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Cancer vaccines based on antigens derived from self molecules rather than pathogens have been under basic and clinical investigations for many years. Up until very recently, they had been tested primarily in the setting of metastatic disease with the goal to engage the immune system in slowing down disease progression. Many therapeutic vaccine trials, either investigator initiated or led by pharmaceutical companies, have been completed and many are currently ongoing, following the FDA Guidance on therapeutic cancer vaccines published in 2011. In recent years, the target of cancer vaccines is being shifted to early cancer and even premalignant disease with the goal of preventing cancer. Although some issues addressed in the FDA Guidance on therapeutic vaccines apply to preventive vaccines, many do not. Here, we discuss a set of recommendations for revising the current Guidance to also cover preventive vaccines, or to include in a new Guidance dedicated specifically to vaccines for cancer prevention. Cancer Prev Res; 8(11); 1011–6. ©2015 AACR.

Pathogen-associated molecular patterns (PAMP) are stand-alone innate and adaptive immunomodulators and critical vaccine components. We present a strategy of sequential intratumoral (i.t.) and intramuscular (i.m.) injections of the stabilized dsRNA viral mimic and PAMP, polyinosinic–polycytidylic acid-polylysine-carboxymethylcellulose (poly-ICLC, Hiltonol; Oncovir). We report the first treated patient, a young man with an exceptionally advanced facial embryonal rhabdomyosarcoma with extension to the brain. After treatment, the patient showed tumor inflammation consistent with immunotherapy, followed by gradual, marked tumor regression, with extended survival. Sequential i.t. and i.m. poly-ICLC injections mimicking a viral infection can induce an effective, in situ, personalized systemic therapeutic “autovaccination” against tumor antigens of a patient. We postulate a three-step immunomodulatory process: (i) innate-immune local tumor killing induced by i.t. poly-ICLC; (ii) activation of dendritic cells with Th1 cell– and CTL–weighted priming against the released tumor antigens; and (iii) i.m. poly-ICLC maintenance of the systemic antitumor immune response via chemokine induction, facilitation of CTL killing through the induction of costimulators such as OX40, inflammasome activation, and increase in the T-effector/Treg ratio. These results support the use of certain simple and inexpensive i.t. PAMPs to favorably stimulate effective immunity against solid cancers. A phase II clinical trial testing the hypothesis presented has begun accrual (clinicaltrials.gov, NCT01984892). Cancer Immunol Res; 2(8); 720–4. ©2014 AACR.

The existing cell phone certification process uses a plastic model of the head called the Specific Anthropomorphic Mannequin (SAM), representing the top 10% of U.S. military recruits in 1989 and greatly underestimating the Specific Absorption Rate (SAR) for typical mobile phone users, especially children. A superior computer simulation certification process has been approved by the Federal Communications Commission (FCC) but is not employed to certify cell phones. In the United States, the FCC determines maximum allowed exposures. Many countries, especially European Union members, use the "guidelines" of International Commission on Non-Ionizing Radiation Protection (ICNIRP), a non governmental agency. Radiofrequency (RF) exposure to a head smaller than SAM will absorb a relatively higher SAR. Also, SAM uses a fluid having the average electrical properties of the head that cannot indicate differential absorption of specific brain tissue, nor absorption in children or smaller adults. The SAR for a 10-year old is up to 153% higher than the SAR for the SAM model. When electrical properties are considered, a child's head's absorption can be over two times greater, and absorption of the skull's bone marrow can be ten times greater than adults. Therefore, a new certification process is needed that incorporates different modes of use, head sizes, and tissue properties. Anatomically based models should be employed in revising safety standards for these ubiquitous modern devices and standards should be set by accountable, independent groups.

This article endeavors to clarify the current requirements and status of regulatory approval for chemoprevention (risk reduction) drugs and discusses possible improvements to the regulatory pathway for chemoprevention. Covering a wide range of topics in as much depth as space allows, this report is written in a style to facilitate the understanding of nonscientists and to serve as a framework for informing the directions of experts engaged more deeply with this issue. Key topics we cover here are as follows: a history of definitive cancer chemoprevention trials and their influence on the evolution of regulatory assessments; a brief review of the long-standing success of pharmacologic risk reduction of cardiovascular diseases and its relevance to approval for cancer risk reduction drugs; the use and limitations of biomarkers for developing and the approval of cancer risk reduction drugs; the identification of individuals at a high(er) risk for cancer and who are appropriate candidates for risk reduction drugs; business models that should incentivize pharmaceutical industry investment in cancer risk reduction; a summary of scientific and institutional barriers to development of cancer risk reduction drugs; and a summary of major recommendations that should help facilitate the pathway to regulatory approval for pharmacologic cancer risk reduction drugs. Cancer Prev Res; 4(3); 311–23. ©2011 AACR.

