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Purpose: Immunotherapy treats some cancers, but not ovarian cancer. Regulatory T cells (Tregs) impede anti-ovarian cancer immunity but effective human Treg-directed treatments are lacking. We tested Treg depletion with denileukin diftitox (DD) ± IFNα as ovarian cancer immunotherapy. Patients and Methods: Mice with syngeneic ID8 ovarian cancer challenge were treated with DD, IFNα, or both. The phase 0/I trial tested one dose-escalated DD infusion for functional Treg reduction, safety, and tolerability. The phase II trial added IFNα2a to DD if DD alone failed clinically. Results: DD depleted Tregs, and improved antitumor immunity and survival in mice. IFNα significantly improved antitumor immunity and survival with DD. IFNα did not alter Treg numbers or function but boosted tumor-specific immunity and reduced tumor Treg function with DD by inducing dendritic cell IL6. DD alone was well tolerated, depleted functional blood Tregs and improved immunity in patients with various malignancies in phase 0/I. A patient with ovarian cancer in phase 0/I experienced partial clinical response prompting a phase II ovarian cancer trial, but DD alone failed phase II. Another phase II trial added pegylated IFNα2a to failed DD, producing immunologic and clinical benefit in two of two patients before a DD shortage halt. DD alone was well tolerated. Adding IFNα increased toxicities but was tolerable, and reduced human Treg numbers in blood, and function through dendritic cell–induced IL6 in vitro. Conclusions: Treg depletion is clinically useful but unlikely alone to cure ovarian cancer. Rational treatment agent combinations can salvage clinical failure of Treg depletion alone, even when neither single agent provides meaningful clinical benefit.

Background: Exosomes function as an intercellular communication system conveying messages from donor to target cells in nearby or distant tissues. Many aspects of exosome trafficking remain unresolved, however. Here, we investigated uptake of ten radiolabeled murine or human exosomes of various cellular origins by the liver, kidney, spleen, and lung of male CD-1 mice. Methods: We radioactively labeled 10 exosomes from mouse or human cancerous or non-cancerous lines, injected them intravenously into male CD-1 mice, and studied their tissue uptake. We examined the ability of wheatgerm agglutinin (WGA), mannose-6 phosphate (M6P), and inflammation induced by lipopolysaccharide (LPS) to modulate uptake. We measured uptake rate using multiple-time regression analysis and used heat mapping and path analysis to correlate tissue and exosomal influences on uptake. Results: Except for the uptake of SCCVII exosomes by kidney, all exosomes were taken up by all tissues, although the uptake levels varied broadly among exosomes and tissues. The liver/serum uptake ratio for exosomes from primary human T-cells was the highest at 4,500 mL/g. Species of origin (mouse vs human) or source (cancerous vs noncancerous cells) did not influence tissue uptake. The uptake of some exosomes was altered by WGA and LPS but not by M6P, except for uptake inhibition of J774A.1 exosomes by liver, suggesting use of the M6P receptor. WGA or LPS treatments enhanced uptake of exosomes by brain and lung but inhibited uptake by liver and spleen. Response to LPS was not, however, predictive of response to WGA. No evidence for a universal binding site controlling exosome uptake was obtained. Applying path analysis and heat map analysis to the data, including our published results for brain, we found that exosome uptake patterns for lung and brain responded similarly to WGA or to LPS, whereas those for liver and spleen clustered together. In path analysis, the 10 exosomes clustered into distinct groups, suggesting that their bindings sites are similarly clustered. Conclusions: Uptake of exosomes by peripheral tissues is differentially regulated by both exosomes and target tissues and is dependent on the number and types of mutually interactive binding sites.

Summary Exosomes in plasma of head and neck squamous cell carcinoma (HNSCC) patients comprise subsets of vesicles derived from various cells. Recently, we separated CD3(+) from CD3(−) exosomes by immune capture. CD3(−) exosomes were largely tumour-derived (CD44v3+). Both subsets carried immunosuppressive proteins and inhibited functions of human immune cells. The role of these subsets in immune cell reprogramming by the tumour was investigated by focusing on the adenosine pathway components. Spontaneous adenosine production by CD3(+) or CD3(−) exosomes was measured by mass spectrometry, as was the production of adenosine by CD4+CD39+ regulatory T cells (Treg) co-incubated with these exosomes. The highest level of CD39/CD73 ectoenzymes and of adenosine production was found in CD3(−) exosomes in patients with the stages III/IV HNSCCs). Also, the production of 5′-AMP and purines was significantly higher in Treg co-incubated with CD3(−) than CD3(+) exosomes. Consistently, CD26 and adenosine deaminase (ADA) levels were higher in CD3(+) than CD3(−) exosomes. ADA and CD26 levels in CD3(+) exosomes were significantly higher in patients with early (stages I/II) than advanced (stages III/IV) disease. HNSCC patients receiving and responding to photodynamic therapy had increased ADA levels in CD3(+) exosomes with no increase in CD3(−) exosomes. The opposite roles of CD3(+) ADA+CD26+ and CD3(−)CD44v3+ adenosine-producing exosomes in early versus advanced HNSCC suggest that, like their parent cells, these exosomes serve as surrogates of immune suppression in cancer.

Head and neck squamous cell carcinoma (HNSCC) is a highly immunosuppressive malignancy. Exosomes in HNSCC patients' plasma are enriched in inhibitory cargo and mediate immunosuppression. As these exosomes are products of various cells, the cellular origin of immunoregulatory proteins they carry is unknown. To test whether tumour‐ or T cell‐derived exosomes in patients' plasma are immunosuppressive and impact upon disease activity, we separated CD3((–)) from CD3((+)) exosomes by immunocapture using anti‐CD3 antibodies. The exosome protein cargo was evaluated for immunoregulatory proteins using on‐bead flow cytometry. Tumour protein‐enriched CD3((–)) exosomes were CD44v3((+)). Surprisingly, mean levels of programmed death ligand 1 (PD‐L1), cytotoxic T lymphocyte antigen 4 (CTLA‐4) and cyclooxygenase‐2 (COX‐2) were similar in CD3((+)) and CD3((–)) exosomes, although the latter induced higher (P

For head and neck squamous cell cancer (HNSCC), standard therapy consists of surgery, radiation, and/or chemotherapy. Antineoplastic immunotherapy could be an option in an adjuvant setting and is already in palliation. A functional immune system is a prerequisite for successful immunotherapy. However, effects of the standard-of-care therapy on the patients’ immune system are not fully understood. Peripheral blood mononuclear cells (PBMC) were collected from patients with HNSCC (n = 37) and healthy controls (n = 10). PBMC were stimulated with staphylococcal enterotoxin B (SEB). Simultaneous expression of various cytokines was measured in CD4+ and CD8+ T cells by multicolor flow cytometry, and polyfunctional cytokine expression profiles were determined on a single-cell basis. Expression levels of all measured cytokines in CD4+ T cells were higher in patients after chemoradiotherapy (CRT) as compared to untreated HNSCC patients or normal controls. After CRT, the frequency of polyfunctional CD4+ T cells, which simultaneously expressed multiple cytokines, was significantly increased as compared to untreated patients (p

For head and neck squamous cell cancer (HNSCC), standard therapy consists of surgery, radiation, and/or chemotherapy. Antineoplastic immunotherapy could be an option in an adjuvant setting and is already in palliation. A functional immune system is a prerequisite for successful immunotherapy. However, effects of the standard-of-care therapy on the patients' immune system are not fully understood.Peripheral blood mononuclear cells (PBMC) were collected from patients with HNSCC (n = 37) and healthy controls (n = 10). PBMC were stimulated with staphylococcal enterotoxin B (SEB). Simultaneous expression of various cytokines was measured in CD4Expression levels of all measured cytokines in CD4CRT increases polyfunctionality of CD4

