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ObjectiveTumour-infiltrating lymphocytes (TILs) favour survival in human colorectal cancer (CRC). Chemotactic factors underlying their recruitment remain undefined. We investigated chemokines attracting T cells into human CRCs, their cellular sources and microenvironmental triggers.DesignExpression of genes encoding immune cell markers, chemokines and bacterial 16S ribosomal RNA (16SrRNA) was assessed by quantitative reverse transcription-PCR in fresh CRC samples and corresponding tumour-free tissues. Chemokine receptor expression on TILs was evaluated by flow cytometry on cell suspensions from digested tissues. Chemokine production by CRC cells was evaluated in vitro and in vivo, on generation of intraperitoneal or intracecal tumour xenografts in immune-deficient mice. T cell trafficking was assessed on adoptive transfer of human TILs into tumour-bearing mice. Gut flora composition was analysed by 16SrRNA sequencing.ResultsCRC infiltration by distinct T cell subsets was associated with defined chemokine gene signatures, including CCL5, CXCL9 and CXCL10 for cytotoxic T lymphocytes and T-helper (Th)1 cells; CCL17, CCL22 and CXCL12 for Th1 and regulatory T cells; CXCL13 for follicular Th cells; and CCL20 and CCL17 for interleukin (IL)-17-producing Th cells. These chemokines were expressed by tumour cells on exposure to gut bacteria in vitro and in vivo. Their expression was significantly higher in intracecal than in intraperitoneal xenografts and was dramatically reduced by antibiotic treatment of tumour-bearing mice. In clinical samples, abundance of defined bacteria correlated with high chemokine expression, enhanced T cell infiltration and improved survival.ConclusionsGut microbiota stimulate chemokine production by CRC cells, thus favouring recruitment of beneficial T cells into tumour tissues.

Immunotherapy has emerged during the past two decades as an innovative and successful form of cancer treatment. However, frequently, mechanisms of actions are still unclear, predictive markers are insufficiently characterized, and preclinical assays for innovative treatments are poorly reliable. In this context, the analysis of tumor/immune system interaction plays key roles, but may be unreliably mirrored by in vivo experimental models and standard bidimensional culture systems. Tridimensional cultures of tumor cells have been developed to bridge the gap between in vitro and in vivo systems. Interestingly, defined aspects of the interaction of cells from adaptive and innate immune systems and tumor cells may also be mirrored by 3D cultures. Here we review in vitro models of cancer/immune cell interaction and we propose that updated technologies might help develop innovative treatments, identify biologicals of potential clinical relevance, and select patients eligible for immunotherapy treatments.

Cancer immunotherapy is increasingly accepted as a treatment option for advanced stage disease. The identification of tumour-associated antigens in 1991 has prompted the development of antigen-specific immunotherapeutic strategies for a variety of cancers. Many of them result in some immunological responses in cancer patients; however, clinical results were not observed concomitantly with immunological responses; therefore, further improvements in the field of immunotherapy are urgently needed. Virosomes are lipidic envelopes devoid of genetic information, but which retain the antigenic profile and fusogenic properties from their viral origin. Virosomes are versatile antigen carriers and can be engineered to perform various tasks in cancer immunotherapy. Preclinical data have fostered the development of innovative clinical protocols. Hence, immunopotentiating reconstituted influenza virosomes will be assessed in breast and melanoma immunotherapy, and may contribute to the development of clinically effective cancer vaccines and ultimately improve patient outcomes. The objective of this review is to provide an overview of the potential clinical applications of virosomes as innovative and potentially effective reagents in active specific cancer immunotherapy.

Recombinant poxviruses expressing immunomodulatory molecules together with specific antigens represent powerful vaccines for cancer immunotherapy. Recently, we and others have demonstrated, in vitro and in vivo, that coexpression of CD80 and CD86 costimulatory molecules enhances the immunogenic capacity of a recombinant vaccinia virus (rVV) encoding different tumor-associated antigens. To further investigate the capacity of these vectors to provide ligands for different costimulatory pathways relevant in the generation of T cell responses, we constructed a recombinant virus (rVV) expressing CD40 ligand or CD154 (CD154rVV). Upon binding the CD40 receptor expressed on antigen presenting cells (APC), this molecule, physiologically expressed on activated CD4+ T cells, increases their antigen presentation and immunostimulatory capacities.Therefore, we evaluated the effects of CD154rVV infection on APC activation and its consequences on T cell stimulation. CD154rVV infection of autologous fibroblasts, monocytes, or iDC promoted the expression of a number of cytokines, including GM-CSF, TNF-α, and IL-15 in iDC. Most importantly, IL-12 p40 gene expression and protein secretion were induced by CD154rVV but not by wild-type VV (WT VV) in either CD14+ cells or iDC, and these effects could be blocked by anti-CD40 monoclonal antibodies. Furthermore, phenotypic characterization of CD154rVV infected iDC revealed enhanced expression of CD83 and CD86 surface markers as compared with wild-type vaccinia virus infection. As expected, VV infection triggered cytokines gene expression in cultures including APC and T cells from VV immune donors. However, cytokine genes typically expressed by T cell receptor triggered T cells such as those encoding IL-2 and IFN-γ, or T cell proliferation, were detectable to a significantly higher extent in CD154rVV infected cultures, as compared with WT VV.Activation of specific CD8+ T cells was then investigated using MART-1/Melan-A27–35 epitope as the model of tumor-associated antigen (TAA). In the presence of CD154rVV activated APCs, significantly higher numbers of specific cytotoxic CD8+ T cells were detected, as compared with cultures performed in the presence of WT VV or in the absence of virus.Taken together, these data indicate that functional CD154 expression from rVV infected cells promotes APC activation, thereby enhancing antigen-specific T cell generation. Such a recombinant vector might help bypass the requirement for activated helper cells during CTL priming, thus qualifying as a potentially relevant vector in the generation of CD8+ T cell responses in cancer immunotherapy.

The cancer testis (CT) family of antigens are expressed in certain malignant neoplasms and are silent in normal adult tissues, except for the testis. Expression of 2 members of this family, MAGE-A4 and NY-ESO-1, has been described recently in germ cell tumors, malignant melanomas, certain carcinomas and sarcomas. Our study is the first to describe the expression pattern of CT antigens in uterine neoplasms. Ninety-eight cases of uterine neoplasms, including 41 endometrioid, 19 papillary serous and 7 clear cell carcinomas, 22 carcinosarcomas and 9 endometrial stromal sarcomas were studied. Immunohistochemistry was carried out with the 57B monoclonal antibody that recognizes predominantly the MAGE-A4 antigen in paraffinized tissues and the D8.38 antibody that recognizes NY-ESO-1. MAGE-A4 expression was found to be present in 12% of the endometrioid adenocarcinomas, 63% of the papillary serous carcinomas and 91% of the carcinosarcomas. Within the tumor population the extent of MAGE-A4 expression was highest in the carcinosarcomas. In 12 of 22 positively staining carcinosarcomas more than 50% of the tumor cells expressed MAGE-A4. NY-ESO-1 expression was seen in 19% of the endometrioid adenocarcinomas, 32% of the papillary serous carcinomas and in 45% of the carcinosarcomas. CT antigen immunoreactivity was observed in both the carcinomatous and sarcomatous components of the carcinosarcomas and strong correlation between MAGE-A4 and NY-ESO-1 expression was present in individual cases. In summary, strong MAGE-A4 expression and to a lesser degree NY-ESO-1 expression is characteristic of the vast majority of uterine carcinosarcomas and a major subset of papillary serous carcinomas. These results suggest that CT antigen expression by these tumors may represent a novel target for immunotherapy. © 2002 Wiley-Liss, Inc.

