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Immune checkpoint inhibitors (ICIs) show limited clinical activity in patients with advanced soft-tissue sarcomas (STSs). Retrospective analysis suggests that intratumoral tertiary lymphoid structures (TLSs) are associated with improved outcome in these patients. PEMBROSARC is a multicohort phase 2 study of pembrolizumab combined with low-dose cyclophosphamide in patients with advanced STS (NCT02406781). The primary endpoint was the 6-month non-progression rate (NPR). Secondary endpoints included objective response rate (ORR), progression-free survival (PFS), overall survival (OS) and safety. The 6-month NPR and ORRs for cohorts in this trial enrolling all comers were previously reported; here, we report the results of a cohort enrolling patients selected based on the presence of TLSs (n = 30). The 6-month NPR was 40% (95% confidence interval (CI), 22.7-59.4), so the primary endpoint was met. The ORR was 30% (95% CI, 14.7-49.4). In comparison, the 6-month NPR and ORR were 4.9% (95% CI, 0.6-16.5) and 2.4% (95% CI, 0.1-12.9), respectively, in the all-comer cohorts. The most frequent toxicities were grade 1 or 2 fatigue, nausea, dysthyroidism, diarrhea and anemia. Exploratory analyses revealed that the abundance of intratumoral plasma cells (PCs) was significantly associated with improved outcome. These results suggest that TLS presence in advanced STS is a potential predictive biomarker to improve patients' selection for pembrolizumab treatment.

Background Soft tissue sarcomas (STS) form a group of rare cancers which accounts for around 1% of tumours. Although up to 15% of patients respond in immunotherapy trials, there are no biomarkers predicting response of STS to checkpoint blockade therapies yet. Methods We analysed transcriptomic data of 4 publicly available cohorts, accounting for more than 600 STS. We used MCP-counter, a deconvolution method to estimate the tumour microenvironment (TME) composition Based on MCP-counter estimates, we established a robust immune classificationof STS tumors into 5 Sarcoma Immune Classes, labelled A, B, C, D and E. These classes exhibited different type and extents of TME. We validated the profiles of these 5 groups on a 72-patients cohort using immunohistochemichal (IHC) stainings for CD3, CD8, CD20 and CD34. Results One group (A: 23.3% of tumours) exhibits a very low to low immune infiltrate for all TME cell types. Another class (C: 14.5% of all tumours) displays moderate immune infiltrate and a strong presence of endothelial cells. Finally, another group (E: 15.6%) is highly infiltrated by all immune cell types. The two remaining groups (B: 27.4% and D: 19.4%) are heterogeneous, respectively rather highly and lowly infiltrated. The immune high group E is associated with an overexpression of several immune checkpoint genes: PDCD1 (PD-1), CD274 (PD-L1), PDCD1LG2 (PD-L2), LAG3, HAVCR2 (TIM-3), CTLA4. On 72 patients, we showed that the immune-high group could be identified by the IHC-visible presence of tertiary lymphoid structures (TLS), defined as T cell aggregates juxtaposing B cell aggregates. The immune-high group also exhibited prolonged overall survival as compared with other groups. Using data from a phase II clinical trial with pembrolizumab, we show that responders can be identified as class E tumours, therefore allowing patient selection. Conclusions We have defined a novel immune-based classification of STS into 5 classes, among which an immune-high group characterized by a strong infiltration by all immune cell and expression of immune checkpoints, presence of TLS and longer overall survival. This class groups responders to PD-1 blockade in a phase II clinical trial. Legal entity responsible for the study INSERM. Funding Institut National de la Sante et de la Recherche Medicale, the University of Paris, Sorbonne University, the Programme Cartes d’Identite des Tumeurs (CIT) from the Ligue Nationale Contre le Cancer, Institut National du Cancer (HTE-INSERM plan cancer, C16082DS), Association pour la Recherche sur le Cancer (ARC), Cancer Research for Personalized Medecine programme, “FONCER contre le cancer” programme, Labex Immuno-Oncology, the National Institute of Health, Moon Shot program at MD Anderson Cancer Center, Ministry of Education and Ministry of Science and Technology of Taiwan, National Taiwan University, Merck, Inc, SARC, Sarcoma Foundation of America, and the QuadW Foundation. Disclosure T.W. Chen: Advisory / Consultancy, Research grant / Funding (self): Eisai; Advisory / Consultancy: Lilly. M.A. Burgess: Advisory / Consultancy: EMD Serono; Advisory / Consultancy: Immune Design; Advisory / Consultancy: Eisai. J. Wargo: Advisory / Consultancy: Merck; Advisory / Consultancy: BMS; Advisory / Consultancy: Novartis; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Roche; Advisory / Consultancy: Genentech; Advisory / Consultancy: Illumina. H.A. Tawbi: Advisory / Consultancy, Research grant / Funding (self): BMS; Advisory / Consultancy, Research grant / Funding (self): Merck; Advisory / Consultancy, Research grant / Funding (self): Genentech; Research grant / Funding (self): Celgene; Research grant / Funding (self): GSK. W.H. Fridman: Advisory / Consultancy: MedImmune; Advisory / Consultancy: Novartis; Advisory / Consultancy: Servier; Advisory / Consultancy: Pierre Fabre. All other authors have declared no conflicts of interest.