Purpose The National Cancer Institute (NCI) has historically evaluated the participation of underserved minorities within University of Pittsburgh Cancer Institute (UPCI) clinical trials in relation to the proportion of African Americans in the general population of the UPCI primary service area of Allegheny County (12%). This standard seemed to be unrealistically high as a result of a younger age distribution of African Americans within the county. Methods The proportions of African Americans within the following four separate county populations were compared using data from 2000 to 2004: general population; invasive cancer patients; invasive cancer patients diagnosed or treated at UPCI-affiliated facilities; and patients enrolled onto UPCI's clinical therapeutic trials. Results Although the proportion of African Americans within the general population was approximately 13%, only 9.8% of patients diagnosed with invasive cancers were African American. Approximately 9.5% of all cancer patients diagnosed or treated at UPCI facilities were African American, which is comparable to the county-wide percentage of African American cancer patients. Recruitment rate of African Americans to oncology clinical trials from within the UPCI patient population was 7.6%. The NCI benchmark did not reflect the actual invasive cancer incidence rate in African American patients. By comparing the percentage of African Americans contributing to cancer incidence with the percentage of African American cancer patients treated at research-affiliated institutions, a more appropriate benchmark was derived. Conclusion The method developed by UPCI is recommended as a useful mechanism for benchmarking recruitment of African American cancer patients to clinical therapeutic trials at other cancer centers.

The taxol resistance gene TRAG-3 was initially isolated from cancer cell lines that became resistant to taxol in vitro. TRAG-3 is a cancer germline Ag expressed by tumors of different histological types including the majority of melanoma, breast, and lung cancers. In the present study, we report that patients with stage IV melanoma and breast cancers developed spontaneous IFN-γ-producing CD4+ T cell responses against a single immunodominant and promiscuous peptide epitope from TRAG-3 presented in the context of multiple HLA-DR molecules. The TRAG-3-specific CD4+ T cells and clones were expanded in vitro and recognized not only peptide pulsed APCs but also autologous dendritic cells (DCs) loaded with the TRAG-3 protein. All stage IV melanoma patients with TRAG-3-expressing tumors developed spontaneous CD4+ T cell responses against TRAG-3, demonstrating its strong immunogenicity. None of these patients had detectable IgG Ab responses against TRAG-3. TCRβ gene usage studies of TRAG-3-specific CD4+ T cell clones from a melanoma patient and a normal donor suggested a restricted TCR repertoire in patients with TRAG-3-expressing tumors. Altogether, our data define a novel profile of spontaneous immune responses to cancer germline Ag-expressing tumors, showing that spontaneous TRAG-3-specific CD4+ T cells are directed against a single immunodominant epitope and exist independently of Ab responses. Because of its immunodominance, peptide TRAG-334–48 is of particular interest for the monitoring of spontaneous immune responses in patients with TRAG-3-expressing tumors and for the development of cancer vaccines.

In addition to their cytotoxic activities, natural killer (NK) cells can have immunoregulatory functions. We describe a distinct “helper” differentiation pathway of human CD56+CD3− NK cells into CD56+/CD83+/CCR7+/CD25+ cells that display high migratory responsiveness to lymph node (LN)–associated chemokines, high ability to produce interferon-γ upon exposure to dendritic cell (DC)- or T helper (Th) cell–related signals, and pronounced abilities to promote interleukin (IL)-12p70 production in DCs and the development of Th1 responses. This helper pathway of NK cell differentiation, which is not associated with any enhancement of cytolytic activity, is induced by IL-18, but not other NK cell–activating factors. It is blocked by prostaglandin (PG)E2, a factor that induces a similar CD83+/CCR7+/CD25+ LN-homing phenotype in maturing DCs. The current data demonstrate independent regulation of the “helper” versus “effector” pathways of NK cell differentiation and novel mechanisms of immunoregulation by IL-18 and PGE2.