TAAs of the MAGE family are mostly studied as targets of specific immune responses. Their potential relevance as tumor markers has also been underlined. We used a MAb, 57B, recognizing MAGE-A4 protein in paraffin-embedded sections, to evaluate its expression in bladder cancers by employing TMA including 2,317 samples from 1,849 patients. In 2,090/2,317 cases (90.2%), immunostaining yielded interpretable results. Since for some patients more than 1 sample was available, only interpretable first biopsies (n = 1,628) were considered. MAGE-A4 protein was expressed at significantly (p < 0.001) higher frequency in squamous (25/55, 45.5%) than in adeno (4/15, 26.7%), sarcomatoid (4/14, 28.6%), small cell (5/20, 25%) or transitional cell (281/1,522, 18.5%) carcinomas. In TCCs, overall MAGE-A4 positivity was significantly correlated with invasive phenotype (p < 0.001) and high tumor grade (p < 0.0001). Clinical data from 908 TCC patients were retrospectively evaluated, revealing that strong 57B staining was highly significantly associated with decreased tumor-specific survival (p < 0.0001). These data suggest that evaluation of MAGE-A4 protein expression is useful in the identification of groups of TCCs characterized by severe prognosis, thus possibly providing indications for early MAGE TAA-targeted immunotherapy. © 2002 Wiley-Liss, Inc.

Type I IFNs are immunomodulatory factors that possibly influence the properties of tissue‐resident dendritic cells. Here, we have investigated the capacity of IFN‐α2a to enhance DC chemoattractive and stimulatory capacity toward CD8+T lymphocytes. Phenotypically, IFN‐α2a‐treated DC (IFN‐DC) showed an increased expression of costimulatory and antigen‐presenting molecules, maintained even after withdrawal of the cytokine. IFN‐α2a enhanced DC stimulatory capacity toward CD8+T cells, as assessed by increased MLR responses and induction of MART‐126–35‐specific CTLs in vitro. No functional CCR7 chemokine receptor could be induced. Instead, high amounts of IP‐10/CXCL10 and MIG/CXCL9 chemokines were produced. Freshly isolated CD8+RO+cells and PHA‐activated CD8+T cells migrated efficiently in response to IFN‐DC‐conditioned medium, and the migration could be inhibited by neutralizing the CXCR3 receptor on responder cells. These results suggest that type I IFNs could enhance the elicitation of class I‐restricted effector functions in vivo in the periphery by modulating DC chemoattractive properties.

A specific cellular immune response directed against a panel of three defined tumor-associated antigen (TAA) epitopes was induced in metastatic melanoma patients by a prime-boost strategy taking advantage of an innovative recombinant vaccinia virus as evaluated by quantitative assessment of cytotoxic T lymphocytes (CTLs) with corresponding specificity. The immunization protocol consisted of the administration of psoralen-UV-treated and replication-incompetent recombinant vaccinia virus encoding the three immunodominant HLA-A*0201-restricted epitopes Melan-A27-35, gp100280-288, and tyrosinase1-9 together with two costimulatory molecules, B7.1 and B7.2, in the context of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. Boosts were subsequently applied with corresponding synthetic nonapeptides and GM-CSF. Specific CTL induction was assessed by tetramer staining and CTL precursor (CTLp) frequency evaluation. Within 12 days of injection of the recombinant vector, cytotoxic T cell responses specific for engineered epitopes were detectable in three of three patients. During the vaccination treatment, antigen-specific CTLp frequencies exceeding 1:10,000 peripheral CD8+ T cells could be observed. Tetramer staining also revealed significant increases in specific CD8+ T cell numbers. We conclude that active specific antitumor vaccination can raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells with heterogeneous antigen expression. Data from this study emphasize the potency of a recombinant vaccinia virus vector encoding multiple minigenes and costimulatory molecules in the context of exogenously administered GM-CSF. Clinical effectiveness of this immunologically active protocol should therefore be explored in appropriately selected groups of patients.

Malignant hyperthemia (MH) is a pharmacogenetic disease triggered by volatile anesthetics and succinylcholine in genetically predisposed individuals. The underlying feature of MH is a hypersensitivity of the calcium release machinery of the sarcoplasmic reticulum, and in many cases this is a result of point mutations in the skeletal muscle ryanodine receptor calcium release channel (RYR1). RYR1 is mainly expressed in skeletal muscle, but a recent report demonstrated the existence of this isoform in human B-lymphocytes. As B-cells can produce a number of cytokines, including endogenous pyrogens, we investigated whether some of the symptoms seen during MH could be related to the involvement of the immune system. Our results show that (i) Epstein-Barr virus-immortalized B-cells from MH-susceptible individuals carrying the V2168M RYR1 gene mutation were more sensitive to the RYR activator 4-chloro-m-cresol and (ii) their peripheral blood leukocytes produce more interleukin (IL)-1β after treatment with the RYR activators caffeine and 4-chloro-m-cresol, compared with cells from healthy controls. Our result demonstrate that RYR1-mediated calcium signaling is involved in release of IL-1β from B-lymphocytes and suggest that some of the symptoms seen during an MH episode may be due to IL-1β production.

Testicular germ cell tumors are the most common malignancy in young males, and the frequency of these tumors has risen dramatically over the last century. Because it is known that the MAGE genes are expressed in a wide variety of tumors but are expressed only in the mitotic spermatogonia (germ cells) and in the primary spermatocytes in the normal testis, the authors screened the expression of MAGE-A4 in a panel of testicular germ cell tumors. Monoclonal antibody 57B raised against MAGE-A4 was tested immunohistochemically on 12 classical seminomas, 5 anaplastic seminomas, 10 various specimens of nonseminomatous germ cell tumors (NSGCTs), 2 combined tumors containing seminoma components, 1 Sertoli cell tumor, 2 Leydig cell tumors, and 15 carcinomas in situ (CIS). In addition, monoclonal antibody 57B was tested on embryonic gonad (age 8 weeks) and fetal gonads (ages 15 weeks, 17 weeks, and 28 weeks). Classical seminomas uniformly and specifically expressed MAGE-A4 compared with anaplastic seminomas and NSGCTs, which were negative for this antigen. Specific expression of MAGE-A4 also was seen in subpopulations of CIS cells, providing additional evidence for heterogeneity of the phenotype of these cells, in which it is believed that differentiation and proliferation generate seminomas and NSGCTs. Finally, MAGE-A4 was expressed in the fetal precursors of the stem germ cells from 17 weeks of gestation onward, in accordance the fact that CIS can arise from prespermatogonia in the fetus. MAGE-A4 can be considered a potential specific marker for normal premeiotic germ cells and germ cell tumors and can be used to characterize classical seminomas. Cancer 2001;92:2778–85. © 2001 American Cancer Society.

