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.

Phase 1 study in patients with metastatic melanoma of immunization with dendritic cells presenting epitopes derived from the melanoma-associated antigens MART-1 and gp100.

Dendritic cells (DCs) have been shown to enhance anti-tumor immune responses in several preclinical models. Furthermore, DC-like function can be elicited from peripheral blood monocytes cultured in vitro with interleukin-4 and granulocyte-macrophage colony-stimulating factor. For this reason, a phase 1 study was initiated at the Surgery Branch of the National Cancer Institute to test the toxicity and biological activity of the intravenous administration of peripheral blood monocyte-derived DCs. The DCs were generated by 5- to 7-day incubation in interleukin-4 (1,000 U/mL) and granulocyte-macrophage colony-stimulating factor (1,000 U/mL) of peripheral blood monocytes obtained by leukapheresis. Before administration, the DCs were pulsed separately with the HLA-A*0201-associated melanoma epitopes MART-1(27-35) and gp-100-209-2M. The DCs were administered four times at 3-week intervals. A first cohort of patients (n = 3) was treated with 6 x 10(7) DCs and a second cohort (n = 5) with 2 x 10(8) DCs (in either case, one half of the DCs were pulsed with MART-1(27-35) and the other half was pulsed with gp-100-209-2M). In a final cohort under accrual (n = 2) 2 x 10(8) DCs were administered in combination with interleukin-2 (720,000 IU/kg every 8 hours). The recovery of DCs after in vitro culture ranged from 3% to 35% (mean, 15%) of the original peripheral blood monocytes. Administration of DCs caused no symptoms at any of the doses, and the concomitant administration of interleukin-2 did not cause toxicity other than that expected for interleukin-2 alone. Monitoring of patients' cytotoxic T lymphocyte reactivity before and after treatment revealed enhancement of cytotoxic T lymphocyte reactivity only in one of five patients tested. Of seven patients evaluated for response, one had a transient partial response with regression of pulmonary and cutaneous metastases. A relatively large number of DCs can be safely administered intravenously. The poor clinical outcome of this study perhaps could be explained by the type of protocol used for DC maturation, the route of administration, or both. For this reason, this clinical protocol was interrupted prematurely, whereas other strategies for DC preparation and route of administration are being investigated at the authors' institution.

Threshold levels of gene expression of the melanoma antigen gp100 correlate with tumor cell recognition by cytotoxic T lymphocytes.

The level of expression of melanoma antigens (MA) may modulate the host immunologic response. Thus, the accurate measurement of MA expression may allow proper patient selection for antigen-specific therapies and yield important information for the evaluation of clinical results. In this study, we measured the absolute levels of MA messenger ribonucleic acid (mRNA) in tumor cell lines utilizing real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). mRNA levels of MART-1, gp100, tyrosinase, TRP-1 and TRP-2 melanoma differentiation antigens and MAGE-1, MAGE-3 and ESO-1 cancer testis (CT) antigens were compared in 24 early-passage (<5 passages in culture) and 12 archival melanoma cell lines. MA mRNA expression was extremely variable among cell lines, occasionally reaching levels comparable to ribosomal RNA (rRNA). gp100 and MART-1 mRNA levels correlated with protein expression measurement obtained by FACS analysis. More significantly, a threshold of gp100 mRNA expression required for T-cell stimulation and target-cell killing was identified. This threshold level corresponded to approximately 500 mRNA copies per 10(8) copies of rRNA. Our results suggest that the measurements of MA mRNA levels may yield useful information relevant to the interpretation of clinical outcome during antigen-specific treatments.

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-alpha-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-gamma 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.

Expansion of tumor-T cell pairs from fine needle aspirates of melanoma metastases.