Natural killer (NK) cells and dendritic cells (DCs), two important components of the immune system, can exchange bidirectional activating signals in a positive feedback. Myeloid DCs, the cell type specialised in the presentation of antigen and initiation of antigen-specific immune responses, have recently been documented to be involved in supporting innate immunity, promoting the production of cytokines and cytotoxicity of NK cells, and enhancing their tumouricidal activity. Natural interferon-producing cells/plasmacytoid DCs (IPCs/PDCs) play an additional role in NK cell activation. Reciprocally, NK cells, traditionally considered to be major innate effector cells, have also recently been shown to play immunoregulatory 'helper' functions, being able to activate DCs and to enhance their ability to produce pro-inflammatory cytokines, and to stimulate T helper (Th) 1 and cytotoxic T lymphocyte (CTL) responses of tumour-specific CD4+ and CD8+ T cells. Activated NK cells induce the maturation of myeloid DCs into stable type-1 polarised DCs (DC1), characterised by up to a 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. In addition, the ability of NK cells to kill tumour cells may facilitate the generation of tumour-related antigenic material, further accelerating the induction of tumour-specific immunity. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumour-related suppressive factors, and demonstrate a strongly enhanced ability to induce Th1 and CTL responses in human in vitro and mouse in vivo models. Compared with the standard mature DCs that are used in clinical trials at present, human NK cell-induced DC1s act as superior inducers of anticancer CTL responses during in vitro sensitisation. This provides a strong rationale for the combined use of NK cells and DCs in the immunotherapy of patients with cancer and patients with chronic infections that are resistant to standard forms of treatment. Stage I/II clinical trials that are being implemented at present should allow evaluation of the immunological and clinical efficacy of combined NK-DC therapy of melanoma and other cancers.

Early detection of ovarian cancer might improve clinical outcome. Some studies have shown the role of cytokines as a new group of tumor markers for ovarian cancer. We hypothesized that a panel comprised of multiple cytokines, which individually may not show strong correlation with the disease, might provide higher diagnostic power. To evaluate the diagnostic utility of cytokine panel, we used a novel multianalyte LabMAP profiling technology that allows simultaneous measurement of multiple markers. Concentrations of 24 cytokines (cytokines/chemokines, growth, and angiogenic factors) in combination with cancer antigen-125 (CA-125), were measured in sera of 44 patients with early-stage ovarian cancer, 45 healthy women, and 37 patients with benign pelvic tumors. Six markers, i.e., interleukin (IL)-6, IL-8, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), and CA-125, showed significant differences in serum concentrations between ovarian cancer and control groups. Out of this group, IL-6, IL-8, VEGF, EGF, and CA-125, were used in a classification tree analysis that resulted in 84% sensitivity at 95% specificity. The receiver operator characteristic curve created using the combination of markers produced sensitivities between 90% and 100% in the area of 80% to 90% specificity, whereas the receiver operator characteristic curve for CA-125 alone resulted in sensitivities of 70% to 80%. The classification tree analysis for discrimination of benign condition from ovarian cancer used CA-125, granulocyte colony-stimulating factor (G-CSF), IL-6, EGF, and VEGF resulting in 86.5% sensitivity and 93.0% specificity. The presented data show that simultaneous testing of a panel of serum cytokines and CA-125 using LabMAP technology may present a promising approach for ovarian cancer detection.

Activated, adherent natural killer (A-NK) cells represent a distinct subpopulation of interleukin (IL)-2-stimulated NK cells, which are selectively endowed with the increased expression of integrins and ability to adhere to solid surfaces, migrate into, infiltrate, and destroy cancerous tissues. The present study defines the phenotype and functions of precursors of A-NK (pre-A-NK) cells in humans. Peripheral blood pre-A-NK cells, in contrast to the rest of NK cells, express a novel epitope of CD56 neuronal cell adhesion molecule, termed ANK-1, and increased cell-surface levels of integrins. Pre-A-NK cells also express low levels of CD56 and CD161, and some express CD162 receptor, do not express CD25 or activation markers, and are effective mediators of NK cytotoxicity. Thus, pre-A-NK cells are generally similar to CD56dim NK cells. However, pre-A-NK cells differ from the main NK cell subpopulation by having a lower expression level of CD16 and a lower ability to mediate redirected antibody-dependent, cell-mediated cytotoxicity. More importantly, pre-A-NK cells are preferentially endowed with the ability to rapidly respond to IL-2 by integrin-mediated adherence to endothelial cells, extracellular matrix, and plastic. This early, specific response of pre-A-NK cells to IL-2 is followed by their activation, vigorous proliferation, and differentiation into phenotypically and functionally similar A-NK cells. Pre-A-NK cells represent only ∼26% of peripheral blood NK cells but encompass the majority of NK cells in normal and cancerous, solid tissues. We conclude that pre-A-NK cells represent a distinct subset of resting, mature NK cells with the characteristics indicative of their ability to migrate and reside in solid tissues.

