In Brief: Myeloid-derived suppressor cells in cancer.

The role of myeloid-derived suppressor cells (MDSCs) in cancer development has become clear over recent years, and MDSC targeting is an emerging opportunity for enhancing the effectiveness of current anticancer therapies. As MDSCs are not only able to limit anti-tumour T-cell responses, but also to promote tumour angiogenesis and invasion, their monitoring has prognostic and predictive value. Herein, we review the key features of MDSCs in cancer promotion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Clinical implication of tumor-associated and immunological parameters in melanoma patients treated with ipilimumab.

Ipilimumab, the first immune-checkpoint inhibitor extending overall survival (OS) in metastatic melanoma patients, has a survival benefit only in a proportion of patients and the development of reliable predictive biomarkers is still an unmet need. To meet this request, we used a multivariate statistical approach to test whether myeloid-derived suppressor cells (MDSC) or other tumor-associated and immunological parameters may serve as predictive or prognostic biomarkers in melanoma patients receiving ipilimumab. By using a standardized approach to determine the circulating levels of four MDSC subsets, we observed a significant expansion of three MDSC subsets at baseline, as compared to controls and, upon treatment, that high levels of CD14+/IL4Rα+ MDSCs were an independent prognostic factor of reduced OS. On the contrary, longer OS was associated to low levels of the proinflammatory proteins IL-6 and CRP and tumor-associated factors S100B and LDH both at baseline and after treatment. Increasing number of total T cells and especially of PD-1+/CD4+ T cells were associated with better prognosis, and upregulation of PD-1+ expression on CD4+ T cells upon treatment was associated with lower toxicity. As several parameters were associated to OS, we included these factors in a multivariate survival model, and we identified IL-6 and ECOG PS as independent biomarkers associated with improved OS, whereas high levels of LDH and CD14+/IL4Rα+ MDSCs were negative independent markers of reduced OS.

MAGE, BAGE and GAGE gene expression in human rhabdomyosarcomas.

MAGE, BAGE and GAGE genes encode tumor-associated antigens that are presented by HLA class I molecules and recognized by CD8(+) cytolytic T lymphocytes. These antigens are currently regarded as promising targets for active, specific tumor immunotherapy because MAGE, BAGE and GAGE genes are expressed in many human cancers of different histotype and are silent in normal tissues, with the exception of spermatogonia and placental cells. MAGE, BAGE and GAGE gene expression has been extensively studied in different tumors of adults but is largely unknown in many forms of pediatric solid cancer. Using RT-PCR, we analyzed MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, BAGE, GAGE-1,-2 or -8 and GAGE-3,-4,-5,-6 or -7b gene expression in 31 samples of pediatric rhabdomyosarcoma, the most frequent form of malignant soft tissue tumor in children. MAGE genes were expressed in a substantial proportion of patients (MAGE-1, 38%; MAGE-2, 51%; MAGE-3, 35%; MAGE-4, 22%; MAGE-6, 35%), while expression of BAGE (6%); GAGE-1, GAGE-2 and GAGE-8 (9%); and GAGE-3, GAGE-4, GAGE-5, GAGE-6 and GAGE-7B (16%) was less frequent. Overall, 58% of tumors expressed at least 1 gene, and 35% expressed 3 or more genes simultaneously. Our data suggest that a subset of rhabdomyosarcoma patients could be eligible for active, specific immunotherapy directed against MAGE, BAGE and GAGE antigens.

MAGE, BAGE, and GAGE gene expression in patients with esophageal squamous cell carcinoma and adenocarcinoma of the gastric cardia.

BACKGROUND: The MAGE, BAGE, and GAGE gene families code for distinct, tumor specific antigens that are recognized by cytotoxic T lymphocytes in the context of HLA molecules. The purpose of this study was to analyze MAGE, BAGE, and GAGE gene expression in the two major histologic types of esophageal carcinoma, squamous carcinoma (ESCc) and adenocarcinoma (CAc), and to correlate their expression patterns with the principal prognostic parameters and long term survival. METHODS: Gene expression was analyzed in surgical samples from 24 patients with ESCc and 24 patients with CAc by reverse transcriptase-polymerase chain reaction amplification (RT-PCR). None of the patients had received preoperative chemotherapy or radiotherapy, and all were followed until death or for a minimum of 4 years. RESULTS: Sixteen ESCc samples (67%) and 9 CAc samples (37.5%) expressed at least one of the genes under study. The expression of each MAGE gene in the two histologic types was not significantly different, with the exception of MAGE-4, which was expressed more in ESCc samples than in CAc samples. BAGE and GAGE expression was rather low and, in every case, was associated with the expression of at least one MAGE gene. CONCLUSIONS: In the group as a whole, and in both ESCc and CAc subgroups, no significant correlation emerged between the expression of any gene and prognostic parameters, such as pathologic tumor, lymph node, or disease stage. Nevertheless, BAGE or GAGE expression was related significantly to a poor prognosis, whereas the expression of MAGE genes (in the absence of BAGE and GAGE expression) was related significantly to a good prognosis.