Public perception of organ donation critically affects the availability of organ transplantation in the Western world.

In this study, a computer-assisted reverse immunology approach was utilized in order to identify potentially antigenic peptides derived from the differentiation antigen TRP-2, a melanosomal protein frequently expressed in melanoma. Among the seven peptides complying with HLA-A2.1-binding motifs, two induced specific CD8⁺ cytotoxic T lymphocytes. HLA-A2.1⁺ melanoma cells expressing TRP-2 were lysed by clones specific for TRP-2360–368 (TLDSQVMSL) peptide, thus identifying it as a naturally processed epitope. Other T-cell clones directed against TRP-2476–484 (VMGTLVALV) were unable to lyse HLA-matched TRP-2⁺ cell lines. The role of intracellular proteolytic processing in the generation of this epitope was investigated by transfecting mini-genes encoding the TRP-2476–484 peptide alone or carrying N- or C-terminal extensions. Specific T-cell clones recognized target cells expressing the cytotoxic T-lymphocyte (CTL)-defined epitope or its C-terminally extended precursor, but failed to recognize cells expressing the N-terminally extended TRP-2476–484 peptide, suggesting the presence of a negative processing signal (NPS). Regarding C-terminus-flanking regions, mutational analysis indicates that the GLY485 residue plays a key role in the processing of the TRP-2476–484 epitope. Interestingly, proteasome inhibitors preventing the generation of the MART-1/Melan-A27–35 immunodominant melanoma tumor-associated antigen (TAA) promoted detectable presentation of TRP-2476–484 epitope in HLA-A2.1⁺ and TRP-2⁺ tumor lines, as witnessed by cytokine release by specific T-cell clones. Int. J. Cancer 87:241–246, 2000. © 2000 Wiley-Liss, Inc.

Monoclonal antibody (Mab) 57B, which was raised against a recombinant MAGE-A3 protein, was tested for its ability to stain cells expressing various members of the MAGE-A gene family. COS-7 cells transfected with cDNAs encoding MAGE-A1, A2, A3, A4, A6, or A12 were stained, whereas those transfected with MAGE-A8, A9, A10, or A11 cDNAs were not. However, in tissue sections, we observed a different pattern of staining: the antibody effectively stained the tumors that expressed MAGE-A4 and only these tumors, regardless of the expression of the other MAGE-A genes. It seems, therefore, that at the level of MAGE gene expression found in tumors, a level clearly lower than that observed in transfected COS cells, only the MAGE-A4 protein can be reliably detected. We conclude that the 57B Mab should be useful for tumor diagnosis related to therapeutic vaccination involving MAGE-A4. Int. J. Cancer 86:835–841, 2000. © 2000 Wiley-Liss, Inc.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that can be used for vaccination purposes, to induce a specific T-cell response in vivo against melanoma-associated antigens. We have shown that the sequential use of early-acting hematopoietic growth factors, stem cell factor, IL-3 and IL-6, followed by differentiation with IL-4 and granulocyte-macrophage colony-stimulating factor allows the in vitro generation of large numbers of immature DCs from CD34⁺ peripheral blood progenitor cells. Maturation to interdigitating DCs could specifically be induced within 24 hr by addition of TNF-α. Here, we report on a phase I clinical vaccination trial in melanoma patients using peptide-pulsed DCs. Fourteen HLA-A1⁺ or HLA-A2⁺ patients received at least 4 i.v. infusions of 5 × 10⁶ to 5 × 10⁷ DCs pulsed with a pool of peptides including either MAGE-1, MAGE-3 (HLA-A1) or Melan-A, gp100, tyrosinase (HLA-A2), depending on the HLA haplotype. A total of 83 vaccinations were performed. Clinical side effects were mild and consisted of low-grade fever (WHO grade I–II). Clinical and immunological responses consisted of anti-tumor responses in 2 patients, increased melanoma peptide-specific delayed-type hypersensitivity reactions in 4 patients, significant expansion of Melan-A- and gp100-specific cytotoxic T lymphocytes in the peripheral blood lymphocytes of 1 patient after vaccination and development of vitiligo in another HLA-A2⁺ patient. Our data indicate that the vaccination of peptide-pulsed DCs is capable of inducing clinical and systemic tumor-specific immune responses without provoking major side effects. Int. J. Cancer 86:385–392, 2000. © 2000 Wiley-Liss, Inc.

Dendritic cells (DC) are professional antigen presenting cells (APC) whose proliferation and functional differentiation can be induced by hematopoietic growth factors including GM-CSF and FLT3 ligand (FL). Colorectal cancers are known to be infiltrated by dendritic cells (DC) and neoplastic cells have been shown to produce GM-CSF. In this work we investigated FLT3 ligand (FL) gene expression and protein production in human colorectal cancer cell lines and clinical tumor specimens. Using reverse transcription polymerase chain reaction (RT-PCR), 6 out of 6 established tumor lines were found to express to variable extents FL gene. In 1 of them, SW480, FL immunoreactivity could be observed by taking advantage of specific antibodies. In contrast, soluble FL could not be detected in any culture supernatant. FLT3 receptor (FR) gene was not expressed and exogenous addition to the cultures of recombinant FL (rFL) did not affect the proliferation of the tumor lines. FL gene expression was investigated using a densitometry-assisted, semiquantitative RT-PCR in clinical tumor specimens. Specific FL gene transcripts were amplified from 12 of 12 surgical samples. In these cases, FL gene expression of significantly lower intensity was also detected in healthy mucosa sampled in the vicinity (2 cm) or at a distance (10 cm) from neoplastic outgrowth. Immunohistochemical studies identified FL-positive cancer cells in 5 of 5 cases tested. No positivity was detected in healthy mucosa epithelia at a distance from the tumor or in stromal cells. FL content in preoperative sera from colorectal cancer patients (n = 13) did not exceed the levels detected in healthy donors (≤ 100 pg/ml). Int. J. Cancer 86:238–243, 2000. © 2000 Wiley-Liss, Inc.

Genes of the MAGE, BAGE, GAGE, and LAGE-1/NY-ESO-1families encode antigenic peptides that are presented by HLA class I molecules and that are recognized on human tumors by autologous cytolytic T lymphocytes. These genes are expressed in many solid tumor types but not in normal tissues, except male germline cells. Because the latter cells are devoid of HLA molecules, the derived antigens are strictly tumor-specific and should constitute safe immunogens for cancer immunotherapy. We detected a significant expression of these genes in a high proportion of bone marrow samples from patients with advanced multiple myeloma. This observation provides a basis for clinical trials aimed at inducing a cellular immune response directed at malignant plasma cells in advanced myeloma patients.

Genes of the MAGE, BAGE, GAGE,and LAGE-1/NY-ESO-1families encode antigenic peptides that are presented by HLA class I molecules and that are recognized on human tumors by autologous cytolytic T lymphocytes. These genes are expressed in many solid tumor types but not in normal tissues, except male germline cells. Because the latter cells are devoid of HLA molecules, the derived antigens are strictly tumor-specific and should constitute safe immunogens for cancer immunotherapy. We detected a significant expression of these genes in a high proportion of bone marrow samples from patients with advanced multiple myeloma. This observation provides a basis for clinical trials aimed at inducing a cellular immune response directed at malignant plasma cells in advanced myeloma patients.

