Cancer/testis antigens in non small cell lung cancer : expression and immunogenicity

Lung cancer is the leading cause of cancer-related mortality in the world, whereby Non Small Cell Lung Carcinomas (NSCLC) constitute 80% of all lung tumors. Whereas in Stage I and II NSCLC surgical resection with or without adjuvant chemotherapy currently represents the most frequently applied treatment, in late stage NSCLC, representing 70% of all cases, chemotherapy or radiotherapy are mainly palliative. Thus, the poor prognosis and the limited therapeutic options available urge the development of new approaches. Among these, active specific immunotherapy targeting Cancer/Testis Antigens (CTA) might represent a valuable additional treatment in NSCLC. CTA have been shown to represent promising targets in different types of cancer as they are silent in healthy adult tissues except in testis and placenta. These tissues do not present antigenic epitopes as they are deficient in MHC expression. Moreover, CTA are expressed by various tumors of different histology, stage and grade, and in some tumors, expression has been found to be correlated with poor disease specific survival. In this study first the prevalence and expression patterns of several CTA (MAGE-A1, -A2, -A3, -A4, -A10, -A12 and NY-ESO-1) in freshly excised NSCLC were investigated at gene and protein level. Tumor specimens (12 adeno-, 17 squamous cell and 4 large cell carcinomas) were obtained from HLA-A*0101 and/or HLA-A*0201 positive patients. CTA expression was detected in five adeno-, eight squamous cell and in two large cell carcinoma samples (45.5%). MAGE-A10 and -A12 were the most frequently (10/15 and 12/15 specimens, respectively) and MAGE-A1, -A4 and NY-ESO-1 the least frequently expressed genes (6/15, 6/15 and 4/15 specimens, respectively). In 10/15 positive cases at least four CTA genes were concomitantly expressed. These results at gene level were widely confirmed by protein detection, the few discrepancies being explained by focal CTA expression limited to defined tumor areas. Immune responsiveness towards MAGE-A1 and -A3 (HLA-A*0101 restricted), MAGE-A4, -A10, multi-MAGE-A (an epitope shared by several MAGE-A antigens) and NY-ESO-1 (HLA-A*0201 restricted) epitopes was evaluated in cancer patients to assess whether a specific cellular response could be detected or generated upon ex vivo stimulation. Induction of CTL was performed on expanded CD8+ T lymphocytes infiltrating the tumors (TIL), possibly enriched in activated specific T cells, eventually due to the presence of antigen. After successful expansion, CD8+ cells were repeatedly stimulated with autologous mature IL-4-DC pulsed with CTA peptides and/or infected with a recombinant vaccinia virus (rVV) encoding the corresponding epitopes together with the gene encoding human CD80. These vectors were constructed during the present study to provide highly effective immunogenic reagents with the perspective of possible clinical application. CTA specific CTL response could be observed in 7/26 populations. In six cultures, cytotoxic activity was low and did not correlate with expression of specific CTA in the original tumor specimens. These CTL responses could possibly be attributed to a primary in vitro sensitization. However, in one case stimulation of TIL with rVV infected APC revealed a high level of MAGE-A10 specific CTL response detectable by cytotoxicity assays and multimer staining. The corresponding gene, encoding the target epitope, was highly expressed in the original tumor. In NSCLC, CTA specific CTL sensitization in TIL, as detectable upon repeated stimulation with a panel of well defined peptides and highly effective APC, is rare. On the other hand, strong CTA specific CTL responses could frequently be generated from peripheral blood lymphocyes of healthy donors, upon stimulation of large numbers of effector cells with antigen pulsed DC obtained by GM-CSF/IFNα induction. The concomitant expression of multiple CTA in NSCLC and the possibility of natural CTL responses in these cancers may support the development of specific vaccination protocols using multi antigen vaccine preparations of CTA

Differential responsiveness to IL-2, IL-7, and IL-15 common receptor gamma chain cytokines by antigen-specific peripheral blood naive or memory cytotoxic CD8+ T cells from healthy donors and melanoma patients

Common receptor gamma chain (c-gamma) cytokines (CKs) support proliferation of CD8+ T cells in presence or absence of antigen triggering and help maintaining the immunologic memory. We addressed the effects of low (> or = 5 ng/mL)-dose interleukin (IL)-2, IL-7, or IL-15 on human naive and memory antigen-specific CD8+ T cells. Peripheral blood CD8+ lymphocytes proliferated with decreasing efficiency in response to IL-15, IL-7, and IL-2. Of note, IL-15 preferentially promoted expansion of CD45RA/CD8+ T-cell memory subset. Accordingly, cytotoxic T lymphocytes specific for cytomegalovirus-derived antigens from seropositive donors proliferated in response to IL-15 and, to lesser extent to IL-7, but poorly to IL-2. CD8+ T cells were then pretreated with CK before antigen stimulation using, as read out, specific cytotoxic activity. After the pretreatment with IL-15, but not IL-2, previously experienced viral antigens induced vigorous cytotoxic responses. Minor effects of IL-7 were also detectable. In contrast, IL-2 best supported the cytotoxic T lymphocyte generation from prevailingly naive CD8 T cells from HLA-A*0201 healthy donors, specific for L27Melan-A/MART-126-35 melanoma-associated antigen. Cells from melanoma patients were tested before and after Melan-A/MART-1-targeted antigen-specific immunotherapy. Untreated patients showed heterogeneous patterns of responsiveness to c-gamma CK. However, when naive patients whose CD8+ T cells best responded to IL-2 were vaccinated, a modified responsiveness pattern was detectable. After immunization, cells displayed a significantly higher response to IL-15 than to IL-2 pretreatment. Thus, responsiveness to c-gamma CK is critically influenced by naive or memory status of peripheral blood CD8+ T cells