Introduction Photodynamic therapy (PDT) is a palliative treatment option for patients with head and neck squamous cell carcinoma (HNSCC). We and others have shown that PDT induces a local inflammatory reaction with the potential to initiate antitumor immune responses. Recent studies indicate that cancer cells can change their morphology after PDT due to cytoskeleton alterations and decreased cell adhesion. To see whether the cargo of exosomes released by these cancer cells reflects cellular alterations after PDT, plasma was collected from PDT-treated patients prior to and at time points after therapy for exosome isolation and molecular characterisation. Material and methods HNSCC patients (n=9) were treated with PDT in a palliative setting. All patients had previously undergone several oncologic treatment regimens. Blood samples were taken before and after PDT. Exosomes were isolated from plasma by mini size exclusion chromatography and were co-incubated with cancer cells. Following co-incubation, tumour cell migration, proliferation and chemotaxis were measured. Expression on tumour cells of the EMT markers: Vimentin, EpCAM (by flow cytometry, PCR, immunofluorescence), Snail, Twist, ZEB1, Slug (by PCR), E-Cadherin, N-Cadherin (by flow and PCR) was determined. Total plasma exosomes were separated by immune capture on beads into CD3 +and CD3neg fractions. The CD3neg exosomes enriched in tumor-derived vesicles were tested by on-bead flow cytometry for the presence of E-Cadherin- and N-Cadherin. Results and discussions Exosomes harvested pre- and 24 hour after PDT contained high levels on N-Cadherin. In contrast exosomes isolated on day 7 and 4–6 weeks after PDT contained high levels of E-Cadherin. Exosomes collected before and 24 hour post PDT and co-incubated with tumour cells altered cell morphology and induced mesenchymal features, including co-expression of Vimentin, N-Cadherin, snail, slug, twist and ZEB1. In functional experiments, exosomes collected pre- and 24 hour after PDT significantly enhanced migration, proliferation and chemotaxis of tumour cells relative to exosomes harvested at two later time points. Conclusion We have shown for the first time that PDT can change the mesenchymal character of tumour cells converting it into an epithelial phenotype, and that exosomes in plasma of the PDT –treated patients were responsible for mediating the conversion. In addition, plasma-derived exosomes served as biomarkers of response to PDT, as their molecular cargo reflected the phenotypic changes occurring in the tumour cells during therapy.

CD39 and CD73 are key enzymes in the adenosine (ADO) pathway. ADO modulates pathophysiological responses of immune cells, including B cells. It has recently emerged that a subpopulation of ADO-producing CD39+CD73+ B cells has regulatory properties. Here, we define the CD39high subset of these cells as the major contributor to the regulatory network operated by human B lymphocytes. Peripheral blood B cells were sorted into CD39neg, CD39inter and CD39high subsets. The phenotype, proliferation and IL-10 secretion by these B cells were studied by flow cytometry. 5′-AMP and ADO levels were measured by mass spectrometry. Agonists or antagonists of A1R, A2AR and A3R were used to study ADO-receptor signaling in B cells. Inhibition of effector T-cell (Teff) activation/proliferation by B cells was assessed in co-cultures. Cytokine production was measured by Luminex. Upon in vitro activation and culture of B cells, the subset of CD39high B cells increased in frequency (p < 0.001). CD39high B cells upregulated CD73 expression, proliferated (approximately 40% of CD39high B cells were Ki-67+ and secreted fold-2 higher IL-10 and ADO levels than CD39neg or CD39inter B cells. CD39high B cells co-cultured with autologous Teff suppressed T-cell activation/proliferation and secreted elevated levels of IL-6 and IL-10. The A1R and A2AR agonists promoted expansion and functions of CD39high B cells. CD39 ectonucleotidase is upregulated in a subset of in vitro-activated B cells which utilize ADO and IL-10 to suppress Teff functions. Proliferation and functions of these CD39high B cells are regulated by A1R- and A2AR-mediated autocrine signaling.

Cytokines, chemokines and growth factors regulate inflammation, resistance to infection and tissue repair. Understanding their function within tissues is a priority in evolving therapy for a number of disease processes. Yet, the existence of complex networks of these factors in the tissue microenvironment has made understanding of their interactions difficult. We demonstrate the capability of microdialysis probes to recover small proteins efficiently in vitro. Further we show that microdialysis of human tissues allows for protein recovery from tissue interstitial fluid. This technology, combined with a multiplexed immunoassay, facilitates the simultaneous measurement of cytokines and chemokines in response to injury in the oral mucosa of human subjects in vivo.

Considerable evidence has accumulated indicating that cultured human or rodent tumor cells can be successfully transduced with cytokine genes and selected in the appropriate antibiotic-containing culture media. The selected transductants are generally able to secrete the cytokine coded for by the transduced gene, and in many cases, substantial levels (e.g., ng quantities) of the cytokine are produced. Using retroviral vectors, it has been possible to obtain stably transduced tumor cells with a variety of cytokine genes (1-4). These tumor cells have been used for immunotherapy of cancer in numerous animal models of tumor growth or metastasis, and more recently, in vaccination protocols in patients with cancer. One possible criticism that can be leveled at this type of vaccination approach is that cultured, genetically modified, and selected tumor cells might have phenotypic characteristics that are substantially different from those of unmodified tumor cells. Since retroviral vectors are often used for transduction, it is also possible that viral antigens expressed on transduced tumor cells contribute to the immune response generated as a result of vaccination. Also, primary cultures of human tumor cells are often difficult to establish and maintain.

Adhesive interactions mediated by cell surface receptors have been shown to induce signal transduction pathways that regulate changes in cellular function. We have reported recently that fibronectin (FN) receptors, alpha4beta1 and alpha5beta1 integrins, on NK cells transduce transmembrane signals leading to tyrosine phosphorylation of 60-, 70-, and 120-kDa proteins. In the current study, we have identified a 120-kDa phosphoprotein as the focal adhesion kinase (p125FAK), a structurally unique nonreceptor protein tyrosine kinase that localizes to focal adhesions. Activity of p125FAK was induced by adhesion of NK cells to plastic-immobilized FN, by cross-linking of cell surface-bound FN or FN fragments, FN120 or FN40, with anti-FN mAb, or by cross-linking of alpha4beta1 or alpha5beta1 integrins with alpha-chain-specific Abs. We also observed that enhanced in vitro kinase activity was associated with immunoprecipitates of alpha4beta1 or alpha5beta1 integrins from lysates of FN-adherent NK cells as compared with BSA-treated NK cells. In addition to p125FAK activity, FN-induced kinase activity was also found to be mediated by Fyn, Lyn, and Zap-70, as assessed by in vitro phosphorylation of the immunoprecipitated kinases in the presence of [gamma-32P]ATP. Clustering of FN receptors on NK cells by agonists such as immobilized FN or alpha4- or alpha5-specific Abs also induced association of Fyn and Zap-70 with p125FAK. Our observations indicate that activation and phosphorylation of p125FAK as well as Zap-70 and certain kinases of the src family play an important role in formation of active signaling complexes in response to triggering via beta1 integrins on NK cells. These results also suggest the existence of cross-talk or points of convergence between the beta1 integrin-mediated and other receptor-signaling pathways.

We recently reported that Fc mu R on NK cells is a signal transducing protein that stimulates a rapid increase in the level of cytoplasmic free calcium upon binding of IgM. This study was designed to examine signal transduction via the Fc mu R on NK cells and to characterize intracellular second messengers activated by IgM. Immunoprecipitation of IgM-bound Fc mu R by IgM-specific Ab coimmunoprecipitated the zeta- and Fc epsilon RI gamma-chains. Furthermore, engagement and clustering of Fc mu R by polyclonal IgM induced tyrosine phosphorylation of the zeta- and Fc epsilon RI gamma-chains, indicating their functional association with the Fc mu R-induced signal transduction cascade. Ligand-induced clustering of the Fc mu R also induced activity of src family kinases, Lck, Fyn, Lyn, and Src, as well as their physical interaction with the receptor. Triggering via Fc mu R also induced the activity of Syk and Zap-70, tyrosine kinases demonstrated to associate with zeta and Lck. Phospholipase C-gamma 1 and phosphatidylinositol 3-kinase were identified as substrates phosphorylated on tyrosine, as down-stream components of the signaling pathway activated in NK cells by polyclonal IgM. Although the Fc mu R on NK cells has not yet been biochemically characterized, our results suggest that the zeta- and Fc epsilon RI gamma-chains are functional subunits of this as well as other important cell surface receptors and that the Fc mu R is coupled either directly or indirectly to nonreceptor tyrosine kinases, which phosphorylate and thereby activate regulatory enzymes such as phospholipase C-gamma 1 and phosphatidylinositol 3-kinase.