The cell cycle events accompanying TGF-beta1-induced growth arrest of normal mouse resting B lymphocytes stimulated by LPS were investigated. We showed that TGF-beta1 prevents the retinoblastoma protein (pRb) phosphorylation and induces growth arrest in mid- to late G1. To explore the molecular basis of the effect of TGF-beta1, we analyzed the in vitro kinase activities of cyclin/cyclin-dependent kinase (cdk) complexes involved in the progression through G1 phase and in the G1/S transition, by using the glutathione S-transferase-pRb fusion protein as a substrate. Cdk2-associated kinase activity was strongly induced in mitogen-treated B cells. It was dramatically inhibited by TGF-beta1 as were the cyclin E- and cyclin A-dependent kinase activities. TGF-beta1 treatment had no significant effect on the expression of two G1/S phase proteins, cyclin E and cdk2. In contrast, the appearance of cyclin A, occuring in late G1 phase, was almost totally inhibited by TGF-beta1. We also showed that expression of the cdk inhibitor protein p27Kip1 decreased as cells progressed through the G1 phase. An accumulation of p27 was found in TGF-beta1-treated cells, showing that TGF-beta1 prevented LPS-induced decline of p27. Finally we found that the lack of kinase activity associated with cyclin E/cdk2 complexes was correlated with increased amounts of cdk2- and cyclin E-bound p27. Overall, these results suggest that both cyclin A and cdk2 may be active participants in the TGF-beta1-induced cell cycle arrest in normal mouse B cells and indicate the involvement of p27 in this mechanism.

A quantitative trait locus for increased IgG serum levels in the NOD mouse strain was mapped to distal chromosome 1, close to the fcgr2 locus encoding the low-affinity type II receptor for the Fc portion of IgG (Fc gamma RII). Expression of membrane-inserted (b2) and soluble (b3) isoforms of Fc gamma RII was strongly decreased in macrophages of NOD compared with C57BL/6 (B6) mice. In contrast, B cell-specific (Fc gamma RIIb1) isoform was only slightly decreased and Fc gamma RIII was not altered. This Fc gamma RII regulatory defect was cis-encoded by fcgr2 or by a closely linked locus, occurred at the mRNA level, and was associated with multiple mutations in the fcgr2 gene promoter. In relation with this defect, binding of IgG1- and IgG2b- but not IgG2a-opsonized RBC by macrophages of NOD and congenic B6.NOD-fcgr2 mice was severely impaired, but was normal in macrophages of NOD.B6-fcgr2 congenic mice, indicating that Fc gamma RII plays a nondispensable role in binding of IgG1 and IgG2b isotypes. Likewise, serum levels of IgG1 and IgG2b but not IgG2a were up-regulated in NOD compared with NOD.B6-fcgr2 congenic mice. These findings indicate that macrophage Fc gamma RII may regulate serum IgG1 and IgG2b through their catabolism, and validate the NOD strain as a model to investigate the functions of Fc gamma RII isoforms.

The type III-B Fcgamma receptor (FcgammaRIII-B) is a glycosyl-phosphatidylinositol-linked receptor found on human neutrophils. A soluble form of FcgammaRIII-B (sCD16) corresponding to the extracellular region of the receptor circulates in plasma. In the present work, we have identified membrane receptors for sCD16. Soluble CD16 bound to CR3 (CDllb/CD18)- and CR4 (CDllc/CD18)- positive leukocytes and cell lines, the labeling was inhibited by anti-CD11b, CD11c or CD18 mAbs, and the up-regulation of CR3 and CR4 led to an increased fixation of sCD16. Transfected eukaryotic cells expressing recombinant CD11b/CD18 or CD11c/CD18 heterodimers but not those expressing CD11a/CD18 bound sCD16. Moreover, the lectin-like binding site of CR3 is probably involved in the interaction with sCD16, as suggested by inhibition studies using mAbs against CR3 or sugars such as N-acetyl D-glucosamine, alpha- or beta-methyl D-glucoside, alpha- or beta-methyl D-mannoside, or zymosan. Thus, the complement receptors CR3 and CR4 are membrane receptors for sCD16. Through this interaction, sCD16 induces a CR3-dependent production of IL-6 and IL-8 by monocytes. These results suggest that sCD16 plays a regulatory role in inflammatory processes and provide a molecular basis for the interaction between FcgammaRIII-B and CR3 described on the cell membrane.

F. Pagès, A. Berger, F. Zinzindohoué, A. Kirilovsky, J. Galon, W.-H. Fridman Lymph node dissection is an integral part of the surgical resection of colon cancers; it completes the wide regional resection of tumor and it allows prognostic evaluation through accurate staging. Studies have demonstrated an immune reaction to the tumoral site which attests to an ongoing dialog between the tumor and systemic defenses. The regional lymph nodes constitute an important first line of immune defense where initial host response is initiated or, inversely, they may participate in a local state of immunosuppression. This article reviews current knowledge on intra-tumoral and nodal immune status in colorectal cancers and attempts to evaluate the potential immunologic implications of lymph node dissection.

We have isolated and deduced the structure of the beta Fc gamma RII gene from the mouse. This gene spans approximately 18 kb of DNA; the structure and nucleotide sequence has been determined and potential regulatory sites identified. This gene is composed of 10 exons that give rise to the beta 1 and beta 2 Fc gamma RII cDNA. Four exons encode the 5' untranslated region and leader sequence, one exon for each Ig-binding domain and membrane-spanning region and three exons encoding the cytoplasmic tail and 3' untranslated region. Analysis of the gene structure indicated that the beta 1Fc gamma RII and beta 2Fc gamma RII arise by differential mRNA splicing when a single 141-bp exon (exon 8) is alternately spliced to give rise to these isoforms. Sequence analysis of 2 kbp upstream of the transcription start site indicated the presence of regulatory elements, including three binding sites for the transcription factor Sp1, and Ap-4 binding site, a tandem glucocorticoid response element, and an overlapping tandem repeat. Furthermore, as in the Thy-1 gene, no CAT or TATA consensus sequences were observed. In addition, sites of methylation that regulate expression of this gene were also located at the 5' end of the gene and within several introns. A large intron located between exons 4 and 5 also contained a series of purine/pyrimidine-rich regions and a potential enhancer sequence.