Natural killer (NK) cells were first identified for their ability to kill tumor cells of different origins. They were subsequently found to be able to kill some normal cells, especially those infected by certain viruses. Natural killer cells are a distinct lymphocytic population, with the morphology of large granular lymphocytes, and they lack surface Ig or T-cell markers. Natural killer cytotoxicity is enhanced in vitro in the presence of cytokines such as interleukin-2 and interferon-alpha. However, when used in immunotherapy, these cytokines have not consistently augmented NK activity or shown antitumor effects. One explanation for this divergence is that NK cell activity is suppressed in tumor tissues by various factors, including reactive oxygen species produced by infiltrating monocytes and macrophages. Agents that inhibit the generation of reactive oxygen species, such as histamine, may abrogate the suppression exerted on NK cells by monocytes/macrophages, therefore offering potential therapeutic benefit as immunoadjuvants.

Natural killer (NK) cells are a subset of lymphocytes with a distinct morphologic appearance (large granular lymphocytes [LGL]) and the ability to kill virally infected and tumor targets but to spare most normal cells. NK cells respond to a variety of biologic agents, such as interleukin-2 (IL-2), or interferons, by upregulation of cytolytic, secretory, and proliferative functions. In cancer-bearing hosts, NK cells have been considered to be the major component of antitumor immunity responsible for rapid elimination of malignant cells from the blood. More recently, however, studies have demonstrated the ability of adoptively transferred, IL-2-activated NK cells to selectively localize into solid tumors tissue and to eliminate established tumors. While these findings indicate a role for NK cells in cancer immunotherapy, additional studies are needed in both animal models and in humans to optimize clinical protocols of cancer therapy based on these cells.

Natural killer (NK) cells are important in host defense against viruses and tumors and can induce death of virally infected cells following engagement of cell surface receptors. Human NK cells express receptors for the Fc portion of IgG which stimulate antibody-dependent cell-mediated cytotoxicity and induce cytokine production. We have shown that NK cells from certain individuals can express, in addition to CD16 (FcgammaRIIIa), isoforms of CD32 (FcgammaRIIc1-4). Expression of CD32 on NK cells is dependent on an allelic polymorphism of the FcgammaRIIC gene. We analyzed the expression and function of CD32 on NK cells from 31 normal donors. Fourteen of the 31 (45%) donors expressed CD32 on their NK cells. Molecular characterization of FcgammaRIIc isoforms expressed by the CD32+ donors revealed that the majority of donors expressed the FcgammaRIIc1 isoform. Interestingly, 3 of the 14 positive donors did not express FcgammaRIIc1, and we identified a novel isoform, FcgammaRIIc5, expressed by these individuals. The expression of this isoform was correlated to a second allelic polymorphism that controls exon splicing. One of the three was found to express FcgammaRIIb on the NK cells. Biochemical analysis revealed that CD32+ donors of both types expressed a 40-kDa protein, specifically immunoprecipitated by anti-CD32 monoclonal antibodies. Functionally, only individuals expressing the FcgammaRIIc1 isoform were able to trigger reverse antibody-dependent cell-mediated cytotoxicity via CD32 whereas a CD32+ individual expressing the FcgammaRIIb isoform was unable to trigger this function. These results demonstrate that the presence of multiple allelic polymorphisms in the FcgammaRIIC gene determine the expression and function of CD32 on NK cells.

The receptors on human natural killer 9NK cells which can specifically bind the Fc portion of immunoglobulin molecules (Fc receptors) have been extensively studied. The best known and studied Fc receptor on human NK cells is FcgammaRIIIa. Interactions of NK cells with IgG antibodies via this receptor are well known to induce a signal transduction cascade and lead to antibody-dependent cell-mediated cytotoxicity (ADCC) as well as release of various cytokines. In addition, interactions with monomeric IgG and FcgammaRIIIa have been demonstrated, which result in negative regulation of NK activity and other immunomodulatory effects. Over the past several years, it has also become increasingly appreciated that human NK cells express a variety of other Fc receptors, including FcmuR, which also can mediate effector and immunoregulatory functions. Also, a novel form of FcgammaR has been demonstrated on human NK cells, termed FcgammaRIIc. Recent molecular studies have shown considerable polymorphism in the genes for FcgammaIIc and the functional consequences are being dissected. This appears to include cross-talk between FcgammaRIIIa and at least some forms of FcgammaRIIc, which may have important functional consequences.