In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4+ T cells from the blood of a healthy donor. CD4+ T cells strongly recognized MAGE-3281–295 and, to a lesser extent, MAGE-3141–155 and MAGE-3146–160. Moreover, CD4+ T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4+ T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3–positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4+ T cells recognized MAGE-3281–295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4+ T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4+ T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4+ T cell epitopes.

Eleven patients with advanced cancer were treated in a clinical gene therapy trial by repeated intra- tumoral injections with different doses of xenogenic fibroblasts secreting high amounts of human interleukin-2 (Vero-IL2). Treatments in a total of 14 courses were well tolerated and resulted in clinical responses and measurable biological effects. Together with increases in serum interleukin-2 (IL-2), modifications of the V-β T cell receptor repertoire and induction of intratumoral T-cell infiltration were observed. When the intratumoral expression of endogenous cytokine genes and the persistence of the IL-2 transgene at the application site and in peripheral blood were investigated, rapid disappearance of the transgene at the application site appeared to be the most prominent biological effect. Tests detecting a single Vero-IL2 cell against a background of 105non-transfected cells were not able to demonstrate significant expression of exogenous IL-2 (i.e. the transgene or transgene-carrying cells) in tumor biopsies or blood at different times. Therefore, further studies were performed to evaluate the mechanism(s) involved in the rapid disappearance of xenogenic carrier cells in more detail. We show here that significant in vitro cytotoxicity against transgene-carrying Vero cells can be observed in peripheral blood of all the patients before treatment as well as in healthy controls. “Cold” target inhibition shows that significant killing of Vero-IL2 cells is mediated by natural killer (NK) cells. This was confirmed by showing that established CD3−/CD16+/CD56+peripheral blood NK cell clones kill both K562 and Vero-IL2 target cells. The failure of other mechanisms (complement, antibody-dependent cell cytotoxicity or cytotoxic T lymphocytes) to destroy xenogenic, histoincompatible Vero cells in vitro suggests that NK cells also might be responsible for the killing of Vero-IL2 in vivo and for the failure to detect the transgene at the application site. These results might also be of importance for some aspects of the current discussion of xenotransplantation.

The therapeutic potential of tumor-infiltrating lymphocytes (TIL) is currently under investigation. TIL have been reported to display characteristic functional defects, including impairments of activation mechanisms. In animal models, therapeutic efficacy of adoptive TIL transfer has been correlated with their capacity to produce cytokines rather than with their in vitro cytotoxic potential. In this work we assayed cytokine gene transcription and protein production in eight cultured TIL populations stimulated with recombinant human IL-2 (rhIL-2), solid-phase-bound anti-CD3 monoclonal antibodies (mAb), or a combination of the two. By using a sensitive reverse polymerase chain reaction technique, we observed that transcription of IL-5, GM-CSF, and TNF-α could be detected in unstimulated, IL-2-starved TIL from all cultures, while IL-4 and IFN-γ genes were found to be transcribed in two cultures out of eight. A 50 U/ml dose of rhIL-2 was sufficient to induce IL-4 and IFN-γ gene transcription in the remaining six cultures and in four more TIL populations, respectively. In contrast, rhIL-2 could not induce IL-2 gene transcription in five of eight TIL cultures. Furthermore, it could not induce IL- 10 gene transcription in any TIL population. Anti-CD3 mAb triggering, however, induced transcription of all these cytokine genes. On the other hand, IL-6, IL-7, or TGF-β 2 gene transcription could not be induced by any of the stimuli used, including the combination of anti-CD3 and rhIL-2. Despite detection of their gene transcripts, GM-CSF, IFN-γ, or TNF-α was never detectable, at the protein level, in unstimulated TIL. Stimulation by rhIL-2 induced GM-CSF secretion, albeit to different extents, in all TIL populations. In contrast, rhIL-2 induced IFN-γ and TNF-α production in three and one TIL population, respectively. CD3 triggering however, induced cytokine production in all TIL populations. Addition of rhIL-2 significantly increased production of GM-CSF, but not of IFN-γ and TNF-α. These data underline that stimulation with a moderate dose of rhIL-2 reveals considerable heterogeneity of TIL populations regarding cytokine gene transcription and secretion. On the other hand, triggering of the CD3-T-cell receptor complex induces transcription of an extended panel of cytokine genes in all TIL populations and secretion of GM-CSF, IFN-γ and TNF-α in virtually all cultures. Thus, if stimulated by properly presented antigenic peptides, TIL are capable of mounting an efficient response in terms of cytokine gene expression and protein production. Copyright 1993, 1999 Academic Press

The role of nitric oxide (NO) in the generation of lymphokine-activated killer (LAK) cells was investigated. Here we report that L-arginine analog NG-monomethyl-L-arginine (NMMA), a specific inhibitor of nitric oxide synthase, prevents LAK cell generation from cultured rat splenic cells. Accumulated NO endproduct nitrite (NO-2), as measured in the supernatants of rat splenic cells, correlated well with the generation of LAK cells. In contrast, cell proliferation induced by rIL-2 or by Con A was not affected by NMMA. Similarly, phenotypic expression of CD25 in rIL-2-stimulated cultures was unaffected. Furthermore, we could not observe differences in percentages of CD5-CD8+cells (NK and LAK cell phenotype markers in rats) between rIL-2-stimulated cultures performed in the presence or absence of NMMA. LAK cell generation could no longer be blocked if NMMA was added to the rat cell cultures 24 hr after rIL-2 stimulation. To further confirm the role of NO in LAK cell generation, rat splenic cells were cultured in medium without L-arginine. Under such conditions rIL-2 could not induce LAK cell generation. Hemoglobin, which is a scavenger of NO, also inhibited LAK cell generation. Finally, addition of sodium nitroprusside (SNP) which releases NO in cultures was able to overcome blocking effects of NMMA. To attempt the identification of NO-producing cells, lysosomotropic agent, L-leucine methyl ester (LME), was used. Generation of LAK cell activity was virtually abolished in cell cultures treated with LME. Addition of SNP to cultures, however, sufficed to restore LAK cell generation. These results suggest that LAK cell precursors depend on a exogenous NO supply from other cell types in order to display their full cytotoxic potential. Similar results were also obtained by using mouse splenocytes as responder cells. In contrast, NMMA did not affect generation of LAK cells from human peripheral blood or spleen mononuclear cells.