Fc gamma R expressed by mouse mast cells were characterized as functional binding sites, as membrane proteins, and as products of the two genes known to encode murine Fc gamma RII. Peritoneal mast cells, bone marrow-derived mast cells (BMMC), and the mastocytoma cells P815 were found to bear trypsin-resistant, 2.4G2+, low-affinity receptors binding mouse monoclonal IgG1, IgG2a, and IgG2b, i.e., Fc gamma RII. BMMC and P815 Fc gamma RII appeared as heterogeneous membrane proteins that, when deglycosylated, had m.w. corresponding to those of the three known products of the alpha and beta Fc gamma R genes, and differed by their respective contents in BMMC and P815 cells. Heterogeneous Fc gamma R transcripts were also found in BMMC and in P815 RNA. P815 cells contained alpha, beta 1, and beta 2 Fc gamma R transcripts, whereas BMMC contained alpha and beta 1 Fc gamma R transcripts. These data disclose an unexpected molecular heterogeneity of murine mast cell Fc gamma R. Although they appear as a single population of receptors when viewed by external ligands, mast cell Fc gamma R comprise three Fc gamma RII subtypes, encoded by the three known transcripts of the alpha and beta Fc gamma R genes, and differing by their intracytoplasmic portion. The different distributions of Fc gamma RII transcripts and corresponding Fc gamma RII subtypes in different types of mast cells may be determinant for triggering the various biologic activities of these cells.

Lymphocytes express voltage-activated K+ channels in their membrane. Combining the patch-clamp techniques of recording with immunological methods, we have analyzed the expression and the involvement of these channels during defined steps of LPS-induced B cell activation. We show that the number of K+ channels increased strongly when B cells entered in the G1 phase of the cell cycle. The involvement of ion channels in B cell proliferation was assessed using channel blockers that inhibit the K+ current. It was first found that TEA, but not TMA, quinine and verapamil totally suppressed both K+ current and DNA synthesis by stimulated lymphocytes as measured by [3H]TdR uptake or propiedium iodide staining. The drugs affected neither the induction by LPS of activation markers such as Ag of the murine class II MHC and type II receptor for the Fc region of IgG nor the initial cell enlargement that occur early during activation. These data indicate that functional K+ channels are not essential for the transition from the G0 to the G1 phases. In contrast, the same channel antagonists blocked the induction of transferrin receptor expression, characteristic of the final stages of G1. These drugs acted on cells already in G1, because their addition 30 h after LPS still suppressed DNA synthesis, and because they inhibited the proliferation of purified B cell blasts. The effect of tetraethylammonium was reversible, a lag period of 12 h occurring before the cells start DNA synthesis after drug removal. Taken together, these data demonstrate that the proliferation of LPS-stimulated B cells requires functional ion channels at a critical period in the G1 phase, taking place before transferrin receptor expression and the entry into the S phase. The involvement of voltage dependent K+ channels at this particular point is suggested by the parallel effects of the drugs used on K+ currents and DNA synthesis.

Three subtypes of murine low-affinity receptors for IgG (Fc gamma RII) have been identified. One is encoded by the alpha Fc gamma R gene, two are encoded by the beta Fc gamma R gene. In the present work, we examined whether DNA methylation might control expression of the alpha Fc gamma R gene. We found that, in DNA from a panel of Fc gamma R(+) and (-) cell lines, two MspI sites of the alpha Fc gamma R gene were selectively unmethylated only in the two cell lines containing alpha transcripts. These sites, separated by a distance of 1.2 kb, are located in the 5' region of the gene. All other MspI sites were methylated in all cell lines. Furthermore, 5-azacytidine induced the demethylation and the expression of the alpha Fc gamma R gene in the Fc gamma R(-) thymoma BW5147. Both alpha Fc gamma R gene transcripts and corresponding protein products became detectable in 5-azacytidine-treated cells. The alpha Fc gamma R gene was also demethylated and expressed in mouse spleen cells cultured with human rIL-2. We conclude that a correlation links the unmethylation and the expression of the alpha Fc gamma R gene in murine cell lines as well as in nontransformed lymphoid cells responding to a physiological stimulus.

Complement protein deficiency of the classical pathway or in proteins of the alternate pathway is rare but considerably increase the risk of infection with Neisseria meningitidis. The aim of this study was to determine the clinical criteria of the group at risk.Retrospective study of the clinical and biological data of patients exhibiting complement protein deficiency associated with one or several N. meningitidis infections.Forty cases were studied, including 35 classical pathway protein deficiencies, with a predominance of C7 deficiency, 3 properdin deficiencies and 2 acquired C3 deficiencies. More than 60% of the patients exhibited recurrent N. meningitidis infections. Serogroups of rare strains were isolated in 50% of cases. Properdin deficiency was associated with a fulminating form in 2 cases out of 3. The age at onset of the first manifestations varied from 2 months to 32 years.A deficiency must be systematically searched for in all patients presenting with a N. meningitidis infection before the age of 6 months or after the age of 5 years. Identification of deficient patients permits the proposal of family screening and appropriate prophylaxis, including preventive vaccination.

Summary We conducted a phase I/II clinical trial of the safety and efficacy of intravesical administration of autologous IFN-γ-activated macrophages (MAK) in patients with superficial bladder cancer. Monocyte-derived MAK cells were prepared in vitro and patients received six instillations of 1·4 × 108 to 2·5 × 108 cells, once a week, for five consecutive weeks. Treatment was well tolerated, with seven grade 1 and five Grade 2 protocol-related adverse effects. Nine out of 17 included patients had no recurrences during the year following the first instillation of MAK. The aim of the present study was to search for immune parameters related to local immunostimulation induced by MAK. Monitoring of the patients showed that urinary IL-8, GM-CSF and, to a lesser extent, IL-18 were increased following MAK instillations, with inter-individual differences. The urinary IL-8 level was about 10-fold higher than that observed for other cytokines, and its biological activity was reflected by a concomitant increase of urinary elastase, indicating neutrophil activation and degranulation. We also showed that nine out of 12 patients investigated presented an increase of urinary neopterin, a marker of IFN-γ-activated macrophages, 7 days after MAK instillation, while serum neopterin levels were almost stable. These results are in line with persistence of activated macrophages in the bladder wall after infusions. Moreover, there was evidence of macrophages in urine smears 2 months after the sixth MAK instillation, and the score of macrophages correlated with the quantity of neutrophils in the urine. Overall, this study provides evidence of a local immunostimulation induced by this novel and safe immunotherapeutic approach of MAK instillations in patients with superficial bladder cancer.