We defined a novel constitutive mechanism of cell-mediated cytotoxicity, utilized by nonactivated human peripheral blood NK cells against a variety of tumor cell targets resistant to secretory (i.e., perforin/granzyme-mediated) NK cell killing. Untreated NK cells rapidly induced membrane damage and necrosis in K562 target cells (as determined by 51Cr release assay and confirmed by transmission electron microscopy; TEM), in the absence of DNA fragmentation and apoptosis (as assessed by [3H]thymidine release assay and TEM). Chelation of extracellular Ca2+ or paraformaldehyde fixation completely abrogated NK cell secretory activity and necrotic killing. In contrast, NK cells with either normal or impaired secretion consistently exhibited cytotoxicity against a wide variety of solid tumor cell lines in 1-h [3H]thymidine release, but not in 4-h 51Cr release, assays. Thus, the cytotoxicity was attributable to a nonsecretory, cell membrane-mediated mechanism. It appeared to selectively induce DNA fragmentation and apoptosis without plasma membrane damage and necrosis. This was further demonstrated by a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and confirmed by TEM. Among defined populations of immune cells, only NK cells showed an inherent ability to rapidly induce apoptotic death in solid tissue-derived malignant cells. This study demonstrates that NK cells are unique immune cells constitutively endowed with multiple mechanisms of destroying abnormal cells.

Human carcinomas spontaneously express abundant IL-2R beta but little IL-2R alpha on the cell surface, contain mRNA for IL-2R beta- and IL-2R alpha-chains, and may be inhibited in growth by exogenous IL-2. To study the relationship between IL-2R expression and growth inhibition by IL-2, carcinoma cells were transduced with IL-2R alpha and IL-2R gamma cDNAs or IL-2R beta antisense cDNA. Transfectants with the IL-2R alpha gene expressed high levels of the alpha- and beta-receptor chains and showed increased binding of [125I]IL-2. Exogenous IL-2 at the picometer concentrations inhibited their growth, and Abs to IL-2R alpha- or IL-2R beta-chains reversed the inhibition. After transduction of IL-2R beta antisense cDNA, gastric carcinoma (HR) cells no longer expressed IL-2R beta-chain, and their proliferation was depressed in the absence of exogenous IL-2. Transduction of IL-2R gamma-chain cDNA into tumor cells increased sensitivity to growth inhibition by exogenous IL-2 of a squamous cell carcinoma line, but not of HR or renal cell carcinoma lines. All of the parental and transduced tumor cell lines were found to constitutively express intracellular IL-2, detectable by immunostaining or flow cytometry of permeabilized cells. IL-2 was present on the surface of some tumor cells. Intracellular IL-2R beta and IL-2R gamma proteins were also detectable in tumor cells. Using reverse-transcription PCR combined with Southern blots or in situ hybridization, mRNA for IL-2 was found to be present in parental and transduced tumor cells. Expression on human carcinomas of IL-2R beta, inhibition of their growth by IL-2R beta antisense cDNA, and their ability to constitutively produce IL-2 and its presence on the cell surface, all suggest that endogenous IL-2 may play a role in tumor cell growth.

To determine the role of NK cells in regulation of tissue growth, the phenotype and function of liver-resident NK cells were studied after 70% partial hepatectomy in rats. The process of liver regeneration was generally completed by day 14. In contrast, the number of liver-resident NK cells (NKR-P1bright) was restored as early as day 3 after partial hepatectomy. However, spontaneous functions of liver-resident NK cells, including killing of YAC-1 and P815 targets, Ab-dependent cellular cytotoxicity, and redirected killing via NKR-P1, were continuously suppressed throughout the entire period of liver regeneration (from 3 h to 14 days). Augmentation of NK cytotoxicity against P815 targets and induction of NK cell adherence to plastic following 24 h of IL-2 stimulation showed a similar pattern of suppression. However, IL-2-induced augmentation of YAC-1 killing, proliferation and generation of adherent NK cells, and LAK activity in 5- to 7-day cultures were found to be suppressed only during the first 24 h and increased between days 2 and 7 after hepatectomy. Sorted NK cells (> or = NKR-P1bright) from liver-resident mononuclear leukocytes 24 h after partial hepatectomy showed the same pattern of suppression as unsorted mononuclear leukocytes. In contrast to liver-resident NK cells, no significant changes were detected in peripheral blood or spleen NK cells of rats following partial hepatectomy. Of particular interest, in normal liver, hepatocytes were resistant to NK lysis, while resident NK cells were cytotoxic for various NK-sensitive targets. In contrast, during the early period of liver regeneration, when hepatocytes were sensitive to lysis by liver-resident NK cells of normal rats, NK cells obtained from regenerating liver tissues were unable to mediate cytotoxicity. At the final phase of liver regeneration (days 7-14 after hepatectomy), both resistance of hepatocytes to killing by NK cells and cytotoxicity of liver-resident lymphocytes against hepatocytes from regenerating liver were simultaneously restored. In vivo depletion of NK cells by injection of rats with anti-NKR-P1 mAb resulted in a significant augmentation of liver regeneration subsequent to partial hepatectomy. Our data suggest that liver-resident NK cells may be involved in regulation of the extent of liver regeneration.

We have previously reported that CD7 expressed on resting human NK cells is a signal-transducing molecule, which upon ligation with mAb induces a rapid increase in cytoplasmic free calcium, secretion of IFN-gamma, and augmented NK activity against K562 targets. We now demonstrate that Ab-mediated clustering of CD7 molecules on NK cells results in enhanced phosphorylation on tyrosine residues of intracellular proteins of 60, 70, 80, 97, and 120 kDa. In the presence of genistein, a specific inhibitor of protein tyrosine kinase, the enhanced level of tyrosine phosphorylation was blocked, indicating that CD7 may induce signaling via activation of tyrosine kinases. Cross-linking of CD7 or CD16 molecules with primary and secondary Abs, as well as stimulation of NK cells with phorbol ester (PMA) or with calcium ionophore A23187 also induced beta 1 integrin-mediated adhesion of these cells to fibronectin (FN)-coated plastic surfaces. In contrast, cross-linking of CD2 expressed on the surface of NK cells had no significant effect on NK cell adhesion to FN. This adhesion was not associated with up-regulation of expression of alpha 4 beta 1 or alpha 5 beta 1 FN receptors on NK cells, but it required an intact cytoskeleton. The CD7-induced adhesion to FN was mediated by alpha 4 beta 1 and alpha 5 beta 1 integrins, as it was partially blocked by FN connective segment-1 peptide (EILDVPST), the alpha 4 beta 1-binding domain, as well as by RGD-containing peptides, the alpha 5 beta 1-binding domain, but not by EILEVPST or RGE control peptides. NK cell binding to FN was also partially inhibited by mAb to alpha 4, alpha 5, and beta 1 integrins. The mechanism by which cross-linking of CD7 or CD16 on NK cells induced adhesion to FN appeared to involve both protein tyrosine kinase and protein kinase C, because this adhesion was blocked in the presence of either genistein or a protein kinase C inhibitor, staurosporin. Our data demonstrate that signals transduced via triggering of either CD7 or CD16 molecules are involved in the regulation of the functional activity of beta 1 integrins on NK cells.