A human CTL recognizes a caspase-8-derived peptide on autologous HLA-B*3503 molecules and two unrelated peptides on allogeneic HLA-B*3501 molecules.

A CTL clone that recognizes autologous tumor cells was previously isolated from the blood of a head-and-neck cancer patient. The Ag was identified as peptide FPSDSWCYF presented by autologous HLA-B*3503 molecules. This peptide was encoded by a mutated CASP-8 gene, which is implicated in the triggering of apoptosis. Here, we show that this CTL clone, which expresses a single TCR, also recognizes two unrelated peptides on allogeneic HLA-B*3501 molecules. One peptide, HIPDVITY, is encoded by squalene synthase, and the other one, QFADVIVLF, is encoded by 2-hydroxyphytanoyl-CoA lyase. Both genes are expressed ubiquitously. These antigenic peptides are processed and presented by HLA-B*3501 cells. The two HLA-B35 alleles are closely related. Our results might reinforce the notion that the recognition of allogeneic HLA molecules depends on the presence in their groove of a limited number of peptides processed from ubiquitous proteins.

Molecular cloning and identification of murine caspase-8.

Several caspases are mediators of apoptotic cell death. We describe a novel murine member of this growing protein family. Based on homology and especially on the substrate specificity, this new procaspase is identified as the murine counterpart of human procaspase-8. The protein exhibits a rather low similarity (76%) and identity (70%) to human procaspase-8. Procaspase-8 mRNA is expressed in all adult mouse tissues examined, the highest levels being reached in kidney, liver and lung. Procaspase-8 mRNA expression is highest in seven-day old embryos, but also during later stages of development the expression was fairly high. Both human and murine procaspase-8 are very weak substrates for granzyme B as compared to procaspase-3. Murine procaspases-1, 2, 3, 6, 7, 8, 11/4 and 12 are processed by recombinant murine caspase-8, suggesting a key role in the procaspase activation cascade. In addition, murine caspase-8 induced cell death that was inhibited both by cytokine response modifier A and p35. In vitro experiments demonstrated that p35 inhibits caspase-8 directly.

A CASP-8 mutation recognized by cytolytic T lymphocytes on a human head and neck carcinoma.

Of the antigens recognized on human tumors by autologous cytolytic T lymphocytes, all those defined thus far have been identified on melanoma or renal cell carcinoma. We report here the identification of an antigen recognized by autologous cytolytic T lymphocytes on a human squamous cell carcinoma of the oral cavity. The antigen is encoded by a mutated form of the CASP-8 gene. This gene, also named FLICE or MACH, codes for protease caspase-8, which is required for induction of apoptosis through the Fas receptor and tumor necrosis factor receptor-1. The mutation, which was found in the tumor cells but not in the normal cells of the patient, modifies the stop codon and adds an Alu repeat to the coding region, thereby lengthening the protein by 88 amino acids. The ability of the altered protein to trigger apoptosis appears to be reduced relative to the normal caspase-8. The antigenic peptide is a nonamer presented by HLA-B*3503. The five last amino acids are encoded by the extension of the reading frame caused by the mutation. This, together with previous observations of CDK4 and beta-catenin mutations, suggests that a significant fraction of the point mutations generating a tumor antigen also play a role in the tumoral transformation or progression.

CD45 regulates apoptosis induced by extracellular adenosine triphosphate and cytotoxic T lymphocytes.

Several lines of evidence implicate protein tyrosine phosphatases (PTP) in the regulation of apoptotic cell death. We have evaluated the role of CD45, the major PTP of hematopoietic cells, in apoptosis induced by extracellular ATP (ATPe) and cytotoxic T lymphocytes (CTL). We observed that two CD45- clones obtained by mutagenesis of the Fas- cell line L1210, exhibit a higher susceptibility to apoptosis induced by ATPe, which was also evident in Ca(2+)-free conditions, when compared to the parental cell line or CD45+ variants. The CD45- cells were also more susceptible to death mediated by an alloreactive CTL clone. When the cytotoxic assay was performed in the presence of EGTA, a Ca2+ chelator, which prevents cytotoxic granule exocytosis and perforin polymerization on target cell membranes, only the CD45- target cells were killed by the CTL clone. These results suggest that a cytotoxic pathway other than the secretory or Fas-dependent pathways was responsible for the enhanced susceptibility of CD45- cells to death, and therefore provide further evidence for the role of ATPe as a possible mediator of Ca(2+)-independent target cell destruction by CTL.