Clinical grade ex vivo-generated antigen-presenting cells, macrophage-dendritic cells (MAC-DCs) or macrophage-activated killers (MAKs) were derived from peripheral blood mononuclear cells (PBMCs). Cultures (7 d) were performed in non-adherent conditions in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and either interleukin 13 (IL-13) or dihydroxy-vitamin D3 respectively. MAKs were activated during the last 24 h with interferon gamma (IFNgamma). Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses indicated that IL-1beta and tumour necrosis factor alpha (TNFalpha) were produced by both cells. Higher pro-inflammatory cytokine (IL-1beta and TNFalpha) amounts were detected on average in MAK supernatants. In contrast, IL-12 p40 was found only in MAC-DC supernatants, but the biologically active IL-12 form (p70) was never detected. T-cell cytokines (IL-2, IL-4, IL-10) were not produced in culture conditions in which T cells were nevertheless present. At d 7, TNFalpha or lipopolysaccharide (LPS) upregulated IL-12 p40 production by MAC-DCs, while IL-12 p70 remained undetectable. LPS stimulation also increased TNFalpha production by these cells. Allogeneic mixed lymphocyte reactions (MLR) showed that MAKs are poor stimulatory cells compared with MAC-DCs. The MAC-DC stimulatory capacity was enhanced by LPS, although the expression of HLA class II, CD83, CD80 and CD86 was unmodified. Thus, MAC-DCs represent a tool for triggering adaptative immunity, while MAK should be primarily used as effector killer cells.

A local increase of interleukin-18 (IL-18) expression has been recently demonstrated in Crohn's disease (CD), suggesting a role for mature IL-18 (cleaved by ICE protease) in the induction of proinflammatory cytokines and Th1 polarization observed in CD lesions. The aim of this study was to investigate IL-18 modulation and its potential immune consequences in CD lesions. We showed increased IL-18 production in chronic CD lesions and identified epithelial cells and macrophages as IL-18-producing cells. A twofold increase in ICE alpha, beta, and/or gamma mRNA that encodes for the complete mature peptide was required for ICE activity, and a marked increase in IL-18R-positive immune cells was observed in chronic lesions compared to uninvolved areas or normal control samples. Chronic lesions also displayed intense transcription of IL-18-induced cytokines, IFN-gamma, IL-1beta, TNF-alpha, and IL-8. By contrast, when neither IL-18 nor ICE mRNAs were enhanced (early asymptomatic CD lesions), IL-18-induced cytokines were not up-regulated. These results are in accordance with a putative role of mature IL-18 in the pathogenesis of CD.

IL-18, originally identified as interferon-gamma inducing factor (IGIF), is related to the IL-1 family in terms of its structure, processing, receptor, signal transduction pathway and pro-inflammatory properties. IL-18 is also functionally related to IL-12, as it induces the production of Th1 cytokines and participates in cell-mediated immune cytotoxicity. This review summarizes the recent advances in the understanding of IL-18 structure, processing, receptor expression and immunoregulatory functions, and focuses on the role of IL-18 modulation in tumours, infections, and autoimmune and inflammatory diseases.

Interleukin (IL) 17 is a proinflammatory cytokine secreted mainly by activated human memory CD4 T cells that induces IL-6, IL-8, and nitric oxide. Because IL-6 and IL-8 have been implicated in the pathogenesis of cervical cancer, we investigated the action of IL-17 on human cervical tumor cell lines in vitro and in vivo. We showed that in vitro, IL-17 increases IL-6 and IL-8 secretion by cervical carcinoma cell lines at both protein and mRNA levels. No direct effect of IL-17 on in vitro proliferation of cervical tumor cell lines could be demonstrated. However, two cervical cell lines transfected with a cDNA encoding IL-17 exhibited a significant increase in tumor size as compared to the parent tumor when transplanted in nude mice. This enhanced tumor growth elicited by IL-17 was associated with increased expression of IL-6 and macrophage recruitment at the tumor site. A potential role of IL-17 in modulation of the human cervical tumor phenotype was also supported by its expression on the cervical tumor in patients with CD4 infiltration. IL-17 therefore behaves like a T-cell-specific cytokine with paradoxical tumor-promoting activity. This may partially explain previous reports concerning the deleterious effect of CD4 T cells in cancer.

Phagocytic cells with macrophage or dendritic cell phenotype, able to capture and ingest tumor cells, were derived in large numbers from peripheral blood mononuclear cells using two different activation procedures. Peripheral blood mononuclear cells were stimulated in nonadherent conditions in the presence of human AB serum with either granulocyte-macrophage colony-stimulating factor and dihydroxy-vitamin D3 for 7 days and with interferon-gamma for the last 18 hours to obtain activated macrophages (MAK) or with granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days (with fresh interleukin-13 added on day 4) to obtain macrophage-dendritic cells (MAC-DC). A strong ability of MAC-DC to phagocytose yeasts was observed, in contrast to a low-intermediate phagocytosis capacity by MAK. Both CD14+ FCgammaR+ (FcgammaRI/CD64, FcgammaRII/CD32, FcgammaRIII/CD16) MAK and CD1a+/CD86+, CD14- MAC-DC were able to phagocytose whole tumor cells. However, only MAK phagocytosis was enhanced by FcgammaR engagement. MAK but not MAC-DC could lyse tumor cell in antibody-dependent cell cytotoxicity assays, via FcgammaRI. Thus, MAK as well as MAC-DC may represent valuable tools for different in vivo therapy strategies that do or do not include the use of monoclonal antibodies.