Lymphocytes expanded from excised specimens can be used to characterize intratumoral T cell responses. These analyses, however, are limited to one time point in the natural history of the removed tumor. The expansion of autologous tumor cells and tumor-infiltrating lymphocytes (TIL) from fine needle aspirates (FNA) of tumors potentially allows a dynamic evaluation of T cell responses within the same lesion at moments relevant to the disease course or response to therapy. Fourteen TIL cultures and 8 tumor cell lines were generated from 18 FNA (12 patients). Five of six TIL that could be tested against autologous tumor demonstrated specific reactivity. Two additional TIL for which no autologous tumor was available demonstrated recognition of HLA-matched melanoma cell lines. Serial FNA of the same lesions were performed in five HLA-A*0201 patients vaccinated with the emulsified melanoma Ag (MA) epitopes: MART-1:27-35; tyrosinase:368-376(370D); gp100:280-288(288V); and gp100:209-217 (210M). FNA material was separately cultured for a short time in IL-2 (300 IU/ml) after stimulation with irradiated autologous PBMC pulsed with each peptide or FluM1:58-66 (1 micromol/ml). No peptide-specific TIL could be expanded from prevaccination FNA. However, after vaccination, TIL specific for gp100:280(g280), gp100:209 (g209), and MART-1:27-35 (MART-1)-related epitopes were identified in three, three, and two patients, respectively. No Flu reactivity could be elicited in TIL, whereas it was consistently present in parallel PBMC cultures. This excluded PBMC contamination of the FNA material. This analysis suggests the feasibility of TIL expansion from minimal FNA material and localization of vaccine-specific T cells at the tumor site.

Development and characterization of melanoma cell lines established by fine-needle aspiration biopsy: advances in the monitoring of patients with metastatic melanoma.

The establishment of melanoma cell lines from fine-needle aspiration biopsies (FNAB) has allowed for an enhanced understanding of the complex interactions that occur between T cells and tumor cells. The technique of FNAB offers the advantage of providing a sequential analysis of the same tumor nodules throughout treatment. The expression of melanoma antigens (MAs) was assessed in fresh melanoma FNAB samples and from tumor cell lines derived from these samples using several different approaches. Cytospin preparations of freshly isolated tumor cell explants were analyzed by immunocytochemistry (ICC), while the daughter cell line was analyzed by fluorescent activated cell sorting (FACS) analysis, and semiquantitative and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR, qRT-PCR). As assessed by these methods, the level of MA expression by the original tumor cell explants correlated with the expression in established in vitro cell lines. Molecular analysis of the established cell lines utilizing PCR technology improved the sensitivity of detection of MA expression. Thus FNAB of melanoma is an efficient and effective method of tissue procurement, capable of generating, sequentially and from the same lesion, fresh tumor cells, tumor infiltrating lymphocytes (TIL), and long-term melanoma cell lines.

A novel cytolysis assay using fluorescent labeling and quantitative fluorescent scanning technology.

A novel cellular cytotoxicity assay using Calcein acetoxymethyl (Calcein-AM), a cytoplasmic fluorescent label, has been developed as an alternative to the standard 51Chromium (Cr)-release. Target cells were loaded with Calcein-AM and then co-incubated with effector cells. An additional reagent, FluoroQuench, is added to extinguish fluorescence of dying target cells and of the culture media. Assay plates are read on a quantitative fluorescent scanner for determination of viable target cells. Percent lysis is calculated as one minus the percent viable cells as compared to fluorescent-labeled targets-only wells. The assay was tested to demonstrate the lytic activity of cytotoxic T lymphocyte (CTL) cultures, lymphokine-activated killer (LAK), and natural killer (NK) cell line effectors against peptide-pulsed and melanoma targets. In addition to the acquisition of results comparable to the 51Cr-release assay, the Calcein assay reliably measures cell-mediated cytotoxicity with little variance among replicates. The fluorescent assay represents a simple and useful alternative to the use of radioactive materials and adds the additional benefit of digital images and analysis.

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.

Functional dissociation between local and systemic immune response during anti-melanoma peptide vaccination.

Peptide vaccination against tumor Ags can induce powerful systemic CTL responses. However, in the majority of patients, no tumor regression is noted. To study this discrepancy, we analyzed CTL reactivity in a melanoma patient (F001) vaccinated with g209-2M peptide, a single residue variant of gp100(209-217). G209/g209-2M-reactive CTL were identified in post- but not prevaccination PBL. Limiting dilution analysis identified one predominant CTL clone (C1-35), with TCR Vbeta6s2, recognizing g209/HLA-A*0201-expressing targets. Additionally, two autologous melanoma lines (F001TU-3 and -4) and 20 separate tumor-infiltrating lymphocyte cultures were generated from a fine needle aspirate of a metastatic lesion progressing after initial response to vaccination. Both F001TU did not express gp100 and were not recognized by C1-35. Loss of gp100 by F001TU correlated with a marked reduction of gp100 expression in the same metastatic lesion compared with prevaccination. Thus, ineffectiveness of C1-35 and tumor progression could be best explained by loss of target Ag expression. Interestingly, 12 of 20 tumor-infiltrating lymphocyte cultures recognized F001TU, but none demonstrated g209/g209-2M reactivity, suggesting a functional dissociation between systemic and local immune response. This study suggests that vaccination effects must be analyzed in the target tissue, rather than in the systemic circulation alone.