Anti-L-selectin monoclonal antibody treatment in mice enhances tumor growth by preventing CTL sensitization in peripheral lymph nodes draining the tumor area.

To examine the in vivo contribution of L-selectin in the sensitization of tumor-specific CTL, we investigated the effects of treatment with the anti-L-selectin monoclonal antibody (MAb) MEL-14 on the immune response to Moloney-murine sarcoma virus (M-MSV)-induced tumors, which exhibit spontaneous regression following generation of a strong virus-specific CTL response. Daily systemic administration of MEL-14 for 10 days to M-MSV-injected mice gave rise to larger sarcomas that persisted for a longer time, compared with those arising in control mice injected with virus only. The enhanced tumor growth could not be attributed to cytotoxic activity on leukocytes by MEL-14 since no reduction in the total cell number was detected in peripheral blood and spleen of MAb-treated mice. Evaluation of the immunological response in MAb-treated animals revealed a strong reduction in the generation of virus-specific CTL precursors (CTLp) in tumor-draining peripheral lymph nodes (PLN) 10 and 15 days after M-MSV injection, while in spleen, where lymphocyte localization is independent of L-selectin expression, CTLp generation was only delayed. By day 20, when tumors had begun to regress, the CTLp number showed a marked increase in both spleen and local PLN, where naive recirculating CTL could now enter because L-selectin was no longer down-regulated or blocked by the injected MAb. Our findings indicate that functional inactivation of L-selectin by MEL-14 treatment prevented migration of naive L-selectin+CTL through high endothelial venules (HEV) and their accumulation in PLN draining the tumor area, thereby precluding the initiation of a tumor-specific CTL response that takes place primarily at this site.

A peptide encoded by the human MAGE3 gene and presented by HLA-B44 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE3.

The human MAGE3 gene is expressed in a significant proportion of tumors of various histological types, but is silent in normal adult tissues other than testis and placenta. Antigens encoded by MAGE3 may therefore be useful targets for specific antitumor immunization. Two antigenic peptides encoded by the MAGE3 gene have been reported previously. One is presented to cytolytic T lymphocytes (CTL) by HLA-A1, the other by HLA-A2 molecules. Here we show that MAGE3 also codes for a peptide that is presented to CTL by HLA-B44. MAGE3 peptides containing the HLA-B44 peptide binding motif were synthesized. Peptide MEVDPIGHLY, which showed the strongest binding to HLA-B44, was used to stimulate blood T lymphocytes from normal HLA-B44 donors. CTL clones were obtained that recognized not only HLA-B44 cells sensitized with the peptide, but also HLA-B44 tumor cell lines expressing MAGE3. The proportion of metastatic melanomas expressing the MAGE3/HLA-B44 antigen should amount to approximately 17% in the Caucasian population, since 24% of individuals carry the HLA-B44 allele and 76% of these tumors express MAGE3.

Protein tyrosine kinases and phosphatases control apoptosis induced by extracellular adenosine 5'-triphosphate.

Extracellular ATP (ATPo) induces apoptosis and osmotic lysis in several cell lines. We investigated the role of protein tyrosine kinases (PTKs) and phosphatases (PTPases) in ATPo-induced apoptosis. The PTK inhibitor genistein prevented DNA fragmentation due to ATPo without affecting cell lysis. Comparison of western blot analysis and in vitro kinase assays of anti-phosphotyrosine immunoprecipitates indicated that ATPo activated PTKs whose activity was tightly regulated by PTPases. In fact, an early increase in tyrosine kinase activity was observed after ATPo-treatment and was prevented by specific PTPase inhibitors. In addition, a rapid dephosphorylation of phosphotyrosyl residues on several proteins was detected in ATPo-treated cells. Accordingly, inhibitors of PTPases, but not of serine/threonine phosphatases, were as effective as PTK-inhibitors in blocking ATPo-mediated DNA fragmentation. We describe the early events occurring in ATPo-induced apoptosis and suggest a role for PTPases in cell death.

Role of anti-LFA-1 and anti-ICAM-1 combined MAb treatment in the rejection of tumors induced by Moloney murine sarcoma virus (M-MSV).