While it is generally accepted that natural killer (NK) cells, by killing tumor cells in the circulation, represent a first line of defense against metastases, their therapeutic activity against established tumors has been limited. In this review, we describe studies to improve the therapeutic effectiveness of activated NK cells in both animal models and clinical trials to better understand the biological problems that limit their effectiveness.

Although 14.5-day murine fetal liver (FL) has few, if any, mature natural killer (NK) cells, culture of FL with recombinant human IL-2 (rhIL-2) and stroma from irradiated NK longterm bone marrow cultures (NK-LTBMC) allows proliferation and differentiation of NK cell progenitors. Using this system, NK cell progenitors were found in both CD34+ and CD34- sorted subpopulations of FL. The CD34 antigen was expressed by 14+/-1.3% of whole FL cells, while mature NK cells cultured from NK cell precursors in FL did not express the CD34 antigen. Anti-TER-119 mAb reacted with 84%+/-10.3% of the FL cells, and NK cell progenitors were enriched in the TER-119- subpopulation. After coculture with rhIL-2 and stroma, neither TER-119- nor TER-119+ cells expressed antigens associated with T cells (CD3, CD4, and CD8) or myeloid cells (Gr-1 and Mac-1). Only the TER-119 subpopulation generated NK1.1+ (77%) and B220+ (87%) cells. Within the TER-119 subpopulation, both CD34+ and CD34- cells generated cytolytic and NK1.1+ cells after culture. By a limiting dilution assay (LDA) of the Lin (i.e., negative for NK1.1, CD3, CD4, CD8, B220, Gr-1, and TER-119) CD34 positive or negative subpopulations, the calculated mean frequency of NK cell progenitors was about 1/100 for the CD34+Lin- subpopulation and about 1/(200-300) for the CD34-Lin- subpopulation. In kinetic studies, we found that NK1.1 antigen expression continued to increase with time in culture for both the CD34+Lin- and CD34-Lin- fractions. In contrast, the percentage of CD34+ cells decreased rapidly and produced CD34- cells, and the CD34- population remained CD34-. These data suggest that both CD34+ and CD34- subpopulations of FL can differentiate into NK cells when cocultured for 13 days with irradiated NK-LTBMC stroma and rhIL-2, and that CD34+ progenitors differentiate to CD34- precursors, which in turn differentiate to CD34- mature NK cells.

PURPOSE To compare the toxicity, pharmacokinetics, and efficacy seen in ovarian cancer patients treated with escalating doses of intraperitoneal (I.P.) interleukin-2 (IL-2) by two different infusion schedules. PATIENTS AND METHODS Forty-five patients were sequentially entered onto a phase I/II study in groups of four at fixed dosage tiers of 6 x 10(4), 6 x 10(5), 6 x 10(6), and 3 x 10(7) IU/m2/d in either of two schedules: (A) intermittent weekly infusions of 24 hours' duration; or (B) alternating continuous 7-day infusions followed by 7-day intervals without therapy. Eligibility criteria included > or = six courses of prior platinum-based chemotherapy and laparotomy-confirmed persistent or recurrent ovarian cancer. RESULTS Forty-one eligible patients received I.P. IL-2 and were assessable for toxicity, but six patients were not assessable for response, which left 35 patients assessable for response. Significant locoregional dose-limiting toxicity was seen with the 7-day infusions (including bowel perforation), with 600,000 IU/m2 as the maximum-tolerated dose (MTD), but catheter infection was the only significant complication seen with the 24-hour infusions, for which an MTD was not established. Among 35 assessable patients, there were six laparotomy-confirmed complete responses (CRs) and three partial responses, for an overall response rate of 25.7% (nine of 35). The median survival time of the cohort was 13.7 months and the overall 5-year survival probability was 13.9%. For the nine patients who demonstrated responses (six on the 24-hour infusion and three on the 7-day infusion), the median survival time has not been reached (range, 27 to 90+ months). CONCLUSION I.P. IL-2 is better tolerated as a weekly infusion as compared with a 7-day infusion and demonstrates evidence of possible long-term efficacy in a modest number of patients. A randomized trial is indicated to determine if the prolonged survival seen in this study is a due to I.P. IL-2 therapy or other factors that cannot be controlled for in a single-arm study.