The CD7 molecule, one of the earliest T-lymphocyte Ag expressed during ontogeny, has recently been demonstrated to facilitate activation of T cells and to preferentially activate TCR-gamma/delta + subset of T cells. The CD7 Ag is also expressed on human NK cells, but its function has not been determined. In this study, expression and function of CD7 Ag on highly enriched NK cells (94 +/- 3% mean +/- SD, n = 12) obtained by negative selection from peripheral blood of normal donors were investigated. The CD7 Ag was found to be expressed at a significantly (p < 0.002) higher level on fresh NK cells than on IL-2-activated, NK cells. CD7 on human NK cells was found to be a signal-transducing molecule with a rapid increase in cytoplasmic free calcium observed on binding of anti-CD7 mAb to the surface of NK cells. Cross-linking of CD7 induced expression of surface activation molecules such as CD25, CD71, HLA-DR, CD69, and CD54. Activation by anti-CD7 mAb cross-linked to plastic or through goat anti-mouse Ig also induced a variety of NK cell functions: it stimulated secretion of IFN-gamma, led to proliferation of NK cells, as measured by [3H]thymidine incorporation, and significantly enhanced cytotoxicity of NK cells against K562 targets (p < 0.03). However, CD7 on NK cells did not seem to transduce a lytic signal, because it neither mediated redirected killing of Fc gamma R+ murine mastocytoma P815 cells nor triggered lysis of a hybridoma expressing the antibody in a membrane-bound form. CD7 molecules appeared to have a regulatory role in adhesion of NK cells to fibronectin, because cross-linking of CD7 on resting NK cells significantly augmented their adhesion to fibronectin-coated plastic surfaces. However, this induced adhesion was not associated with increased expression of beta 1-integrins on NK cells. Thus, CD7-mediated signals appear to augment function of adhesion molecules on NK cells, which may be involved in NK cell activation by providing both anchorage and costimulatory triggering.

After treatment with human normal IgM, 78 +/- 8% of purified CD3-CD56+ resting human NK cells and 93 +/- 6% of IL-2-activated NK cells selected by adherence to plastic reacted with FITC-goat anti-human IgM. Binding of IgM to the FcR for IgM (Fc mu R) on human NK cells was not species specific because mouse myeloma IgM also bound to these cells. The percentage of CD56+ cells binding IgM after incubation with anti-CD16 mAb was similar to that of cells incubated with medium alone (95 +/- 1% vs 93 +/- 4%). Binding of anti-CD16 mAb to Fc gamma RIII on NK cells was unaffected by pretreatment with IgM (65 +/- 12% vs 69 +/- 4%). The CD7 molecule has been reported to be the Fc mu R on the surface of T cells. Two-color flow cytometry showed that 94 +/- 3% of CD3-CD56+ resting NK cells and 71 +/- 16% of activated NK cells were CD7+. Preincubation of NK cells with three anti-CD7 mAb (Leu-9, 8H8-1, and LAU-A1) failed to block the binding of IgM to the Fc mu R. Modulation of the CD7 molecule off the cell surface (CD7+ = 1.5% +/- 0.3) did not reduce IgM binding, thus excluding the possibility that IgM anti-CD7 might bind to different epitopes of the same molecule. These data indicate that the Fc mu R is a specific Ig-binding structure, distinct from the Fc gamma RIII (CD16) or CD7. The Fc mu R on NK cells functions as a signal-transducing molecule because the addition of 0.2 mg/ml IgM to R-NK cells caused a rapid increase in [Ca2+]i (delta = 40 nM). One of the early events that followed signaling through the Fc mu R was the down-modulation of IFN-gamma gene expression and IFN gamma production in NK cells. The presence of IgM during culture of NK cells consistently decreased the expression of HLA-DR (16% vs 40% in control). Thus, the Fc mu R, a constitutively-expressed receptor on human NK cells, seems to be an important functional molecule, which delivers negative regulatory signals to NK cells.

In vitro effects of human recombinant IL-6 (1-1000 U/ml) on highly enriched human NK CD3-CD56+ cells (94% +/- 2; mean +/- SEM; n = 8), obtained from PBL were studied. IL-6 induced low levels of NK cell proliferation (7- to 30-fold during 6-day incubation), which was IL-2-independent, because IL-6 did not induce detectable IL-2 production by NK cells. Two-color flow cytometry analysis demonstrated that incubation of NK cells with IL-6 at the optimal concentration of 250 U/ml for 6 days significantly increased the proportion of NK cells expressing the following activation Ag: CD25 (26% +/- 17, mean +/- SEM vs 4% +/- 1 in control, n = 5), CD54 (44% +/- 17 vs 9% +/- 3), HLA-DR (29% +/- 13 vs 12% +/- 4), CD69 (45% +/- 7 vs 12% +/- 3), and CD71 (34% +/- 17 vs 6% +/- 2). The mean fluorescence intensity of these activation Ag was increased as well. IL-6 induced expression of CD49b (alpha-chain of VLA-2, 20% +/- 11 vs 2% +/- 1) and CD49c (alpha-chain of VLA-3, 43% +/- 17 vs 5% +/- 3), which are not expressed on resting NK cells. IL-6 also enhanced the fluorescence intensity of beta 1 integrins, CD49d, CD49e, and CD49f, expressed on NK cells. IL-6-stimulated NK cells showed significantly increased integrin-mediated adhesion to fibronectin- or laminin-coated plates (26 +/- 3 mean % cells adhering +/- SEM vs 15 +/- 4 in control for FN and 19 +/- 1 vs 11 +/- 1 for LM, p < 0.05 for both) as determined in a 3 h binding assay. As assessed by inhibition of adhesion using mAb to the VLA-2, -3, -4, -5, and -6, NK cell adhesion to fibronectin was mediated by VLA-4 and 5, and their adhesion to laminin by VLA-3 and -6. NK cells incubated in the presence of IL-6 were found to produce a factor cytostatic to WEHI-164 clone 13 target cells. This effect was partly, although significantly, blocked by neutralizing antibodies to TNF-alpha or TNF-beta. Our data demonstrate that IL-6 can directly activate human NK cells, but is a less potent NK cell activator, for all activation and functional parameters studied, than IL-2.

SUMMARYInterferon-alpha (IFN-α) has been reported to be beneficial in the treatment of chronic active hepatitis occurring as a result of hepatitis B virus (HBV) infection. Treatment with IFN-a has been proposed as a means of reducing the high rate of allograft infection in clinical liver transplantation in patients transplanted for HBV-related chronic active hepatitis and cirrhosis who are positive for hepatitis B surface antigen (HBsAg). We obtained resected whole livers from two groups of patients who received liver transplants. Group A consisted of 11 patients who were HBsAg+ but were not treated with IFN-α, and group B consisted of 10 patients who were also HBsAg+ but received IFN-α therapy for 29.4 ± 5.6 days prior to orthotopic liver transplantation. No differences between the two groups existed in terms of a variety of demographic and clinical characteristics. The liver tissue was stained with monoclonal antibodies to cell surface antigens unique to different mononuclear cell populations by the avidin-biotin-immunoperoxidase technique to determine the effect of IFN-α on the lymphocyte subsets as well as HLA antigen expression on liver-infiltrating mononuclear cells, The number of HLA-DR+ lymphocytes in the liver was significantly increased (P < 0.005) within the portal areas in group B compared with that found in group A (84 ± 14 versus 33 ± 5 per one high-power field). Moreover, the intensity of the HLA-DR antigen expression on lymphocytes in the portal areas (P < 0.02) and in the hepatic lobule (P < 0.05) was greater in group B than in group A. The number of natural killer (NK) cells was increased in the portal areas (P < 0.05) of group B compared with group A. These alterations in the lymphocyte and NK cell populations present in the liver in response to IFN-α therapy presumably reflect an IFN-α-induced enhancement of the immune response to virus-infected cells.

SUMMARY Activated T lymphocytes often accumulate in the‘ower dermis of patients with systemic selerosis (seleroderma) any may play a role in the development of dermal libresis. We propagated and cloned these cells directly from skin biopsies in four of eight cases of early, antreated systemic selerosis with diffuse seleroderma. The cloning frequency estimates were f – 0.20 and f – 0.48 for T cells derived from the skin of two patients versus f - 0.68 and f - 0.96 for autologous blood T lymphocytes, All but one of 24 skin-derived selerederma clones were CD4+. Clonal analyses performed with CD4+ clones from patients and normal controls showed that all but one skin-derived clones synthesized either laterferon-gamma (60%), glycosantinolgycan-stimulatory factor (26%) or both (9%) when induced in vitro by a nitrogen, concanavalin A, but not by antologous dermal fibroblasts. In contrast, blood-derived clones had a different functional phenotype. All skin-derived clones produced tumour accrosis factor-alpha. Our results demonstrate that T lymphecytes obtained from the skin of patients with systemic selerois synthesized cytokoines which could modulale functions of human dermal fibroblasts.