We investigated the effect of combined treatment with anti-LFA-1 and anti-ICAM-1 monoclonal antibodies (MAbs) in the immune reaction to Moloney-murine-sarcoma-virus(M-MSV)-induced tumors, which spontaneously regress due to the generation of a strong virus-specific cytotoxic-T-lymphocyte(CTL) response. Repeated systemic administration of both MAbs to M-MSV-injected mice enhanced tumor growth and delayed regression, while treatment with a single MAb had a similar, though less pronounced, effect. The immune depression achieved could not be attributed to lymphocyte depletion, because no reduction in the total number of leukocytes was detected in the peripheral blood or spleen of these mice. However, anti-LFA-I MAb, alone or in combination with anti-ICAM-I MAb, prevented lymphocyte homing in tumor-draining lymph nodes. Cytofluorimetric analysis disclosed a profound down-modulation of LFA-I and ICAM-I molecule expression on T cells following in vivo MAb treatment. Moreover, in anti-LFA-I MAb-treated mice, the receptor was coated to saturation, while anti-ICAM-I MAb treatment brought about ICAM-I-molecule-coating levels below saturation. Evaluation of M-MSV-specific CTL precursor (p) frequency in lymphoid organs of mice receiving combined MAb treatment showed that CTL generation was greatly reduced 10 days after M-MSV injection, and returned to control levels by day 15. Our findings indicate that systemic administration of MAbs to LFA-I and ICAM-I molecules brings about a strong immune suppressive effect which is mainly due to a block in T-lymphocyte re-circulation, and activation by tumor cells. However, this immune-depressive effect is only temporary, and strictly dependent on continuous MAb administration. Thus, our data suggest that treatment with anti-LFA-I and anti-ICAM-I MAbs combined is unable to induce T-cell tolerance in a highly immunogenic system.

Inhibition of protein tyrosine phosphorylation prevents T-cell-mediated cytotoxicity.

Several lines of evidence point to a central role for protein tyrosine kinases (PTKs) in the signal transduction cascade initiated by T-cell receptor (TCR) engagement. In cytotoxic T lymphocytes (CTL), TCR crosslinking leads to activation of the lytic process which includes conjugate formation, lethal hit delivery, and events leading to target cell death. We studied the role of PTKs in antigen-specific cytotoxicity exerted by both in vivo activated and in vitro maintained CTL. We found that the PTK inhibitors herbimycin A and genistein blocked T-cell-mediated lysis in a dose-dependent manner. Lack of cytotoxic function was not due to abrogation of conjugate formation, but was associated with inhibition of both granule exocytosis and phosphatidylinositides turnover, thus indicating that PTK activity is an obligatory event for the activation of antigen-specific CTL effector function.

Adoptive transfer of lymphokine-activated killer cells loaded with 4'-deoxy-4'-iododoxorubicin: therapeutic effect in mice bearing lung metastases.

We studied the potential use of lymphokine-activated killer (LAK) cells loaded with 4'-deoxy-4'-iododoxorubicin (IDX) in adoptive immunotherapy experiments. Because LAK cells preferentially locate in the lung, we evaluated the therapeutic effect of IDX-loaded LAK cells in mice bearing lung metastases induced by B16F1 tumor cell injection. In vitro studies showed that LAK cells rapidly incorporated IDX, with maximum uptake at 15 min, followed by a plateau; drug efflux was initially rapid and then continued at a much slower rate. Evaluation of LAK cell cytotoxic activity against relevant target cells showed a 30% decrease after IDX treatment that progressed with time over the next 6 h. P388 tumor cell growth was inhibited by coculture with IDX-loaded LAK cells, thus demonstrating that the released IDX maintained its pharmacological activity. Finally, high performance liquid chromatography analysis of tissue IDX concentration revealed a considerably higher and long-lasting concentration in the lungs of mice receiving IDX-loaded LAK cells, compared to mice given injections of a comparable amount of free drug. Moreover, adoptive transfer of IDX-loaded LAK cells into tumor-bearing mice caused a significant reduction in the number of lung metastases versus control mice given injections of even higher doses of free drug.

Synergistic effect of extracellular adenosine 5'-triphosphate and tumor necrosis factor on DNA degradation.