The effects of methionine enkephalin (met-enkephalin) on human immune function are reviewed. This pentapeptide functions to upregulate, or enhance, immune function in the majority of donor samples at low doses and suppresses at high doses. The influence of this molecule is shared by the central nervous, neuroendocrine, and immune systems. Cells from each of these systems possess receptors for met-enkephalin and have the ability to process met-enkephalin from its prohormone, proenkephalin A. Studies have shown that this molecule is capable of enhancing immune function in patients with cancer or AIDS. It is proposed that this molecule be classified as a cytokine.

Cytophilia is one of the biologic properties of the Fc region of immunoglobulins operationally defined as the ability of an antibody molecule to attach to certain cell types before combination with antigen. By attachment of cytophilic antibody to membrane Fc receptors, cells become "armed" with antibodies and able to interact specifically with soluble or particulate antigens. In contrast to this cytophilia, the so-called opsonic antibody binds to FcR of various cell types only after it has complexed with antigen. In spite of knowledge of cytophilic properties of immunoglobulins for more than 30 years and an impressive accumulation of facts regarding the structure and function of cell-surface FcR binding IgG, IgE, or IgA molecules, less progress has been made in understanding the molecular basis of cytophilia. Our extensive studies of the structural and functional aspects of the interaction of IgG in monomeric form with human natural killer cells, via the type IIIA IgG Fc receptor (Fc gamma RIIIA), provide a focal point for revisiting this major pathway of direct and continuous communication between the humoral and cellular compartments of the immune system.

The Fc gamma receptor (R)IIIA (CD 16) plays an important role in regulating the cytotoxic and non-cytotoxic functions of human natural killer (NK) cells. Some anti-CD 16 monoclonal antibodies (mAb) have been shown to stimulate NK activity, while human monomeric (m) IgG induces dose-dependent inhibition of NK activity. To explore further these interactions mediated via Fc gamma RIIIA, purified NK cells were cultured for 2-3 days in the presence of mIgG, 3G8 mAb, interleukin-2 (IL-2) or a combination of mIgG or 3G8 with IL-2. Binding of mIgG or 3G8 to Fc gamma RIIIA induced divergent effects of functions of cultured NK cells: 3G8 mAb + IL-2 induced dose-dependent inhibition of proliferation attributable to apoptosis; in contrast, mIgG + IL-2 significantly increased NK cell proliferation. Incubation of NK cells in the presence of mIgG up-regulated expression of surface activation markers (CD69, IL-2R alpha, ICAM-1), cytotoxicity, cytokine production (IL-1 beta, IFN-gamma and TNF-alpha) and release of soluble IL-2R. Thus, mIgG binding to Fc gamma RIIIA induced stimulatory signals in human NK cells, leading to up-regulation of IL-2R alpha expression, cell proliferation and cytokine release.

Influenza A virus (FLU) is an important pathogen in humans. Although many features of the antiviral immune response have been elucidated in murine and human models of disease, little is known about the role of NK cells, which provide natural, innate immunity. The effects of experimental intranasal FLU (H1N1) inoculation on NK cells and other immune parameters were studied in 18 healthy, adult volunteers during the acute and convalescent phases of infection. Peripheral blood mononuclear cells (PBMNC) were assayed at baseline and on Postinoculation Days 1, 3, 4, 6, 7, 23, and 44. FLU infection and pathophysiologic upper airway responses were documented in all subjects, and there was no mortality. During both the acute (Days 1-3) and the convalescent (Days 23 and 44) stages of the FLU infection, significant increases in NK activity and decreases in the number of activated NK cells were observed. Reductions in the absolute number of T lymphocytes and in PBMNC proliferation to FLU virus antigen and mitogen were also observed. The current investigation extended those findings to include reductions in the number of CD4+ and CD8+ T lymphocytes and increases in the number of activated T lymphocytes. These results document that FLU infection was accompanied by enhancement of natural immunity and, as expected, suppression of most of the other measured parameters of cellular immunity. The normal response to FLU infection in humans may involve sequential modulation of the different components of the cellular immune system.