Biologic therapies able to induce or up-regulate anti-tumor immune responses could represent a complementary approach to improve the conventional treatment of squamous cell carcinomas of the head and neck (SCCHN). Patients with SCCHN are frequently immunocompromised due to the elimination and dysfunction of critical immune effector cells. Therefore, it might be necessary to restore these immune functions to allow for the generation of effective anti-tumor host responses. Simultaneously, to prevent tumor escape from immunological recognition and destruction, it might also be necessary to alter antigenic and immunogenic attributes of the malignant cells. The present overview summarizes general aspects, historical data, and recent advances in the field of immunotherapy of SCCHN, including non-specific immune stimulation, transfer of immunocompetent cells, gene therapy, use of monoclonal antibodies, and anti-cancer vaccines.

Apoptosis of circulating CD8+T cells seen in patients with squamous cell carcinoma of the head and neck (HNSCC) suggests a possibility of lymphocyte imbalance. Therefore, absolute numbers and percentages of T lymphocyte subsets were examined in the peripheral blood of patients with HNSCC and age-matched controls. Venous blood was obtained from 148 patients with HNSCC and 54 normal volunteers. Absolute numbers of CD3+, CD4+ and CD8+ T lymphocytes were determined using fluorobeads in a flow-cytometry-based technique. Percentages of T lymphocyte subsets were also evaluated by flow cytometry. The patients were grouped, at the time of blood draw (active vs. no evident disease, NED), type of therapy administered and the length of follow-up. Patients with HNSCC were found to have significantly lower absolute numbers of CD3+, CD4+and CD8+T cells than normal controls (NC). However, no differences in the percentages of T cell subsets between patients and NC were observed. Patients with active disease had significantly lower CD3+ and CD4+ T cell counts than those with NED. Patients with NED after surgery and radiotherapy had lower T cell counts than those treated by surgery alone. Patients who remained without evident disease for more than 2 years did not recover their T cell counts, and the T cell imbalance was evident many years after curative surgery. Patients with recurrent disease at the time of blood draw tended to have the lowest CD4+T cell counts. The TNM stage or site of the disease were not related to the absolute T cell count. Our data indicate that patients with HNSCC have altered lymphocyte homeostasis, which persists for months or years after curative therapies.

Therapeutic vaccines administered to patients with cancer are expected to induce and sustain tumour-specific immune responses. However, anti-tumour responses are often compromised in such patients, and a new approach to evaluating their immune competence before vaccination is needed. Spontaneous apoptosis of activated anti-tumour effector cells, including CD8+ Tetramer+ T cells, occurring with a high frequency in the circulation of such patients may be one reason for low frequencies of peptide-specific T cells detectable after vaccination. For patients treated with anti-tumour vaccines, monitoring strategies, including a broad range of antibody-based and cellular assays, have been developed. Single-cell assays, using circulating PBMC and capable of detecting fewer than 1/10,000 of antigen-specific T lymphocytes in the tested population are currently in demand. ELISPOT assay, cytokine flow cytometry (CFC) and tetramer binding were recently compared by us as part of a dendritic cell-based multi-peptide vaccination trial in patients with metastatic melanoma. All three assays performed under GLP conditions in an experienced monitoring laboratory detected a low frequency of CD8+ peptide-responsive T cells and were not found to be concordant in measuring pre- to post-vaccine changes in the frequency of these T cells. The selection of an assay for monitoring of anti-cancer vaccines is an important decision, which has to be undertaken rationally with a complete understanding of the assay limitations. The assay performance has to be validated in terms of sensitivity, specificity, inter- and intra-assay variability, and its coefficient of variation established. Performance of an assay with fresh vs. frozen/thawed specimens has to be evaluated carefully. The challenge of serial monitoring in patients receiving therapeutic vaccines is best met by using experienced reference laboratories.

Dendritic cells (DCs) are antigen-presenting cells with a unique ability to cross prime T cells and generate strong antitumor responses. This study evaluates the presence and prognostic significance of DCs as well as functional T cells, which accumulate in the microenvironment in patients with oral squamous cell carcinoma (OSCC).Immunohistochemistry for S-100 positive or p55 positive DCs and for T-cell receptor (TcR)-associated zeta-chain expression in tumor-infiltrating lymphocytes (TILs) was performed in 132 paraffin embedded specimens from patients with primary OSCC. The median clinical follow-up for the patients was 50 months. The numbers of intratumoral DCs or TILs expressing the zeta chain were determined microscopically and compared with clinical and pathohistologic prognostic parameters, including disease stage, T stage or tumor grade, lymph node involvement, as well as disease free survival and overall survival.Immunostaining identified DCs in the epithelial compartment of the tumors (S-100 positive) as well as interdigitating reticular DCs (p55 positive) in peritumoral areas. Based on S-100 staining, intratumoral DC infiltrates were low (10 DCs per high-power field [HPF]) in 20% of OSCC specimens, intermediate (10-20 DCs per HPF) in 42% of OSCC specimens, and high (20 DCs per HPF) in 37% of OSCC specimens. The number of S-100 positive DCs was positively and significantly correlated with that of p55 and of TILs with normal zeta-chain expression. A low number of infiltrating S-100 positive DCs was more predictive of poor survival (hazard ratio, 7.95) than lymph node involvement (hazard ratio, 3.36) or late T stage (hazard ratio, 2.92). A significant but weaker association of p55 positive DC infiltration with survival was observed. Low density of DCs and low or absent expression of the zeta chain in TILs correlated with each other and predicted the poorest survival and the greatest risk.The number of DCs infiltrating the tumor is a highly significant prognostic parameter in patients with OSCC. Furthermore, the absence or paucity of DCs is strongly linked to abnormalities in the TcR-associated zeta chain in TILs. The two biomarkers, zeta-chain expression in TILs and the number of S-100+ DCs in the tumor, independently predict overall survival, disease free survival, and time to disease recurrence in patients with OSCC.

Expression of T-cell receptor- or Fcgamma receptor III-associated signal-transducing zeta chain is important for the functional integrity of immune cells. We found that significantly higher proportions of circulating CD3+ T cells as well as natural killer cells had low or absent expression of the zeta chain in patients with advanced melanoma than in normal donors (P0.0005). Decreased zeta expression was always observed in a small subset of circulating CD3+ T cells that were in the process of apoptosis, i.e., bound Annexin V or were terminal deoxynucleotidyl transferase-mediated nick end labeling positive. Up to 80% of T cells in the peripheral blood of patients with melanoma were Fas+, with the mean percentage of Fas+CD3+ cells significantly higher in patients (P0.004) than normal controls. These Fas+CD3+ T cells were found to preferentially undergo apoptosis. Annexin V binding, the loss of Fas expression from the cell surface as well as zeta down-regulation, which are associated with early apoptosis, were detected in a proportion of circulating Fas+CD3+. In Jurkat cells incubated with agonistic anti-Fas antibody (CH-11), a rapid loss of Fas expression from the cell surface coincided with Annexin V binding and preceded the loss of zeta chain during early apoptosis. In a subset of Jurkat cells coincubated with human melanoma cells, Annexin V binding and zeta degradation as well as DNA fragmentation were observed, indicating that the tumor induced T-cell death. Triggering of death receptors expressed on activated T lymphocytes was accompanied by the loss of zeta expression. On the other hand, soluble factors secreted by melanoma cells induced down-regulation but no apoptosis in activated normal T cells. In the circulation of patients with melanoma, apoptosis of immune effector cells may be related to the state of chronic activation, resulting in the up-regulation of death receptors and increased susceptibility to apoptosis.