Induction of cytokine gene transcription by recombinant human IL-2 (rhIL-2) in peripheral blood mononuclear cells (PBMC) from healthy donors was studied by qualitative polymerase chain reaction. In all donors tested, optimal doses of rhIL-2-induced transcription of genes encoding for IL-5, GM-CSF, IFN-γ and TNF-α whereas transcription of IL-1-α, IL-3, IL-4, and IL-6 genes could only be detected in about half of the donors. Moreover, we observed that different doses of rhIL-2 were needed to induce transcription of different cytokine genes. In contrast, transcription of IL-2 and IL-10 genes was only observed in a minority of donors, irrespective of the concentration of rhIL-2 used. Since IL-10 displays a well-characterized inhibitory activity on the synthesis of cytokines possibly involved in the generation of lymphokine-activated killer (LAK) cells, we asked whether the absence of IL- 10 gene transcription plays a role in the induction of LAK cells. Thus, we tested the effects of different doses of rhIL-10 on the rhIL-2-driven generation of LAK activity. Interestingly, rhIL-10 dose-dependently inhibited the production of IFN-γ and TNF-α induced by IL-2, but had no effects on PBMC proliferation and generation of LAK activity. Similarly, purified CD3-/CD16+lymphocytes, the precursors of LAK effector cells, could be optimally induced by low doses of rhIL-2 to proliferate and generate MHC-unrestricted cytotoxic activity against NK-resistant targets in the presence of rhIL-10. Altogether, our results indicate that rhIL-2 induces transcription of a preferential pattern of cytokine genes, with the IL-10 gene being infrequently transcribed. On the other hand, rhIL-10 shows diverse effects on rhIL-2-triggered PBMC activation, in that it inhibits IFN-γ and TNF-α production but does not affect PBMC proliferation or generation of LAK activity.

Active specific immunotherapy targeting tumor-associated antigens (TAA) requires reagents of high immunogenicity and safety. To address this issue, we constructed a recombinant vaccinia virus carrying a minigene insert encoding the HLA-A2.1-restricted MART-1/Melan-A27-35 melanoma TAA (rVV-M). To facilitate the entry of the antigenic epitope into the endoplasmic reticulum, a sequence coding for adenovirus E3/19K leader peptide was added. This rVV-M was made replication-incompetent by treatment with psoralen and UV light. Infection with rVV-M rendered HLA-A2.1 EBV-transformed lymphoblastoid cells sensitive to the cytotoxic effects of HLA-class-I-restricted, MART-1/Melan-A27-35-specific cytotoxic T lymphocytes (CTL). The capacity of rVV-M to generate HLA-A2.1-restricted MART-1/Melan A-specific CTL was demonstrated from tumor-infiltrating-lymphocyte (TIL) cultures and from healthy donors' peripheral-blood mononuclear cells (PBMC). MART-1/Melan-A27-35-specific CTL were generated from TIL after 2 weekly stimulation courses. Infection with rVV-M elicited a higher CTL response than addition of exogenous peptide, whereas, when a similar protocol was used to stimulate PBMC of healthy donors, significant and specific cytotoxic activity could be observed only upon rVV-M infection but not upon exogenous peptide addition. All CTL generated upon rVV-M stimulation were also able to efficiently kill melanoma cell lines expressing both MART-1/Melan-A and HLA-A2.1. In addition, TNF-α production could be induced in rVV-M-stimulated CTL upon co-culture with COS-7 cells transiently transfected with MART-1/Melan-A and HLA-A2.1 genes. This safe and highly immunogenic reagent could be of use in TAA-targeted clinical immunotherapy. Int. J. Cancer 71:491-496, 1997. © 1997 Wiley-Liss Inc.

We constructed recombinant vaccinia viruses (recVV) encoding the human T-cell costimulatory molecules B7-1 and B7-2. To abrogate the vaccinia virus transcription termination signal for early genes, the cDNA of B7-1 had to be modified by a T through C sense mutation at position 766. Upon infection with replication incompetent and noncytopathic recVV, several tumor cell lines as well as cultured human fibroblasts expressed the costimulatory molecules. All these cells were capable of providing effective costimulation for proliferation of resting CD4+T-cells after infection with recVV encoding B7 molecules. The costimulatory effect could be blocked with CTLA-4 IgG fusion protein, the soluble ligand for B7. RecVV-induced overexpression of B7 on syngeneic EBV-transformed lymphoblastoid B-cells was able to costimulate the proliferative response of CD4+memory cells against VV antigens. The possibility of easily engineering a variety of human cells using recVV encoding human B7 molecules holds implications for the future design of vaccination strategies.

C-type lectin-like inhibitory receptors are heterodimers consisting of CD94 and NKG2-A-B molecules expressed on NK cells and on a subset of activated T lymphocytes. Their inhibitory effects on NK cytotoxicity and on the NK-like activity of T cell clones have been demonstrated, but no data are currently available on antigen-specific class I-restricted cytotoxic T lymphocytes (CTL). We have generated a panel of HLA-A2.1-restricted CTL clones directed against a nonapeptide derived from a melanoma-associated antigen, dopachrome tautomerase (TRP-2). All clones were CD8⁺ and TCR α β⁺. About half of them expressed a CD94^bright phenotype, whereas the remaining were CD94^dim. Only the CD94^bright CTL expressed the NKG2-A-B gene, consistent with the expression of a C-type, lectin-like, inhibitory CD94/NKG2-A-B heterodimer. Both CD94^bright and CD94^dim clones appeared to require similar amounts of synthetic epitope sensitizing target cells. Addition of anti-CD94 mAb resulted in a significant increase of specific killing by CD94^bright, but not by CD94^dim clones in the presence of suboptimal concentrations of peptide, whereas, when optimal amounts were used, the mAb did not induce a significant modulation of the cytotoxicity. Antigen-induced inward [Ca²⁺]i fluxes were unaffected, but an enhancement of TCR down-modulation could be observed in the presence of anti-CD94 mAb at high concentration of antigenic peptide. The analysis of the TCR-Vβ repertoire of the CTL clones by RT-PCR and immunofluorescence revealed that all clones regardless of CD94 phenotype shared Vβ22 expression. Most importantly, sequence analysis showed that they all expressed identical Vβ22 TCR rearranged with Jβ2.1 and Cβ2. Taken together, these data indicate that different expression of functionally active lectin-like inhibitory receptors can be detected in CTL clones sharing identical TCR sequence and peptide specificity.

Granulocyte-macrophage colony stimulating factor (GM-CSF) gene expression and protein production were investigated in colorectal cancer cell lines and surgical specimens. In 6 of 6 established tumor lines, expression of the GM-CSF gene was observed by reverse transcription polymerase chain reaction (RT-PCR). Furthermore, for 2 of the lines, the cytokine was detectable in ≥100 pg/ml amounts in culture supernatants by enzyme-linked immunosorbent assay tests. Addition of recombinant GM-CSF at doses ranging between 30 pg and 30 ng/ml did not appear to affect the proliferation of colorectal cancer cell lines as measured by ³H-thymidine incorporation. GM-CSF gene expression was then examined in surgical specimens by a densitometry-assisted, semi-quantitative RT-PCR technique. In the 10 samples analyzed, significantly higher expression was detectable in tumors, as compared with autologous healthy mucosa sampled in the vicinity (2 cm) or at distance (10 cm) from the neoplastic focus. Immunohistochemistry studies performed on 13 specimens led to the identification of intracytoplasmic GM-CSF in tumor cells in 9 samples. In 6 of these, positivity of stromal fibroblasts and lymphocytes adjacent to the tumor was also observed. In contrast, intracellular GM-CSF was only detectable in 2 cases in untransformed epithelial cells, close to the neoplasm, but never in healthy mucosa at distance from the tumor. Infiltration by dendritic cells (DC) was also investigated. In 5 of 8 colorectal cancers tested, DC aggregates accounted for more than 10% of stromal cells. Lower numbers were detectable in healthy mucosa. However, DC infiltration could not be correlated with the presence of GM-CSF-positive neoplastic cells in the tumor specimens. Remarkably, cultured DC were unable to exert significant cytotoxic activity against colorectal cancer cells in vitro. Int. J. Cancer 77:378–385, 1998. © 1998 Wiley-Liss, Inc.