Fc gamma RIIB are single-chain low-affinity receptors for IgG that bear an immunoreceptor tyrosine-based inhibition motif in their intracytoplasmic domain and that negatively regulate immunoreceptor tyrosine-based activation motif-dependent cell activation. They are widely expressed by cells of hematopoietic origin. We investigated here whether Fc gamma RIIB could also negatively regulate protein tyrosine kinase receptor (RTK)-dependent cell proliferation. As an experimental model, we used growth factor-dependent mast cells that constitutively express Fc gamma RIIB and c-kit, an RTK prototype. We found that anti-c-kit Abs mimicked the effect of stem cell factor and induced thymidine incorporation in Fc gamma RIIB-/-, but not in wild-type (wt) mast cells unless Fc gamma RIIB were blocked or anti-c-kit F(ab')2 were used. When coaggregated with c-kit by intact Abs in wt mast cells, Fc gamma RIIB inhibited thymidine incorporation, as well as cell proliferation, and inhibition was correlated with an arrest of cells in G1 during the cell cycle. The coaggregation of c-kit with Fc gamma RIIB did not affect ligand-induced c-kit phosphorylation and induced the tyrosyl-phosphorylation of Fc gamma RIIB, which selectively recruited the Src homology 2 domain-bearing inositol 5-phosphatase SHIP. Our results indicate that IgG Abs to growth factors or growth factor receptors may control RTK-dependent proliferation of a variety of cells that express Fc gamma RIIB.

On the basis of compelling preclinical data in cats and dogs we initiated a clinical gene therapy study in nine patients with advanced solid tumors using xenogeneic fibroblasts secreting human IL-2 (Vero-IL-2 cells). Cohorts of three successive patients with tumors accessible to CT- or ultrasound-guided injection were treated repeatedly with 5 x 10(5), 5 x 10(6), or 5 x 10(7) Vero-IL-2 cells. Endpoints of the study were feasibility, toxicity, and clinical and biological effects of this novel approach to immunotherapy of cancer. Histopathological, immunological and molecular analyses were performed on biopsy specimens of tumors and blood samples from before, during and after treatment. Low levels of serum antibodies to Vero cells developed in 2/9 patients. Analysis of tumor biopsies showed increased expression of CD3 mRNA and enhanced tumor infiltration with varying lymphocyte subpopulations after treatment. In addition, monoclonal alterations of the TCR repertoire of blood and tumor lymphocytes were observed. Treatment was well tolerated and toxicity consisted of transient fever in one patient and short-lived, mild itching and erythema in two others. One patient with soft tissue sarcoma showed a more than 90% and more than 50% reduction of the volume of two distant, non-injected metastases, respectively, lasting for 22+ months. Four other patients showed stabilization of their disease for three to nine months, among whom was a patient with melanoma who developed marked vitiligo. We conclude that repeated injection of up to 5 x 10(7) Vero-IL-2 cells was safe and showed biological and clinical activity in heavily pretreated patients with advanced solid tumors. Further evaluation of intratumoral application of Vero-IL-2 seems warranted.

Fas and Fas ligand (FasL) are implicated in programmed cell death or apoptosis. In the immunological field, they are particularly important in auto-immunity, graft rejection and anti-tumoral response. Fas ligand expression on thymocytes, activated T lymphocytes, and in sites of immune privilege, suggests the importance of Fas/FasL interactions in negative control of the immune response. The recent description of FasL expression by tumoral cells, represents a new mechanism of immune escape for different cancer, and has been well studied in colon adenocarcinoma.

Fc gamma RIIB are low-affinity receptors for IgG whose intracytoplasmic domain contains an immunoreceptor tyrosine-based inhibition motif (ITIM). Fc gamma RIIB inhibit cell activation triggered by receptors that signal via immunoreceptor tyrosine-based activation motifs. This inhibition requires ITIM tyrosyl phosphorylation and is correlated with the binding of SH2 domain-containing phosphatases that may mediate the inhibitory signal. In the present work, we investigated the mechanism of Fc gamma RIIB phosphorylation and its consequences in mast cells. We demonstrate that the phosphorylation of Fc gamma RIIB requires coaggregation with Fc epsilon RI and that, once phosphorylated, Fc gamma RIIB selectively recruit the inositol polyphosphate 5 phosphatase SHIP, in vivo. In vitro, however, the phosphorylated Fc gamma RIIB ITIM binds not only SHIP, but also the two protein tyrosine phosphatases, SHP-1 and SHP-2. We show that the coaggregation of Fc gamma RIIB with Fc epsilon RI does not prevent Fc epsilon RI-mediated activation of lyn and syk. Both kinases can phosphorylate Fc gamma RIIB in vitro. However, when coaggregated with Fc epsilon RI, Fc gamma RIIB was in vivo phosphorylated in syk-deficient mast cells, but not in lyn-deficient mast cells. When Fc epsilon RI are coaggregated with Fc gamma RIIB by immune complexes, Fc epsilon RI-associated lyn may thus phosphorylate Fc gamma RIIB. By this mechanism, Fc epsilon RI initiate ITIM-dependent inhibition of intracellular propagation of their own signals.

Since the elucidation of the structure of antibodies, it has been recognized that immunoglobulins (Ig) are bifunctional molecules which assemble the products of several genes, allowing the almost infinite diversity of antigen recognition and a large array of effector and regulatory functions. Families of V genes encode the variable regions of Ig heavy (H) and light (L) chains whereas a set of C genes encodes the H and L constant regions which define Ig isotypes. Associations of the variable regions form the antigen-binding site while the C terminal constant regions form the Fc part of the Ig molecule and bear the sites for functional activities. The use of proteolytic enzymes has allowed separation of the Fc region from the rest of the molecule. An Fc-less Ig is a pure antigen-binding unit (and has therefore be called F(ab) when monovalent and F(ab)′ 2 when divalent) whereas intact molecules, after binding to antigen, exert multiple effector and regulatory functions [1,2].