Previous studies have indicated that interaction of Fc gamma RIIIA on natural killer (NK) cells with various immunoglobulin ligands or monoclonal antibodies (mAbs) can have either stimulatory or inhibitory effects on cytotoxic activity, but the basis for such divergent functional effects has been unclear. We report here that stimulation of NK cells via Fc gamma RIIIA by monoclonal anti-human CD16 (3G8), monomeric IgG (mIgG), or dimeric IgG (dIgG), used either alone or cross-linked by secondary Ab (goat anti-mouse IgG or goat anti-human IgG), resulted in different phosphotyrosine protein patterns. These results suggest that distinct substrates are involved in signaling pathways activated via various agonists of the same triggering surface molecule. Three protein tyrosine kinases, i.e., LCK, LYN, and SYK, were activated by occupancy of the Fc gamma RIIIA, and only LCK activity showed a divergence in effects induced by the various ligands, with strong autophosphorylation induced by mIgG upon cross-linking. We observed no ligand-induced activation of p59fyn, p60c-src, or p62c-yes, src-related protein tyrosine kinases which are expressed in NK cells. Activity of phosphatidylinositol 3-kinase (PI 3-kinase) induced by receptor-specific antibodies or IgG ligands had different kinetics while the level of cytoplasmic free calcium was greatest upon 3G8-induced stimulation. Although the changes in kinase activities associated with Fc gamma RIIIA-mediated regulation of NK cells are complex, it appears that the patterns induced varied with the nature of the ligand and the direction of the regulation of NK activity.

In the past year, a subset of natural killer cells designated ‘A-NK cells’ has been characterized. These immune cells appear to be able to enter solid tissues, migrate to sites of metastasis and eliminate malignant tissue cells, but spare normal tissue cells. They appear to be ideal surveillance cells, readily capable of upregulating antitumor functions in response to local activation signals.

To examine supernatants (SNs) of human squamous cell carcinoma of the head and neck cell lines for soluble tumor-derived factors capable of inducing activation and proliferation of human immune cells.The SN of squamous cell carcinoma of the head and neck cell line PCI-50 was cultured in serum-free medium and tested for the ability to induce expression of activation antigens, proliferation, cytotoxicity against tumor cell targets and cytokine production by purified human natural killer (NK) and CD4+ T cells.Supernatant of PCI-50 promoted expression of the following activation markers on NK and T cells: CD25 (interleukin-2R-alpha), HLA-DR (major histocompatibility complex class II), CD54 (ICAM-1), CD71 (transferrin receptor), and CD69 (activation-inducing molecule). In addition, SN induced and significantly sustained (P.01) proliferation of human unseparated peripheral blood lymphocytes and NK or T cells in culture. Purified human NK or T cells cultured in the presence of the SN and IL-2 (120 IU/mL) had significantly higher antitumor cytotoxicity than that mediated by NK or T cells cultured in AIM-V medium and IL-2. The SN induced cytokine (interferon gamma, tumor necrosis factor alpha, interleukin-6) production in purified NK or T cells. When the SN was fractionated by molecular weight-based filtration into fractions greater and less than 30 kd, the growth- and cytotoxicity-promoting activities were consistently detectable in the greater than 30-kd fraction.Culture SN of squamous cell carcinoma of the head and neck cell lines contain a soluble factor(s) capable of activating NK and CD4+ T cells and of promoting growth and antitumor cytotoxicity of these lymphocyte subsets in vitro.

Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer. © 2011 Fox et al; licensee BioMed Central Ltd.

Viewing the immune system as part of an adaptive communication network, rather than merely a coalition of effectors, we argue that the alterations in the immune system that occur in HIV-infected patients, generally considered to be "abnormalities," cannot be attributed directly to deleterious effects of the virus on CD4+ T cells or other particular cells. Rather, many of the functional changes that occur during the asymptomatic phase reflect a normal mode of immune resistance to chronic infection, different from the "stereotypic" immune response, whereby patterns of signals are recognized and classified and evoke selective activities. The relative stability of the virus-host relationship in this phase involves a degree of mutual adaptation. However, an excessively perturbed microenvironment is the core of unstable cellular organization in which the resistance to infection gradually deteriorates. We suggest that this is due to "overadaptation" of lymphocytes and accessory cells to the infectious agent(s). We further speculate that a key factor underlying this process is a reduced rate of replacement of CD4+ T cells, which are sequestered at the sites of infection, by fresh unprimed or memory T cells. Direct and local viral effects are amplified and propagated by "affected" cells, which are not necessarily infected. The collective profile of gene expression in various types of affected cells might adequately reflect tissue organization and the overall functional status of the immune system and thus could serve as a guide to therapy. This would require collection of a more extensive array of immunologic data than is now gathered, and novel approaches to analyzing such data.

