant cause of morbidity after allogeneic stem cell transplantation. Adoptive immune therapy using CTLs stimulated with immune dominant CMV pp65 peptides may be effective in preventing CMV disease, but immune dominant CMV peptides have been identified for only a few HLA class I molecules. The purpose of this study was to use a novel molecular system to establish a rapid and precise method to identify new HLA-restricted CMV epitopes. Cytomegalovirus pp65 peptides expected to bind to the HLA-24 molecule were identified with a computer algorithm. Five candidate peptides were screened by direct ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) from CMV-seropositive HLA-A*2402 individuals, and quantitative real time PCR (qRT-PCR) was used to evaluate CTL responses by measuring interferon-&ggr; (IFN-&ggr;) transcripts. One of the five candidate peptides, pp65341–350 (QYDPVAALFF), induced significant quantities of IFN-&ggr; mRNA production after 3 hours. PBMCs from CMV-seropositive HLA-A*2402 individuals sensitized in vitro with pp65341–350 also recognized CMV-infected targets. In conclusion, the measurement of IFN-&ggr; mRNA by qRT-PCR can be used to detect CTL responses 3 hours after peptide stimulation of a small quantity of PBMCs. This method has an advantage over other methods used to identify immune dominant epitopes in that it does not require in vitro expansion of CTLs with cytokines or virally infected targets. As a result, this method measures naturally induced immune reactions.

Comprehensive method for the typing of HLA-A, B, and C alleles by direct sequencing of PCR products obtained from genomic DNA

Molecular testing is gradually replacing standard typing techniques in the field of HLA because it allows higher resolution, which has significant functional implications. Although several techniques have been so far described for this purpose, the definitive means to determine which alleles are present in a particular sample is to identify their sequence. We describe a simplified method for typing HLA-A, B, and C alleles by direct sequencing of polymerase chain reaction (PCR) products amplified from genomic DNA that could allow large-scale handling of samples for clinical use. The template is the product of a nested PCR. A first round of PCR amplifications from genomic DNA is performed with three different sets of primers, one pair specific for each locus. The PCR products encompass exons 2 and 3, the regions of interest to determine the allele present. These fragments are a mixture of both alleles present in one locus. In a second round of PCRs using the first fragment as template, exons 2 and 3 are separately amplified and simultaneously tailed with sequences corresponding to fluorescent-labeled commercial primers. The sense and antisense sequence of each exon is obtained and compared with a database of all known HLA-A, B, or C alleles. Heterozygous positions are determined and the most probable alleles assigned. This simplified procedure has the practical advantage of allowing high-resolution typing of clinical material by utilizing the same genomic DNA used for standard molecular typing of HLA class I.