Soluble FcγR (sFcγR) were discovered in supernatants of cells of the immune system during the 1970s, and called immunoglobulin G-binding factors. Twenty years later, although the structure and mechanisms of generation of sFcγR have been largely clarified [see References 1–5 for reviews], questions remain regarding their biological role. In this chapter, we will review available evidence showing that these molecules can regulate immune reactions in vitro. The question of their in vivo role will be addressed on the basis of studies performed in various pathological situations and with transgenic animals.

gp130 acts as a common transducing signal chain for all receptors belonging to the interleukin (IL)-6 receptor family. The IL-6-related cytokines [IL-6, IL-11, oncostatin M (OSM), leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotrophin] often modulate tumor phenotype and control the proliferation of many tumor cell lines. We demonstrate that melanoma cell lines release, in vitro and in vivo (when transplanted in nude mice), soluble gp130 (sgp130), a potential antagonist of cytokines from the IL-6 family. Biochemical analysis revealed that sgp130 derived from melanoma patients' sera or from culture supernatants of melanoma cell lines is a Mr 104,000 protein that resolved after deglycosylation as a Mr 58,000 protein. PCR and Northern blot analysis only identified one gp130 membrane mRNA, suggesting that the soluble form of gp130 is generated by proteolytic cleavage. OSM reproducibly increases sgp130 released by melanoma cell lines, whereas leukemia inhibitory factor stimulates the production of sgp130 in only one of three cell lines tested. This tumor-derived sgp130 is functional because it binds in solution to the IL-6-soluble IL-6 receptor (gp80) complex. Recombinant sgp130 inhibits the growth inhibitory activity of the IL-6-soluble IL-6 receptor complex and OSM on some melanoma cell lines. Therefore, this soluble gp130 represents a natural antagonist of cytokines from the IL-6 family.

Fc gamma RIII (CD16), a low affinity FcR which binds IgG-containing immune-complexes, exists under membrane-associated forms and under a soluble form (sFc gamma RIII). The latter, present in biological fluids (serum, saliva), is generated by proteolytic cleavage of the two membrane-associated Fc gamma RIII isoforms, Fc gamma RIII-A (expressed by macrophages and NK cells) and Fc gamma RIII-B (expressed exclusively by neutrophils). Herein we demonstrate that dendritic cells (DCs), generated by culturing monocytes with GM-CSF and IL-4, bind biotinylated recombinant sFc gamma RIII. This binding is specific and involves the complement receptor CR3 (CD11b/CD18) and CR4 (CD11c/CD18). Indeed, preincubation of DCs with anti-CD11b and anti-CD11c mAbs decreased by 52% and 62% respectively the binding with sFc gamma RIII. Moreover, electron microscopy showed that binding of gold-labeled sFc gamma RIII to DCs maintained at 4 degrees C occurred within clathrin-coated pits. Once internalized, at 37 degrees C, sFc gamma RIII entered the endocytic pathway and reached the MHC class II compartments. Furthermore, DCs incubated for 48 h with multivalent sFc gamma RIII expressed increased levels of CD40, CD80, CD86, CD54, CD58, HLA class I and class II molecules and decreased levels of CD23 and CD32. These effects result in an increased capacity of DCs to trigger proliferative responses by CD4+ CD45RA+ allogeneic T cells. RT-PCR amplification demonstrated that incubation of DCs for 20 h in the presence of multivalent sFc gamma RIII induced the appearance of GM-CSF and IL-12 p40 mRNA. Among the cytokines constitutively expressed, IL-1 beta and IL-8 were strongly up-regulated whereas IL-6 and IL-12 p35 mRNA were increased to a lesser extent and the expression of MIP-1 alpha mRNA remained constant. Finally, ELISA tests demonstrated that DCs incubated with multivalent sFc gamma RIII secreted the cytokines IL-1 beta, IL-6, IL-8, GM-CSF and IL-12 p75. Thus, while becoming internalized sFc gamma RIII could affect the capacity of DCs to present antigens and, via the induction of accessory molecules and the release of the IL-12 p75 protein, could initiate Th1 type immune response.

FcγRIII (CD16), a low affinity FcR which binds IgG-containing immune-complexes, exists under membrane-associated forms and under a soluble form (sFcγRIII). The latter, present in biological fluids (serum, saliva), is generated by proteolytic cleavage of the two membrane-associated FcγRIII isoforms, FcγRIII-A (expressed by macrophages and NK cells) and FcγRIII-B (expressed exclusively by neutrophils). Herein we demonstrate that dendritic cells (DCs), generated by culturing monocytes with GM-CSF and IL-4, bind biotinylated recombinant sFcγRIII. This binding is specific and involves the complement receptor CR3 (CD11b/CD18) and CR4 (CD11c/CD18). Indeed, preincubation of DCs with anti-CD11b and anti-CD11c mAbs decreased by 52% and 62% respectively the binding with sFcγRIII. Moreover, electron microscopy showed that binding of gold-labeled sFcγRIII to DCs maintained at 4°C occured within clathrin-coated pits. Once internalized, at 37°C, sFcγRIII entered the endocytic pathway and reached the MHC class II compartments. Furthemore, DCs incubated for 48 h with multivalent sFcγRIII expressed increased levels of CD40, CD80, CD86, CD54, CD58, HLA class I and class II molecules and decreased levels of CD23 and CD32. These effects result in an increased capacity of DCs to trigger proliferative responses by CD4+ CD45RA+ allogeneic T cells. RT-PCR amplification demonstrated that incubation of DCs for 20 h in the presence of multivalent sFcγRIII induced the appearance of GM-CSF and IL-12 p40 mRNA. Among the cytokines constitutively expressed, IL-lβ and IL-8 were strongly up-regulated whereas IL-6 and IL-12 p35 mRNA were increased to a lesser extent and the expression of MIP-lα mRNA remained constant. Finally, ELISA tests demonstrated that DCs incubated with multivalent sFcγRIII secreted the cytokines IL-1β, IL-6, IL-8, GM-CSF and IL-12 p75. Thus, while becoming internalized sFcγRIII could affect the capacity of DCs to present antigens and, via the induction of accessory molecules and the release of the IL-12 p75 protein, could initiate Thl type immune response.