Extracellular ATP (ATPo) was recently considered a possible mediator of cell-mediated cytotoxicity since it is secreted by effector cells following appropriate stimulus and causes lysis as well as DNA degradation of susceptible target cells. This hypothesis however is not readily reconciled with the finding that ATPo-resistant cells are fully susceptible to intact effector cells, which instead suggests that a necessary step in cell-mediated cytotoxicity is the interaction between different molecules released by a cytotoxic cell. By combining ATPo with TNF or lymphotoxin (LT), cytokines which induce late DNA damage, we observed a synergistic effect on target cell death. Under these conditions, the phenomenon of DNA degradation also appeared early, with a kinetics reminiscent of that observed during target cell incubation with intact effector cells. Target cells which are resistant to one of the two molecules exhibited an enhanced rate of cell death when exposed to their association. Target cell lysis and DNA degradation were also Ca(2+)-independent events as they took place following external Ca2+ chelation by EGTA addition and under experimental conditions in which little or no Ca2+ was released from target cell intracellular stores. These findings suggest that ATPo might represent a further mediator which is responsible for the alternative Ca(2+)-independent cytolytic pathway in association with other molecules released by effector cytotoxic lymphocytes.

Role of adhesion molecules in the immune reaction to M-MSV-induced tumors.

We have investigated the in vivo role of 2 different adhesion molecules, LFA-1 and LECAM-1, in the immune reaction to Moloney-murine-sarcoma-virus(M-MSV)-induced tumors, which undergo a peculiar spontaneous regression due to generation of a strong virus-specific cytotoxic-T-lymphocyte(CTL) response. Repeated administration of anti-LFA-1 monoclonal antibody (FD441.8 MAb), i.p. or at the site of virus inoculation, enhanced tumor growth and delayed regression, while i.p. administration of anti-LECAM-1 MEL-14 MAb gave rise to tumors that grew progressively and caused host death. Evaluation of the immunological response in MAb-treated mice showed reduced generation of virus-specific CTL precursors (p) in the spleen of animals given FD441.8 MAb i.p.; CTLp frequency in locally treated mice overlapped with that of control mice injected with virus only. FD441.8 MAb treatment did not interfere with CTL homing in the tumor, since the frequency of M-MSV-specific CTLps in sarcomas was similar in treated and control mice. Cytofluorimetric analysis indicated that the majority of tumor-infiltrating lymphocytes (TIL) from MAb-treated mice were covered by anti-LFA-1 MAb, and lacked cytotoxic activity when assayed against target cells bearing relevant tumor antigens. Instead, in mice injected i.p. with MEL-14 MAb, a very low frequency of CTLps was detected in lymph nodes draining the tumor area, and within the tumor. Our results indicate that enhanced tumor growth, depending on the MAb used, is the resultant of an inhibitory effect on different T-lymphocyte functions. Tumor progression in anti-LFA-1 MAb-injected mice is explained mostly by blockage of CTL lytic activity at the tumor site; in mice receiving i.p. MEL-14 MAb treatment, by the failure of naive T lymphocytes to enter peripheral lymph nodes and subsequently by the lack of generation of tumor-specific CTLs.

Antitumour efficacy of lymphokine-activated killer cells loaded with ricin against experimentally induced lung metastases.

Adoptive transfer of tumour-specific T lymphocytes loaded with ricin into tumour-bearing mice exerts a transient therapeutic effect against locally induced tumours [Cerundolo et al. (1987) Br J Cancer 55: 413]. As transferred cells preferentially locate in the lung, we studied the therapeutic effect of ricin-loaded, lymphokine-activated killer (LAK) cells on lung metastases induced by M4 or B16-F1 (F1) tumour cell injection. In vitro studies demonstrated that ricin-treated LAK cells were about 100-fold more efficient than untreated LAK cells in inhibiting growth of the ricin-sensitive M4 tumour cell line. This effect was most likely due to the released ricin, as treated and untreated LAK cells inhibited the relatively toxin-resistant F1 cell line to the same extent. Ricin treatment did not alter the tissue distribution of intravenously (i.v.) injected LAK cells, which selectively localized in the lung early after inoculation, whether or not metastases were present. Adoptive transfer experiments showed that ricin-treated LAK cells were significantly more efficient than untreated LAK cells in inhibiting M4- but not F1-induced lung metastases. These results indicate that LAK cells are able to deliver a therapeutic concentration of antineoplastic compounds directly to the lung.

The in vivo role of leukocyte function-associated antigen-1 in cytotoxic cell activity against tumors induced by Moloney-murine sarcoma/leukemia retroviral complex.

The in vivo role of LFA-1 molecule in the immune reaction to a murine retrovirus-induced tumor was investigated. Local and systemic treatment with high doses of anti-LFA-1 monoclonal antibody led to tumor progression by blocking CTL interaction with tumor cells without interfering with CTL generation and localization in the tumor mass.