Human monocyte-derived dendritic cells (DC) can ingest apoptotic tumor cells (ATC) and present tumor-associated antigens (TAA) to T cells, leading to the generation of tumor-specific cytotoxic effector cells (Cancer Res 2000;60:3542-9). To further augment antitumor effector cell responses, attempts were made to modify antigen presentation and cross-priming of T cells by DC fed with ATC. Proinflammatory cytokines (PC), CD40 ligand (CD40L) and/or interferon-gamma (IFN-gamma) were found to markedly enhance the immunogenicity of TAA presented by DC. While PC upregulated expression of major histocompatibility complex class I/II and costimulatory molecules on the surface of DC, CD40L +/- IFN-gamma increased interleukin (IL)- 12 and to a lesser extent, IL-15 production by DC. Additionally, lactacystin, a specific proteasome inhibitor, significantly abrogated the effects of IFN-gamma and, in part, also those of CD40L or PC. The ability of DC + ATC to cross-prime TAA-inexperienced ("naive") T cells was significantly enhanced by PC and CD40L or CD40L + IFN-gamma, but not by IFN-gamma alone. These results indicate that future vaccines for patients with cancer incorporating DC fed with ATC could be made more effective by the addition of proinflammatory cytokines or CD40L +/- IFN-gamma to improve the DC function.

JBT 1001 is a vaccine used for therapy of prostate cancer (CA), which consists of recombinant prostate-specific antigen (PSA) with lipid A formulated in liposomes. Patients with prostate CA were vaccinated with JBT 1001 emulsified in mineral oil (n = 5) or with the vaccine in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) administered locally at the site of vaccination (n = 5). Frequency of PSA-reactive T cells was measured in peripheral blood mononuclear cells (PBMC) before and after immunization, using an interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay with autologous dendritic cells (DC) as antigen-presenting cells. The hypothesis tested was that PSA-based vaccines induce T cell responses to human PSA.In order to expand precursor cells, in vitro sensitization (IVS) was performed. Microcultures of peripheral blood lymphocytes (PBL) (1 x 10(5)/well) in medium supplemented with interleukin-2 (IL-2) (10 IU/ml) and interleukin-7 (IL-7) (10 ng/ml) were stimulated twice (day 0 and day 7) with monocyte-derived autologous DC, generated by culture with interleukin-4 (IL-4) and GM-CSF and pulsed with PSA (10 microg/ml) at an effector to stimulator ratio of 10:1. ELISPOT assays were performed on day 14 of culture. In addition, PBMC were separated on immunobeads into CD4(+) and CD8(+) subsets for ELISPOT assays performed without IVS.Two patients had PSA-reactive responses before vaccination (frequency range, 1/700-1/4,400). After vaccination, 8/10 patients had measurable PSA-reactive T-cell frequencies, ranging from 1/200-1/1900, using IVS. In contrast, without IVS, but after immunoselection to enrich in CD8(+) and CD4(+) T cells, only 2/10 patients had detectable PSA-reactive T cells after vaccination, at a frequency ranging from 1/2,600-1/4,000.Vaccination with PSA formulated into liposomes induced T-cell responses in 8/10 patients with prostate carcinoma. The frequency of PSA-reactive precursor T cells was relatively low in the blood of these patients, and IVS, leading to amplification of the precursor cells prior to ELISPOT, was necessary for quantification of the PSA-responding T cells. Cellular responses to PSA were predominantly mediated by CD4(+) T lymphocytes.

Major histocompatibility complex (MHC)-peptide tetrameric complexes (tetramers) are valuable tools for detecting and characterizing peptide-specific T cells. Because the frequency of these cells is generally very low, it may be difficult to discriminate between nonspecific and specific tetramer binding.A four-color flow cytometric assay that simultaneously measures tetramer, CD3, CD8, and CD14 was used to investigate the sensitivity and specificity of MHC class I tetramer staining. This was accomplished by using the influenza virus matrix protein peptide, GILGFVFTL (FLU), as a model recall antigen and the human immunodeficiency virus (HIV) reverse transcriptase peptide, ILKEPVHGV (HIV), as a model novel antigen. Peripheral blood mononuclear cells (PBMC) from 31 HLA-A2.1(+) and 10 HLA-A2.1(-) healthy individuals were stained with the tetramers.The lower limit of detection was established at approximately 1/8,000. In HLA-A2(+) PMBC, frequencies of tetramer-positive CD8(+) T cells were log normally distributed and were high for FLU (1/910) but low for HIV (1/6,067). A novel competition assay, in which tetramer binding was shown to diminish subsequent staining with anti-CD3 antibody, was used to confirm the specificity of tetramer binding to the T-cell receptor (TCR) complex. The competition assay was validated by evaluating several anti-CD3 antibodies and showing that in PBMC from HLA-A2(-) subjects, spurious tetramer-positive events (1/20,000) failed to compete with CD3 binding. For the "recall" FLU tetramer, the degree of competition was proportional to the frequency, suggesting a selection of high avidity cells. Although CD3 competition was also highly correlated with the intensity of tetramer staining, competition allowed the identification of false positive cases with relatively high tetramer staining intensity.The data indicate that competition of CD3 binding allows confirmation of the specificity of tetramer binding to the TCR, extending the usefulness of tetramers in the frequency analysis of peptide-specific T lymphocytes.

It has been suggested that dendritic cells (DCs) are capable of ingesting apoptotic tumor cells (ATCs) and presenting tumor-associated antigens to immune cells. We evaluated the potential of human DCs, which have ingested ATCs, to serve as a source of antigenic epitopes for presentation to T cells specific for PCI-13, a squamous cell carcinoma of the head and neck cell line. Immature DCs (DCimm) generated in the presence of interleukin 4 and granulocyte machrophage colony-stimulating factor from peripheral blood monocytes of HLA-A2+ healthy donors were incubated in the presence of ATCs. Uptake of ATCs by DCs was monitored by flow cytometry and confocal microscopy after 2-18 h of coincubation. When DCs were matured (DCmat) in the presence of proinflammatory cytokines, their capacity to uptake ATCs was reduced. Responses of PCI-13-specific CD8+ T cells to unmodified PCI-13 cells and to DCimm or DCmat +/- ATCs or +/- tumor lysates were tested in gamma-IFN enzyme-linked immunospot and cytotoxicity assays. Unmodified tumor cells were found to be the best stimulators of antitumor activity of the established T-cell line, and ATCs alone were minimally stimulatory. However, DCs that ingested ATCs were able to present tumor antigens to CTLs, and DCimm and DCmat were almost equally stimulatory. When DCs plus various tumor-derived preparations were used as antigen-presenting cells with autologous HLA-A2+ T cells obtained from normal donors, DCs that had ingested ATCs were more effective in generating CD8+ CTLs than tumor cells alone or DCs pulsed with tumor lysates. The results indicate that human DCs fed with ATCs and then matured effectively generated T cell-mediated antitumor responses in vitro.

Antitumor effects of antibodies against ganglioside antigens of melanoma have been reported, but neither optimal doses nor mechanisms have been established.This Phase IB trial of the murine immunoglobulin IgG(3) monoclonal antibody R(24) against disialoganglioside GD3 was conducted with 37 patients to define better the dose-response relation and mechanism of action of R(24) in patients with metastatic melanoma.Dose-limiting toxicity consisted of a pulmonary capillary leak syndrome in 3 of 5 patients in the 80 mg/M(2)/day dosage tier. Serial blood and tumor biopsy samples were obtained prior to therapy and on Days 5, 9, and 22 following R(24) infusion. Tumor biopsy-infiltrating lymphocytes were enumerated in peritumoral, endotumoral, and perivascular compartments: endotumoral CD4(+) and CD8(+) T cells and HLA-DR(+) T cells increased over time on R(24) antibody. Endotumoral CD4 lymphoid infiltrate activation (DR expression) and antibody-dependent cytotoxicity were the greatest in the one patient who achieved a complete response.Clinical response was associated with depression in natural killer (CD56(+) and CD56(+)DR(+)) blood cells (P = 0.03) and was associated with R(24) dosage (P = 0.01). A complete response that lasted 2 years and a partial response that lasted 2 months occurred at a dose of 1 mg/M(2)/day. The limited number of clinical responses observed in this trial hampered the correlation of antitumor and immune parameters but provided a rational foundation for the future evaluation of antiganglioside antibodies.