The recent discovery of the molecular structure of tumor antigens expressed by melanoma cells has contributed to the development of therapeutic strategies aimed at stimulating anti-tumor immunity. The first clinical trials in patients with cancer involved systemic administration of interleukin 2 alone or in combination with alpha-interferon or other chemotherapy agents. Objective clinical responses were observed in 15 to 25% of the patients with melanomas or renal cell cancer. In order to improve these encouraging early results, different clinical protocols are being evaluated with the goal of increasing intra-tumoral concentrations of the administered cytokines. Better optimization of methods for immunizing against specific tumor antigens naturally leads to the transition from non-specific stimulation of the human immune system to tumor antigen specific immunotherapy.

The murine receptor for the Fc portion of IgG is a molecule expressed by cells of the immune system. This study suggests the hypothesis that Fc gamma receptor type II B I (Fc gamma RIIB I) functions as a progression-enhancing factor when expressed ectopically on non-lymphoid tumor cells. It has been shown previously that BALB/c 3T3 cells transformed in vitro with polyoma virus (PyV) do not express Fc gamma RII but acquire the expression of this receptor following an in vivo passage in syngeneic mice. The specific Fc gamma RII transcript present in tumor cells was identified in this report as Fc gamma RIIB I (BI). In order to determine whether or not the ectopically expressed Fc gamma RII plays a role in the progression of these transformed cells, PyV-transformed 3T3 cells were transfected with BI-cDNA. The BI transfected cells were tested for their ability to form local tumors in syngeneic mice, as compared to transfected cells which express the co-transfecting neomycine resistance (neores) DNA alone or together with the lacZ gene. Fc gamma RIIB I expressors exhibited a significantly higher tumorigenic phenotype than FcR-negative controls, though both types of cells exhibited the same growth curve in vitro. The ability of Fc gamma RIIB I to act as a potentially tumorgenicity-enhancing factor was also demonstrated as Fc gamma RII was expressed by tumor cells, originating from inoculated Fc gamma RIIB I-transfected cells, or from inoculation of a mixture of receptor-positive and -negative cells. B I-expressing cells dominated the tumor-cell population over non-expressors. This dominance strengthened the hypothesis that FcR plays a role in tumor progression in vivo.

Thyroid dysfunction and abnormalities in corticosteroid regulation were observed after clinical use of cytokines (IL2, IFN alpha), molecules with immunostimulating properties. Interleukin 2 administration induces hypothyroidism often associated with anti-thyroglobulin and anti-thyroid microsomal antibody. All these features recall Hashimoto's thyroiditis. Frequency of thyroid dysfunction during immunotherapy varies according to authors between 20 and 40 percent of all patients treated. A correlation between thyroid dysfunction and response to IL2 therapy was reported. Hypothyroidism and hyperthyroidism with development of anti-TSH receptor antibody are observed after IFN alpha administration. An auto-immune aetiology of these thyroid side effects was suggested because fine needle aspirates of the thyroid of these patients, when available, revealed a mixed cellular infiltrate with lymphocytes and histiocytes and immunocytochemical staining showed strong HLA-DR expression of all thyrocytes. Involvement of lymphokine activated killer (LAK) or direct effect of cytokines on thyroid are also possible. After intravenous injection of IL2, a marked increase in ACTH levels occurred, peaking at 4 hour with a comparable peak in cortisol level at 5 hour. Expression of IL2-Receptor in pituitary cells suggests a direct action of IL2 on anterior pituitary. Nevertheless, indirect action of IL2 via induction of other cytokines or possible secretion of ACTH by activated lymphocytes, cannot be ruled out. Testosterone serum levels decreased significantly after IL2 or IFN alpha administration. It is noteworthy that these endocrine effects induced by cytokines may modulate the clinical efficacy of these substances underlying the bi-directional communication between the immune and endocrine system.

Cancer immunotherapy has been carried out since the early fifties and first involved nonspecific system (BCG, Corynebacterium parvum, levamisole...). More recently, the production of cytokines as interferons or interleukin 2, the introduction of monoclonal antibodies have allowed a new development to cancer immunotherapy. Nevertheless, these new approaches have to be considered as a step in the biological therapy of cancer.

The engagement of the B cell antigen receptor is the first step of antigenic stimulation of B lymphocytes. This step is followed by a series of biochemical events, including the activation of protein-tyrosine kinases, phosphoinositide turnover, and multiple patterns of calcium mobilization, which lead to the regulation of gene transcription and cellular responses. The B cell antigen receptor complex is composed of membrane immunoglobulins (as antigen recognition subunits) and associated chains (Ig-alpha and Ig-beta) that couple the receptor to cytoplasmic protein kinases. To investigate independently the relative signaling capacity of Ig-alpha and Ig-beta, chimeric proteins containing their cytoplasmic domains were expressed in a B cell line. We found that Ig-alpha and Ig-beta activate two distinct intracellular signaling pathways. The engagement of Ig-alpha chimeras induces a complete release of calcium from intracellular stores, followed by transmembrane calcium influx and late cell activation signals, detected by lymphokine secretion. In contrast, Ig-beta chimeras do not induce lymphokine secretion or calcium influx, but induce short oscillatory release of calcium, dependent on the activity of the Ca-ATPase pump of the endoplasmic reticulum. These results provide a structural basis for the diversity of B cell responses.