No abstract.

The surface expression of ß2-integrins was investigated in leukocytes from patients undergoing ischemia induced by tourniquet application for elective hand surgery. Blood samples were obtained before initiation, at the end of ischemia, and after 15 minutes of reperfusion from ischemic and contralateral arms of five patients. Comparable expression of CD18, CD11a, CD11b, and CD11c could be detected by immunofluorescence in leukocytes from samples drawn from either arm before tourniquet application. In contrast, a significant increase in the expression of CD18 was detectable in monocytes, granulocytes, and lymphocytes from the ischemic arm compared with that in the nonischemic contralateral control, at the end of the ischemia time (80 ± 16 minutes). A significantly increased expression of CD11b, but not CD11a or CD11c, determinants was also observed in granulocytes and monocytes. Concomitantly, a significant reduction in the percentages of granulocytes in samples from ischemic areas was detectable. After 15 minutes of reperfusion, differences in the expression of these adhesion molecules were no longer significant. The expression of the genes encoding interleukins IL-1a, IL-1ß, and IL-6 and tumor necrosis factor alpha (TNFa) proinflammatory cytokines was also studied by reverse polymerase chain reaction (rPCR) in peripheral blood mononuclear cells (PBMCs) obtained from the same samples in three patients. IL-1ß or IL-6 gene expression was never observed. Expression of IL-1a and TNFa genes, as detected in two patients, was not related with induction of ischemia. However, in these patients expression of one or both these genes was observed in samples derived from the ischemic but not the control arm after 15 minutes of reperfusion. These data document that overexpression of adhesion molecules and sequestration of leukocytes take place following short ischemia times, as routinely applied clinically for minor surgical procedures. L’expression de surface des intégrins ß2des leucocytes a été analyséc chez les patients opérés de la main ayant une ischémie induite par un garrot appliqué au niveau du bras. On a prélevé du sang aux deux bras, chez cinq patients, avant et 15 minutes après l’ischémie provoquée. Par immunofluorescence on a mis en évidence l’expression des intégrins CD18, CD11a, CD11b et CD11c. L’expression avant l’ischémie était comparable dans les deux bras. Par contre, on a retrouvé à la fin de l’ischémie provoquée (80 ± 16 minutes) une augmentation significative de l’expression de CD18 dans les monocytes, les polynucléaires et les lymphocytes du bras sous garrot, comparé au bras libre contrôle. On a également retrouvé une augmentation de l’expression de CD11b, mais pas de CD11a ou de CD11c, dans les polynucléaires et les monocytes. Dans le même temps, on a détecté une réduction significative du pourcentage des polynucléaires dans les zones ischémiques. Après 15 minutes de reperfusion, les différences dans l’expression de ces molécules d’adhésion n’étaient plus significatives. L’expression des gènes qui codent les cytokines proinflammatoires IL-1-a, IL-1ß, IL-6 and TNF-a a été également étudiée par une réaction en chaîne de polymérase inversée dans les cellules mononucléées du sang périphérique (PBMC) obtenues à partir de ces mêmes échantillons chez trois patients. L’expression des gènes IL-1ß ou IL-6 n’a jamais été observée. L’expression des gènes IL-1a et TNF-a, détectéc chez deux patients, n’était pas en rapport avec l’ischémie. Après 15 minutes de reperfusion, l’expression d’un ou des deux gènes pouvaient être détectée dans les échantillons provenant du bras ischémié mais pas dans le bras contrôle. Ces données confirment que la surexpression des molécules d’adhésion et la séquestration des leucocytes ont lieu après des temps d’ischémie relativement courts, tels que ceux qui sont entraînés par les procédés chirurgicaux mineurs de tous les jours. Se investigó la expresión de superficie de las integrinas-b2 en leucocitos de pacientes sometidos a isquemia por aplicación de un torniquete para cirugía electiva de la mano. En 5 pacientes se tomaron muestras de sangre antes de la iniciación y al final de la isquemia y luego de 15 minutos de reperfusión de la isquemia y en el brazo contralateral. Se detectó expresión comparable de CD18, CD11a, CD11b y CD11c mediante immunofluorescencia en leucocitos de las muestras tomadas de cualquiera de los dos brazos antes de la aplicación del torniquete. En contraste, se detectó un incremento significativo de la expresión de CD18 en monocitos, granulocitos y linfocitos del brazo isquémico, en comparación con el control contralateral no isquémico, al final del período de isquemia (80 B1 16 minutos). También se observó una expresión significativamente aumentada de CD11b, pero no de déterminantes de CD11a o CD11c, en granulocitos y monocitos. En forma concomitante, se detectó una reducción significativa en el porcentaje de granulocitos en muestras de territorios isquémicos. Luego de 15 minutos de reperfusión, la diferencia en la expresión de éstas moléculas de adhesión dejó de ser significativa. La expresión de los genes, que encodan la citocinas proinflamatorias IL-1E0, IL-1á, IL-6 y FNT-E0 también fue estudiada por medio de la técnica de rPCR en células sanguíneas mononucleares periféricas obtenidas a partir de las mismas muestras, en tres pacientes. No se observó expresión del gen IL-1a o IL-6. La expresión de los genes de IL-1E0 y FNT-E0, según fue detectada en dos pacientes, no se relacionó con la inducción de isquemia. Sin embargo, en estos pacientes la expresión de uno o de los dos genes pudo ser observada en muestras derivadas del brazo isquémico pero no en las del brazo control, despuaés de 15 minutos de reperfusión. Estos datos documentan que la sobre-expresión de moléculas de adhesión y el secuestro de leucocitos tienen lugar luego de cortos tiempos de isquemia, tales como se aplican en el curso de procedimientos quirúrgicos menores.

The surface expression of β2-integrins was investigated in leukocytes from patients undergoing ischemia induced by tourniquet application for elective hand surgery. Blood samples were obtained before initiation, at the end of ischemia, and after 15 minutes of reperfusion from ischemic and contralateral arms of five patients. Comparable expression of CD18, CD11a, CD11b, and CD11c could be detected by immunofluorescence in leukocytes from samples drawn from either arm before tourniquet application. In contrast, a significant increase in the expression of CD18 was detectable in monocytes, granulocytes, and lymphocytes from the ischemic arm compared with that in the nonischemic contralateral control, at the end of the ischemia time (80 ± 16 minutes). A significantly increased expression of CD11b, but not CD11a or CD11c, determinants was also observed in granulocytes and monocytes. Concomitantly, a significant reduction in the percentages of granulocytes in samples from ischemic areas was detectable. After 15 minutes of reperfusion, differences in the expression of these adhesion molecules were no longer significant. The expression of the genes encoding interleukins IL-1α, IL-1β, and IL-6 and tumor necrosis factor alpha (TNFα) proinflammatory cytokines was also studied by reverse polymerase chain reaction (rPCR) in peripheral blood mononuclear cells (PBMCs) obtained from the same samples in three patients. IL-1β or IL-6 gene expression was never observed. Expression of IL-1α and TNFα genes, as detected in two patients, was not related with induction of ischemia. However, in these patients expression of one or both these genes was observed in samples derived from the ischemic but not the control arm after 15 minutes of reperfusion. These data document that overexpression of adhesion molecules and sequestration of leukocytes take place following short ischemia times, as routinely applied clinically for minor surgical procedures.