Interferon gamma (IFNgamma) gene transfer of an EMT6 tumor that is poorly responsive to IFNgamma stimulation: increase in tumor immunogenicity is accompanied by induction of a mouse class II transactivator and class II MHC.

Abstract Interferon gamma (IFNgamma) is an important cytokine with immunomodulatory properties that include activation of immune cells and induction of class I and class II major histocompatibility complex antigens. In this study a retroviral vector was used to introduce the IFNgamma gene into EMT6 tumor cells to assess the effect of IFNgamma gene expression on tumor immunogenicity. Transfectants were selected in G418-containing tissue-culture medium and were determined to express the inserted IFNgamma gene by reverse transcriptase/polymerase chain reaction. Flow-cytometric analysis revealed that parental unmodified EMT6 cells constitutively expressed only class I MHC and were poorly responsive to exogenous IFNgamma stimulation, whereas class II MHC was induced in IFNgamma-transfected cells. The induction of class II MHC in IFNgamma-transfected cells correlated with the expression of a mouse class II transactivator gene that was dormant in unmodified or mock-transfected cells. In addition, IFNgamma-gene-transfected tumor cells were found to secrete up to 17 ng IFN (equivalent to 75 units/10(6) cells) by enzyme-linked immunosorbent assay (ELISA). Whereas parental EMT6 cells grew unchecked, the growth of genetically modified tumor cells was significantly inhibited in immunocompetent mice. Rechallenge of animals that rejected an IFNgamma-transfected EMT6 clone (EMT6-B17) with parental EMT6 cells resulted in tumor rejection, suggesting that IFNgamma-transfected EMT6 cells were able to induce long-term immunity. Mixing experiments using gene-transfected and unmodified tumor cells demonstrated that 10% of IFNgamma-transfected cells in the population was sufficient to protect mice against subsequent challenge with tumorigenic EMT6 cells. These studies demonstrate that the immunogenicity of tumor cells that are poorly responsive to exogenous IFNgamma can be enhanced by inserting and expressing the IFNgamma transgene. These findings also suggest a role for class II MHC in reducing tumorigenicity of the EMT6 tumor and inducing long-term tumor immunity.

T lymphocytes infiltrating sites of tumor rejection and progression display identical V beta usage but different cytotoxic activities.

Most tumors grow progressively and overwhelm the host. The rare but documented cases of spontaneous regression of primary tumors are indicative of the potential of tumor-bearing hosts to develop a significant antitumor response. Because most tumors grow progressively in the host, it is not surprising that the majority of studies have focused on T lymphocytes that infiltrate these tumors. Although these studies have generated significant and useful information during the period of tumor growth, they can only speculate on the mechanisms that are involved in tumor rejection. We have used a well developed sponge model of concomitant tumor immunity that allows us to compare the immunologic events that occur during tumor progression vs rejection. In this model, an animal harboring a primary EMT6 mammary tumor is challenged with a secondary tumor implant through a pre-implanted gelatin sponge. During the manifestation of concomitant tumor immunity, the secondary tumor is rejected and the effector cells mediating the response are retained within the sponge matrix. Using this model we analyzed the TCR usage, cytotoxic activity of lymphocytes, and cytokine production at both tumor sites. The data revealed that tumor-rejecting lymphocytes isolated from the site of secondary tumor implant were cytotoxic toward EMT6 cells, whereas tumor-infiltrating lymphocytes isolated from the progressing primary tumor were not. Interestingly, the TCR-V beta repertoire of the tumor-infiltrating lymphocytes and tumor-rejecting lymphocytes were identical with V beta 1 and V beta 8 being predominant at both sites. Furthermore, the rejection site showed higher gene expression of IFN-gamma, TNF-alpha, and IL-10 whereas TGF-beta expression was slightly higher in the progressing tumors. These findings suggest that the disparate effector functions observed during tumor progression vs rejection are not caused by different T cell phenotypes but may be due instead to influences exerted by cytokines produced at the tumor sites.