This study analyzed clinical response induced by TIL in patients with renal cell carcinoma previously treated by interleukin-2. Six patients (4 men, 2 women, mean age 44.3 years) with measurable metastatic localizations have been treated by TIL reinjection (0.02 to 7.6 x 10(10) cells). TIL phenotype was a combination of CD4 and CD8 in 3 patients, predominantly CD4 in two patients and predominantly CD8 in one patient. Previous treatment by interleukin-2 induced one partial response, 2 stabilizations of the disease and 3 tumoral progressions. TIL led to an amelioration for 4 patients: 2 were in complete response, 2 were stabilized and 2 had tumoral progression and decreased. This study shows that CD4 TIL may improve an initially response induced by IL-2 therapy.

Membrane and soluble forms of Fc gamma receptors play important role in immune reactions. Upon interaction with immune complexes, Fc gamma R transduce activation or inhibition signals via their intracytoplasmic portions. Soluble Fc gamma R contain the Fc gamma R ectodomain. They bind to the Fc portion of IgG and inhibit Fc dependent immune reactions. In addition, they bind directly to various cell types and regulate immune reactions.

Soluble Fc gamma receptors have been identified in biological fluids of mice and humans. They are produced either by alternative splicing of the exon encoding the transmembrane region of the receptor (Fc gamma RII) or by proteolytic cleavage at the cell membrane (Fc gamma RII and Fc gamma RIII). They inhibit B cell proliferation and immunoglobulin production. Their concentrations in plasma seem to be modified during the development of certain diseases, as for instance in multiple myeloma, where plasma concentrations of soluble Fc gamma RIII are correlated with the stage of the disease.

The low-affinity receptors for the Fc portion of IgG (Fc gamma RII and Fc gamma RIII) are born by most of the immunocompetent cells and mediate a wide spectrum of biological activities. Macrophages, mast cells and lymphocytes express the type II Fc gamma R whereas the type III Fc gamma R is expressed on macrophages, mast cells and NK cells. In mice, the beta Fc gamma R gene codes for Fc gamma RII and the alpha Fc gamma R gene codes for the ligand-binding Fc gamma RIII alpha-chain. We have previously demonstrated that the methylation of the 5' region of these genes control their expression. In the present paper, we investigate the role of two unmethylated regions of the beta gene, the promoter and the third intron, in the control of its transcription. We show, by using two cell lines representative of B and mast cells, that different promoter fragments determine, in these two cell types, the transcription of the beta Fc gamma R gene. The third intron of the beta Fc gamma R gene contains sequences, which, introduced upstream to homologous or heterologous promoter, inhibit the transcriptional activity of these promoter. Thus, in B cells and in mast cells, the transcription of the beta Fc gamma R gene is controlled by two distinct regions of the gene.

Soluble forms of receptors for the Fc portion of IgG (sFc gamma R) were detected in biological fluids from mice and humans. In mouse bearing tumors, circulating amounts of sFc gamma R increased concurrently with tumor growth. Tumors secreting IgG2a, IgG2b or IgG3 led to a 5- to 10-fold increase in serum sFc gamma R levels whereas tumors secreting IgG1, IgGA or other types of tumors (non Ig B cell tumors, T cell lymphoma and a melanoma) increased 2- to 3-fold the levels of circulating sFc gamma R. In the human, sFc gamma R were also detected in whole unstimulated saliva. Levels of sFc gamma RII and of sFc gamma RIII were variable and did not seem to depend on the dental status of the individuals. Finally, a murine recombinant sFc gamma R (rsFc gamma R) composed of the two extracellular domains of Fc gamma RII was produced by culture of transfected L cells in bioreactors. The purified rsFc gamma R was found to inhibit antibody production in vitro in anti-SRBC responses and by cultures of small B cells stimulated by anti-IgM antibodies in the presence of IL-4 and IL-5. Moreover, the i.p. injection of this material into adult mice immunized with SRBC led to a decrease of IgG antibody production by splenocytes, as measured by a hemolytic plaque assay, and in serum, as measured by antigen-specific ELISA.

Twenty human salivary samples from two groups of patients (with or without dental caries) were tested for the presence of soluble forms of Fc gamma receptors type II (sFc gamma RII): sFc gamma RII were detected by immunoblotting using a radiolabelled monoclonal antibody directed against human Fc gamma RII. These soluble forms specifically interact with the Fc portion of IgG and are produced by cells of the immune system (macrophages, neutrophils, Langerhans cells). This study showed that sFc gamma RII are present in human unstimulated saliva. The statistical analysis indicated that the relative amounts of sFc gamma RII in human unstimulated saliva are variable depending on the patient. No apparent association was found between the sFc gamma RII and the oral status of the patients.

Between April 1988 and December 1990, 37 patients with progressive histologically proven metastatic melanoma were treated with interleukin-2 according to three multicentric successive protocols. Eighteen males and 19 females entered the trial. Mean age was 44 years (range 20-66); none of the patients had severe visceral disease or brain metastasis. Superficial and visceral metastatic sites were equally distributed. Interleukin-2 was administered as a 3 to 5 day continuous intravenous infusion, at a dose varying from 16 to 24 million international units/m2/day, as previously described by West. The second course was given after a 9 to 16 day free interval and one to seven courses were administered (mean three courses). A total of 132 courses has been given to the 37 patients. All are evaluable for toxicity and efficacy. Toxicity was tolerable and not different from that presented in recent reports. Only four patients had to definitively stop therapy for toxicity, one of them for cardiotoxicity; a dose modification or a transient suspension of therapy occurred in 18% of treatment cycles. One hypothyroidism with anti-thyroglobulin and anti-microsome antibodies was observed. We observed eight major responses (21.6%), usually of short duration (2-6 months). Most responses occurred in superficial lesions. One patient remains in complete remission, as therapy is stopped for 40 months. Immunological parameters, although demonstrating induced immunostimulation, did not correlate with clinical outcome. With an overall response rate of 21.6%, we confirm the activity of interleukin-2 in melanoma, as previously reported by others.