Objectives: Analysis of tumor immune infiltration has been suggested to outperform tumor, node, metastasis staging in predicting clinical course of colorectal cancer (CRC). Infiltration by cells expressing OX40, a member of the tumor necrosis factor receptor family, or CD16, expressed by natural killer cells, monocytes, and dendritic cells, has been associated with favorable prognosis in patients with CRC. We hypothesized that assessment of CRC infiltration by both OX40+ and CD16+ cells might result in enhanced prognostic significance.Methods: Colorectal cancer infiltration by OX40 and CD16 expressing cells was investigated in 441 primary CRCs using tissue microarrays and specific antibodies, by immunohistochemistry. Patients’ survival was evaluated by Kaplan-Meier and log-rank tests. Multivariate Cox regression analysis, hazard ratios, and 95% confidence intervals were also used to evaluate prognostic significance of OX40+ and CD16+ cell infiltration.Results: Colorectal cancer infiltration by OX40+ and CD16+ cells was subclassified into 4 groups with high or low infiltration levels in all possible combinations. High levels of infiltration by both OX40+ and CD16+ cells were associated with lower pT stage, absence of peritumoral lymphocytic (PTL) inflammation, and a positive prognostic impact. Patients bearing tumors with high infiltration by CD16+ and OX40+ cells were also characterized by significantly longer overall survival, as compared with the other groups. These results were confirmed by analyzing an independent validation cohort.Conclusions: Combined infiltration by OX40+ and CD16+ immune cells is an independent favorable prognostic marker in CRC. The prognostic value of CD16+ immune cell infiltration is significantly improved by the combined analysis with OX40+ cell infiltration.

No Abstract. Int. J. Cancer 86:749–751, 2000. © 2000 Wiley-Liss, Inc.

No abstract.

ABSTRACTCD40 triggering may result in antitumor effects of potentially high clinical relevance. To gain insights important for patient selection and to identify adequate targeting techniques, we investigated CD40 expression in human cancer tissues and generated a replication-incompetent recombinant vaccinia virus expressing CD40 ligand (rVV40L). Its effects were explored in vitroand in vivoupon direct CD40 targeting on malignant cells or macrophage activation. CD40 expression was analyzed by immunohistochemistry in tumor and stromal cells in a multi-tumor array including 836 specimens from 27 different tumor types. Established tumor cell lines were used to explore the capacity of rVV40L to induce malignant cell apoptosis and modulate functional profiles of polarized macrophages. CD40 expression was detectable in significantly higher numbers of stromal as compared to malignant cells in lung and breast cancers. CD40 ligation following rVV40L infection induced apoptosis in CD40(+) cancer cells, but only in the presence of intact specific signal transduction chain. Importantly, rVV40L infection promoted the induction of TNF-α-dependent antitumor activity of M1-like macrophages directed against CD40(-) targets. CD40-activated M1-like macrophages also displayed enhanced ability to CXCL10-dependently recruit CD8+ T cells and to efficiently present cancer cell intracellular antigens through cross-priming. Moreover, rVV-driven CD40L expression partially “re-educated” M2-like macrophages, as suggested by detectable CXCL10 and IL-12 production. Most importantly, we observed that intra-tumoral injection of rVV40L-infected human macrophages inhibits progression of human CD40(-) tumors in vivo. First evidences of anticancer activity of rVV40L strongly encourage further evaluations.

ABSTRACTInteraction between cancerous, non-transformed cells, and non-cellular components within the tumor microenvironment plays a key role in response to treatment. However, short-term culture or xenotransplantation of cancer specimens in immunodeficient animals results in dramatic modifications of the tumor microenvironment, thus preventing reliable assessment of compounds or biologicals of potential therapeutic relevance.We used a perfusion-based bioreactor developed for tissue engineering purposes to successfully maintain the tumor microenvironment of freshly excised breast cancer tissue obtained from 27 breast cancer patients and used this platform to test the therapeutic effect of antiestrogens as well as checkpoint-inhibitors on the cancer cells.Viability and functions of tumor and immune cells could be maintained for over 2 weeks in perfused bioreactors. Next generation sequencing authenticated cultured tissue specimens as closely matching the original clinical samples. Anti-estrogen treatment of cultured estrogen receptor positive breast cancer tissue as well as administration of pertuzumab to a Her2 positive breast cancer both had an anti-proliferative effect. Treatment with anti-programmed-death-Ligand (PD-L)-1 and anti-cytotoxic T lymphocyte-associated protein (CTLA)-4 antibodies lead to immune activation, evidenced by increased lymphocyte proliferation, increased expression of IFNγ, and decreased expression of IL10, accompanied by a massive cancer cell death in ex vivotriple negative breast cancer specimens.In the era of personalized medicine, the ex vivoculture of breast cancer tissue represents a promising approach for the pre-clinical evaluation of conventional and immune-mediated treatments and provides a platform for testing of innovative treatments.

Colorectal cancer (CRC) infiltration by cells expressing myeloperoxidase (MPO) or CD8 positive T lymphocytes has been shown to be independently associated with favorable prognosis. We explored the relationship occurring between CD8+ and MPO+ cell CRC infiltration, its impact on clinical-pathological features and its prognostic significance in a tissue microarray (TMA) including 1,162 CRC. We observed that CRC showing high MPO+ cell infiltration are characterized by a prognosis as favorable as that of cancers with high CD8+ T cell infiltration. However, MPO+ and CD8+ CRC infiltrating cells did not synergize in determining a more favorable outcome, as compared with cancers showing MPOhigh/CD8lowor MPOlow/CD8highinfiltrates. Most importantly, we identified a subgroup of CRC with MPOlow/CD8lowtumor infiltration characterized by a particularly severe prognosis. Intriguingly, although MPO+ and CD8+ cells did not co-localize in CRC infiltrates, an increased expression of TIA-1 and granzyme-B was detectable in T cells infiltrating CRC with high MPO+ cell density.