Peripheral blood mononuclear cells (PBMCs) obtained from patients with advanced melanoma but not from healthy individuals were found to undergo spontaneous ex vivo apoptosis upon incubation in medium. PBMCs were evaluated for evidence of apoptosis using Annexin V binding, caspase-3 activation, and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). PBMCs of patients with melanoma contained a significantly higher proportion (P = 0.0027) of spontaneously apoptotic cells than PBMCs of controls after 24-h incubation in medium alone. The relative proportion of activated Fas+ and tumor necrosis factor receptor 1-positive (TNFR1+) PBMCs was significantly higher in patients with melanoma than that observed in controls. To demonstrate that the TNF family of receptors and ligands was involved in this type of apoptosis, PBMCs were incubated in the presence of agonistic anti-Fas antibody (CH-11) or TNF-alpha. The proportion of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive PBMCs undergoing spontaneous apoptosis was found to be comparable with that induced by CH-11 antibody or TNF-alpha. Three-color flow cytometry revealed that CD3+ Fas+ T cells were especially sensitive to apoptosis and were preprogrammed in vivo to die. Apoptosis occurred in all subsets of PBMCs but was significantly higher (P = 0.01) in the CD3+ CD8+ T-cell subset in patients relative to controls. In two patients with melanoma, who responded clinically to dendritic cell-based peptide vaccines, the proportion of apoptotic T cells was decreased by half after therapy. In patients who were treated previously with vaccination-based therapies, levels of T-cell apoptosis were lower than in the other melanoma patients. The observed accelerated death of T cells, which are activated and susceptible to apoptosis in patients with melanoma, may contribute to a rapid turnover of immune cells, resulting in a decreased immunocompetence.

Evidence has accumulated indicating that HLA-A2-restricted CTLs specific for human wild-type sequence p53 epitopes lyse tumor cells expressing mutant p53. To explore the possibility that wild-type sequence p53 peptides could also be used in vaccines for patients expressing HLA-A24 antigen, another frequent HLA class I allele, we investigated the induction of HLA-A24-restricted p53-specific CTLs from the peripheral blood lymphocytes of normal donors. Of six p53-derived peptides possessing an HLA-A24 binding motif, the p53 peptide 125-134 (p53(125-134)) was found to have a high binding capacity and induced peptide-specific CTLs from peripheral blood mononuclear cells, using peptide-pulsed autologous dendritic cells and subsequent cultivation with cytokines interleukin 2 and interleukin 7. Bulk CTL populations lysed peptide-pulsed HLA-A24+ targets as well as HLA-A24+ squamous cell carcinoma of the head and neck (SCCHN) cell lines. However, IFN-gamma pretreatment of HLA-A24+ SCCHN cell lines was necessary for lysis, suggesting that a ligand density higher than that normally expressed by tumor cells is required for these CTLs to mediate lysis. Moreover, a cloned CTL, designated TH#99, isolated from the bulk population by limiting dilution, lysed HLA-A24+ SCCHN targets more efficiently than the bulk CTL population. Lysis was inhibited by anti-HLA class I monoclonal antibody but not by anti-HLA-DR monoclonal antibody. These results indicate that HLA-A24-restricted CTLs recognizing the wild-type sequence p53(125-134) can be generated using autologous dendritic cells from precursors present in peripheral blood lymphocytes obtained from normal HLA-A24+ donors. This finding suggests that vaccine strategies targeting wild-type sequence p53 epitopes can be extended to a wider range of cancer patients.

Our recent studies suggest that human squamous cell carcinoma of the head and neck (SCCHN) is capable of activating an intrinsic mechanism of programmed-cell death in interacting lymphocytes in situ and in vitro. The current study used Jurkat T-cell line as a model to investigate intracellular apoptotic events in T cells interacting with SCCHN. Apoptosis induced in T lymphocytes by tumor cells was in part Fas-mediated, since it was partially, but significantly, inhibited in the presence of anti-Fas ligand Ab or in Fas-resistant Jurkat cells. The synthetic caspase inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) and N-benzyloxycarbonyl-Asp-glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK), effectively blocked apoptosis of Jurkat cells co-incubated with SCCHN cell lines, suggesting the involvement of caspases in tumor-induced apoptosis of lymphocytes. Overexpression of CrmA, an inhibitor of caspase-1 and caspase-8, partially inhibited tumor-induced T-cell death. Caspase-8 and caspase-3 were identified as effector molecules in the execution of tumor-induced T-cell death, since the proform enzymes were processed into active subunits during co-incubation of T cells with tumor cells. Furthermore, co-incubation with tumor cells resulted in cleavage of poly(ADP-ribose) polymerase (PARP), a common caspase-3 substrate, and in cleavage of TcR-zeta chain, shown by us to be a T-cell specific caspase-3 substrate. Overexpression of Bcl-2 did not provide protection of T cells from SCCHN-induced DNA degradation. Instead, the Bcl-2 protein was cleaved in the target T cells during their co-incubation with tumor cells. These findings demonstrate that tumor cells can trigger in T lymphocytes caspase-dependent apoptotic cascades, which are not effectively protected by Bcl-2. (Blood. 2000;95:2015-2023)

Recombinant interleukin (IL)-2 administration can mediate regression of solid tumors in patients with melanoma and renal cell carcinoma. A better understanding of the mechanisms of IL-2-mediated antitumor effects has led to the investigation of novel immunotherapeutic approaches. The rationale for these immunotherapeutic approaches and the results of preliminary clinical studies are presented.The therapeutic potential of dendritic cells and the role of FLT3 ligand, a potent hematopoietic growth factor, was investigated in a variety of preclinical models. In addition, a clinical study with autologous dendritic cells pulsed with synthetic melanoma peptides derived from the MART1/ Melan A, gp100, and tyrosinase proteins was conducted. Twenty-eight human leukocyte antigen (HLA)-A2+ melanoma patients received an average of 106 dendritic cells a week for 4 weeks.In a murine liver metastases model, FLT3 ligand administration alone or in combination with IL-12 or IL-2 had significant antitumor effects and resulted in significant infiltration of the tumor border by lymphocytes and dendritic cells, which was associated with an increased number of apoptotic figures. Administration of melanoma peptide-pulsed dendritic cells to 28 patients with advanced metastatic melanoma produced a complete response in two patients and a partial response in one. Significant infiltration of T cells and dendritic cells into melanoma lesions was observed.These studies confirm the feasibility of immunotherapeutic approaches using dendritic cells and FLT3 ligand and demonstrate their potential antitumor activity. These approaches may be effective for patients with metastatic melanoma and other solid tumors and will likely be used to improve the efficacy of IL-2-based immunotherapy.

Development of post-transplant lymphoproliferative disease (PTLD) is a major complication of organ transplantation. While immune mechanisms seem to play a major role in the development of PTLD, how the immune system contributes to the process of PTLD development or its regression remains unknown. Between 1990-1994, 303 organ transplant recipients were enrolled into a prospective study designed to analyze risk factors for PTLD. Using a nested case-control design, 9 PTLD and 18 control patients were matched for age, EBV serological status at the time of transplantation, and, in most cases, for the type of transplanted organ. The immunologic profiles of both groups were compared prior to and following transplantation. Immune measures included absolute numbers of lymphocytes and of subsets of T, B and natural-killer (NK) cells as well as spontaneous NK-cell and in vitro generated LAK-cell activities. A consistent trend for higher levels at baseline as well as following transplantation for almost all immune parameters was observed in patients who developed PTLD. A high absolute count of activated NK cells (CD56+ DR+) at baseline was found to be a significant predictor of PTLD development. The immunologic profile of patients who developed PTLD was consistent with pre- as well as post-transplant chronic immunologic stimulation, and not immunosuppression. In the PTLD group, 3 patients had pre-transplant autoimmune hepatitis and one had primary biliary cirrhosis, which suggests that the underlying presence of certain autoimmune disorders in organ transplant recipients might predispose to PTLD development.

In mammalian cells, the octamer motif (ATGCAAAT) binding proteins, Oct-1 and Oct-2, play an important role in the transcriptional transactivation of several ubiquitously expressed genes as well as cell-specifically expressed genes. To date, a role of the octamer binding proteins in damage-stimulated response is not known. In this report, we demonstrate that DNA-binding activity of Oct-1, as demonstrated by the electrophoretic mobility shift assay, is significantly induced in a dose-dependent manner upon treatment of human head and neck squamous carcinoma cells (PCI-04A) with ionizing radiation (5 Gy: 5-fold; 15 Gy: 11-fold). By comparison, activities of other transcription factors were modestly increased (15 Gy: AP-1, 2.5-fold; NF-kappaB, 2.6-fold; SP-1, 5-fold). Radiation stimulation of Oct-1 activity was also noted in two other human cancer cell lines, albeit to a lesser extent (MDA-MB231 breast carcinoma cells and PC-3 prostate carcinoma cells (5 Gy: approximately 2-fold). These data represent the first report of the activation of an octamer factor DNA binding activity in response to environmental cues and suggest a novel role of Oct-1 in the radiation signaling cascade in these cancer cells.