Recent evidence suggests that natural killer (NK) cells are typically defective in infiltrating solid tumors, with the exception of gastrointestinal stromal tumors (GIST). Interestingly, however, infrequently infiltrating NK cells do not appear to have a direct effect on tumor progression. Here, prompted by the recent evidence that NK cell and T cell crosstalk may trigger, or enhance, tumor antigen-specific immune responses, we have tested the clinical significance of this reciprocal signaling. To this end, a tissue microarray constructed with 1410 colorectal carcinoma (CRC) patient specimens was stained with NK and T cell antigen-specific monoclonal antibodies, utilizing the immunoperoxidase staining technique. Cut-off scores for positive (>4 NK cells) and negative (≤4 NK cells) NK cell CRC patient samples were determined using receiver operating characteristic curve analysis. Using this approach, NK cells were detected in 423 (30%) of the 1410 CRC specimens evaluated. The number of NK cells was >4 in only 132 (9%) of CRC samples. Correlation of the immunohistochemical staining results together with analysis of the clinical course of the disease revealed that the infiltration of colorectal tumors with both NK cells and CD8+ T cells is associated with prolonged patient survival. In contrast, infiltration of tumors with NK cells in combination with CD3+and CD4+T lymphocytes had no detectable effect on the clinical course of the disease. These results suggest that NK cell and CD8+T cell crosstalk in the tumor microenvironment may benefit patient outcome and further, that the enumeration of infiltrating NK and CD8+T cells in CRC tumors may provide useful prognostic information.

The prognostic relevance of innate immune cells infiltrating colorectal carcinoma lesions is highly debated. By evaluating the expression of myeloperoxidase (MPO) as a marker of neutrophil granulocytes in a large cohort of colorectal carcinoma specimens, we have observed that robust tumor-infiltration by MPO+cells correlates with improved patient survival independently of other histopathological parameters, including disease stage.

In immune compromised subjects such as patients undergoing bone marrow, organ transplantation or immunosuppressive therapies, Cytomegalovirus (CMV) infection is associated with significant morbidity until the individual's immune system is completely reconstituted. One method of preventing CMV infection during immune suppression in transplant patients is represented by adoptive administration of CMV peptide-specific cytotoxic T lymphocytes (CTLs) from HLA-matched donors. Despite the strong immunogenicity demonstrated by specific HLA class I peptides, the peptide's responsiveness varies among individuals. Therefore, it is important to identify additional epitopes for each HLA determinant of interest. In this study we report a comprehensive analysis of CD8 T cell-specific activity against two novel CMV pp65 HLA-A*0201 associated peptides in CMV-experienced HLA-A*0201 subjects. PBMCs from CMV seropositive HLA-A*0201 restricted healthy subjects were peptide-stimulated ex vivo and IFN-γ gene expression was analyzed by qrt-PCR. An IFN-γ ELISPOT assay was carried out on peptide-specific elicited CD8 T lymphocytes to confirm the qrt-PCR results. Tetrameric HLA/epitope complexes (tHLA) were used to track the levels of peptide-specific CD8 T cells responsiveness to cognate epitopes. In addition, in vitro peptide-specific expanded populations of CTLs were used in 51Cr release assay. Based on qrt-PCR results, four out of eight HLA-A*0201 peptides identified by computer algorithms were selected. Two are preaviously published peptides: pp65495–503(NLVPMVATV) and pp65347–355(ALFFFDIDL), while two are novel: pp65340–348(RQYDPVAAL) and pp65310–318(LMNGQQIFL). In spite the four peptides induced comparable mRNA IFN-γ transcript production, peptides pp65495–503,pp65340–348and pp65310–318induced a consistent and sustained IFN-γ protein release and specific killing, while the pp65347–355failed to induce IFN-γ protein secretion and killing activity (if we exclude a positive IFN-γ protein release after 2-week in vitro induction in one of the donors tested). Comparative assays carried on the functional activity of the three peptides pp65495–503,pp65340–348and pp65310–318revealed no intrinsic differences in term of IFN-γ protein release and cytotoxic activity save for the CTL affinity to the HLA-A*0201/epitope complexes (pp65495–503~2.6% in nearly 100% of donors vs pp65340–348~0.67% or pp65310–318~0.77% in nearly one third of the donors after 2-week in vitro induction). The tHLA binding results could be possibly ascribed to differencies in the peptide avidity and stability for the HLA class I. Taken together, these results lead to the conclusion that different peptides can induce a variable levels of immune responses ranging between mere cytokine gene expression and effective cytotoxic activity. In addition, the two novel peptides selected here broaden the panel of potential reagents useful for adoptive immune therapy. Thus, in anticipation of a specific epitope-targeted immune intervention, the three HLA-A*0201 peptides described could be used in combination for adoptive transfer of epitope-specific T cells or epitope-specific vaccination.

In immune compromised subjects such as patients undergoing bone marrow, organ transplantation or immunosuppressive therapies, Cytomegalovirus (CMV) infection is associated with significant morbidity until the individual’s immune system is completely reconstituted. One method of preventing CMV infection during immune suppression in transplant patients is represented by adoptive administration of CMV peptide-specific cytotoxic T lymphocytes (CTLs) from HLA-matched donors. Despite the strong immunogenicity demonstrated by specific HLA class I peptides, the peptide’s responsiveness varies among individuals. Therefore, it is important to identify additional epitopes for each HLA determinant of interest. In this study we report a comprehensive analysis of CD8 T cell-specific activity against two novel CMV pp65 HLA-A*0201 associated peptides in CMV-experienced HLA-A*0201 subjects. PBMCs from CMV seropositive HLA-A*0201 restricted healthy subjects were peptide-stimulated ex vivo and IFN-γ gene expression was analyzed by qrt-PCR. An IFN-γ ELISPOT assay was carried out on peptide-specific elicited CD8 T lymphocytes to confirm the qrt-PCR results. Tetrameric HLA/epitope complexes (tHLA) were used to track the levels of peptide-specific CD8 T cells responsiveness to cognate epitopes. In addition, in vitro peptide-specific expanded populations of CTLs were used in 51Cr release assay. Based on qrt-PCR results, four out of eight HLA-A*0201 peptides identified by computer algorithms were selected. Two are preaviously published peptides: pp65495–503 (NLVPMVATV) and pp65347–355 (ALFFFDIDL), while two are novel: pp65340–348 (RQYDPVAAL) and pp65310–318 (LMNGQQIFL). In spite the four peptides induced comparable mRNA IFN-γ transcript production, peptides pp65495–503, pp65340–348 and pp65310–318 induced a consistent and sustained IFN-γ protein release and specific killing, while the pp65347–355 failed to induce IFN-γ protein secretion and killing activity (if we exclude a positive IFN-γ protein release after 2-week in vitro induction in one of the donors tested). Comparative assays carried on the functional activity of the three peptides pp65495–503, pp65340–348 and pp65310–318 revealed no intrinsic differences in term of IFN-γ protein release and cytotoxic activity save for the CTL affinity to the HLA-A*0201/epitope complexes (pp65495–503 ~2.6% in nearly 100% of donors vs pp65340–348 ~0.67% or pp65310–318 ~0.77% in nearly one third of the donors after 2-week in vitro induction). The tHLA binding results could be possibly ascribed to differencies in the peptide avidity and stability for the HLA class I. Taken together, these results lead to the conclusion that different peptides can induce a variable levels of immune responses ranging between mere cytokine gene expression and effective cytotoxic activity. In addition, the two novel peptides selected here broaden the panel of potential reagents useful for adoptive immune therapy. Thus, in anticipation of a specific epitope-targeted immune intervention, the three HLA-A*0201 peptides described could be used in combination for adoptive transfer of epitope-specific T cells or epitope-specific vaccination.