Recent reports have variously described expression of Fas ligand (FasL) or its absence in human tumors. The importance of the Fas-FasL mechanism for the immune evasion by tumors provided a strong rationale for the examination of FasL expression and function in squamous cell carcinoma of the head and neck (SCCHN), which is one of the most immunosuppressive human cancers. Using immunostaining or immunoblotting, SCCHN cell lines and tumor biopsies were examined for the presence of the components of the Fas-FasL pathway and found to express Fas, as well as both the full-length and cleaved forms of FasL. By reverse transcription-PCR, mRNA for FasL and Fas were detected in all SCCHN tested, and cross-hybridization to radioactive Fas and FasL cDNA probes confirmed the specificity of amplification. To demonstrate that FasL expressed on cell surface of SCCHN cells was biologically active, various SCCHN lines were coincubated with the Fas-sensitive Jurkat T-cell lines or activated peripheral blood mononuclear cells. Tumor-induced apoptosis of T cells was dependent on the ratio of tumor cells: lymphocytes. It was significantly but only partially inhibited by neutralizing antibodies to FasL and antagonistic antibodies to FasR. Tumor-induced apoptosis was enhanced by the pretreatment of tumor cells with metalloproteinase inhibitors, which increased expression of FasL on tumor cells. Supernatants of tumor cells transduced with FasL also induced apoptosis of Jurkat cells. Thus, coincubation of SCCHN with Fas-sensitive lymphocytes can induce apoptosis of these lymphocytes, and the Fas/FasL pathway appears to be responsible, at least in part, for tumor-induced lymphocyte death. The data suggest that the Fas/FasL pathway is potentially immunosuppressive and may be involved in the escape of human carcinoma cells from immune destruction.

We have previously reported that immune anti-tumor effector cells, both cytotoxic T lymphocytes (CTLs) and IL-2-activated natural killer (A-NK) cells, are effective at eliminating human head-and-neck cancer (HNC) targets in vitro and in vivo in xenograft models. In this study, these 2 types of human effector cell were compared for the ability to prevent the development of lymph node metastases in a metastasis model of human squamous-cell carcinoma of the head and neck (SCCHN) established in nude mice. A tumor cell line, OSC-19, was injected into the floor of the mouth in nude mice, and the tumor grew progressively and metastasized to cervical lymph nodes by day 21. As effector cells, a human HLA-A2-restricted CTL line recognizing a shared antigen on OSC-19 and human non-MHC-restricted A-NK cells were used. Both types of effector cell mediated high levels of lysis against OSC-19 targets in 4-hr (51)Cr-release assays. Administration of human CTLs or A-NK cells and IL-2 to the site of tumor growth in mice with 7-day OSC-19 tumors resulted in significant reduction of the number of lymph node metastases relative to untreated or sham-operated controls or to mice treated with IL-2 without the effector cells. Our results suggest that in a xenograft model of human SCCHN implanted in the oral cavity of nude mice, the development of lymph node metastases can be successfully controlled by adoptive transfer of human SCCHN-specific CTLs or SCCHN-reactive A-NK cells plus IL-2.

CTLs recognizing the HLA-A2.1-restricted, wild-type sequence p53 epitopes p53(149-157) and p53(264-272) were generated from CD8-enriched populations of nonadherent peripheral blood lymphocytes (PBLs) obtained from healthy donors. The PBLs were restimulated in vitro with peptide-pulsed granulocyte macrophage colony-stimulating factor- and interleukin (IL)-4-induced autologous dendritic cells in the presence of IL-6 and IL-12 and subsequently cultivated with IL-1alpha, IL-2, IL-4, IL-6, and IL-7. Bulk anti-p53(264-272) CTL populations were generated from PBLs obtained from two of five donors. Both CTL populations were cytotoxic against peptide-pulsed HLA-A2+ target cells, but not against untreated target cells. A CD8+ anti-p53 CTL clone designated p264#2 was isolated from one of the bulk populations. It was found to have an intermediate affinity of approximately 10(-9) M for the epitope and to mediate cytotoxicity against several human tumor cell lines, including the squamous cell carcinoma of the head and neck cell line SCC-9, which is known to present the wild-type sequence p53(264-272) epitope. In addition, CTLs reactive against p53(149-157)-pulsed targets as well as a HLA-A2+ tumor cell line were cloned from a bulk population of antitumor CTLs obtained from one of the five normal PBLs restimulated with this epitope. The results indicate that CTLs recognizing wild-type sequence epitopes can be generated from precursors present in PBLs obtained from some normal individuals using autologous dendritic cells as antigen-presenting cells and suggest that vaccine strategies targeting these epitopes can lead to antitumor CTL generation, thereby emphasizing the therapeutic potential of p53-based cancer vaccines.

Proportions of apoptotic (TUNEL+) peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry in patients with head and neck cancer and normal controls at the time of blood draws (0 time) and after 24-h incubation. PBMCs were incubated at 37 degrees C in medium (spontaneous apoptosis) and in the presence of CH-11 antibody (anti-Fas) or tumor necrosis factor (TNF)-alpha, both capable of inducing DNA fragmentation in activated T cells expressing the TNF family of receptors. PBMCs obtained from the patients had significantly higher (P0.0001) proportion of apoptotic cells than PBMCs of controls at 0 time as well as after 24-h incubation. Ex vivo apoptosis included all subsets of PBMCs: CD3+ T cells, CD16+ CD56+ natural killer cells, CD19+ B cells, and CD14+ monocytes, as determined by two-color flow cytometry. However, T cells represented the largest PBMC subset undergoing apoptosis, and lymphocytes rather than monocytes were the major TUNEL+ PBMC population. Among T cells, the level of spontaneous ex vivo apoptosis was nearly as high as that of CH-11 antibody-induced or TNF-alpha-induced apoptosis, indicating that activated Fas+ and TNFR1+ T cells were preprogrammed in vivo to die. Also, elevated levels of spontaneous apoptosis at time 0 in patients with head and neck cancer (P0.0001) indicated that a higher fraction of PBMCs was undergoing apoptosis in vivo in patients than controls. Together, the data suggest that an increased rate of turnover of lymphocytes is associated with cancer and may be responsible for functional lymphocyte imbalance, even in treated patients who have no evident disease.

We recently reported an association between loss in T-cell receptor (TcR) zeta-chain expression and tumor-induced apoptosis of T lymphocytes. In this study, the possibility that zeta-chain serves as a direct substrate for activated caspases was investigated. Here, we report that two DXXD motifs, which are putative recognition sequences for caspase-3-related proteases and are present in the amino acid sequence of the zeta-chain, are cleaved in apoptotic Jurkat T lymphocytes. Cleavage of zeta-chain in Jurkat cells ligated by agonistic anti-Fas antibody was inhibited in the presence of peptide inhibitors of caspases, including the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone, an inhibitor of caspase-3-like activity. Fas-induced cleavage of zeta-chain was also inhibited in Jurkat cells overexpressing the intracellular inhibitors of caspase activity, Bcl-2 or cytokine response-modifier A. In vitro translated zeta-chain was cleaved in a similar fashion by recombinant caspase-3 or caspase-7 in a dose-dependent manner. In the presence of N-benzyloxycarbonyl-AspGlu-Val-Asp-fluoromethyl ketone, no cleavage of in vitro translated zeta-chain was observed. These results suggest that the loss of TcR zeta-chain, previously associated with tumor-induced immune dysfunction and more recently associated with tumor-induced apoptosis of T lymphocytes, is mediated by a direct degradation of the zeta-chain by activated caspases. This is the first report of involvement of caspases in degradation of the zeta protein.