Human natural killer (NK) cells can be functionally subdivided into adherent (A) and non-adherent (NA) subpopulations. In the presence of 22 nM of interleukin 2(IL2), a substantial proportion of resting (R)-NK cells developed adherence to plastic as early as after 5 min of IL2 incubation, and by 1-5 hr of IL2 induction, 16% (range, 4-30%) of NK cells were adherent. Optimal concentration of IL2 for adherence of NK cells was 2-22 nM. This adherence was blocked completely by antibody to IL2 receptor (IL2R)-beta and, partially, by antibodies to beta 1 or beta 2 integrins, ICAM-1, CD2 or LFA3, but not by antibodies to the IL2R-alpha or CD56 antigen. A-NK cells separated from NA-NK cells after 5 hr of incubation in the presence of IL2 were significantly (P < 0.05) enriched in CD56dimCD16dim or -IL2Rp55+ and IL2Rp75+ cells, but were depleted of CD56bright CD16- cells. While surface density of CD56 and CD16 antigens was lower, that of beta 2 integrins (CD18, CD11a, CD11b) was higher on A-NK than on NA-NK cells. In a single-cell cytotoxicity assay, 61% of A-NK vs 37% of NA-NK cells bound, and 24% of A-NK vs 11% of NA-NK cells killed, K562 targets. In 4-day cultures with 0.02 or 2.2 nM of IL2, A-NK cells developed lymphokine-activated killer (LAK) activity later than NA-NK cells. By autoradiography, three to eight times more A-NK than NA-NK cells incorporated [3H]TdR into cell nuclei between 48 and 96 hr of IL2 incubation. In 14-day cultures in the presence of 22 nM of IL2, A-NK cells, which were initially adherent but later grew as single-cell suspensions, proliferated better (30-fold; P < 0.03) and expressed lower membrane density of CD56 than NA-NK cells. In culture, A-NK cells had consistently higher cytotoxicity against K562 targets than NA-NK cells, but cytotoxicity against Daudi was similar for both subsets. The data indicate that short incubation (1-5 hr) of human NK cells in the presence of 22 nM of IL2 allows for selection of a subpopulation which differs from the rest of NK cells not only by properties of rapid adherence to plastic, but also by a characteristic phenotype (CD3-CD56dim or -CD16dim or -beta 2integrinsbrightIL2Rp75+), rapid expression of IL2R-alpha, higher NK activity, delayed development of LAK activity, and ability to respond optimally in the presence of 22 nM of IL2.(ABSTRACT TRUNCATED AT 400 WORDS)

Abnormalities in the p53 gene were studied in a series of cell lines of human squamous-cell carcinoma of the head and neck (SCCHN) and in tumor tissues. Restriction-fragment-length polymorphism (RFLP), quantitative hybridization and immunochemical analysis of mutant p53 proteins were combined to detect and characterize 3 different phases in the p53 gene alteration: mutation (in 9/9 cases), 17p13 deletion (9/10 cases) and amplification of the non-deleted allele (9/31 cases). In SCCHN, deletion of the p53 gene was nearly always accompanied by mutation, only one cell line studied having mutation without deletion. Alterations in the p53 gene are common in SCCHN, and involve a series of genetic events which occur in sequence during tumor progression.

To determine if mononuclear cells (MNC) infiltrating various types of human solid tumours express genes for cytokines, in situ hybridisation with 35S-labelled cDNA antisense probes for interleukin 2 (IL2), interferon gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), interleukin 1-beta (IL1-beta), transforming growth factor beta (TGF-beta) and interleukin 2-receptors (IL2R) was performed. Fresh-frozen tissue samples of ovarian carcinomas (n = 13), breast carcinomas (n = 12), and squamous cell carcinomas of the head and neck (SCCHN, n = 7) were evaluated for the presence and localization in the tumour of MNC positive for cytokine genes. In ovarian tumours and those breast carcinomas producing little or no mucin, only rare positive MNC were observed. In contrast, breast carcinomas producing mucin and all SCCHN contained numerous MNC expressing gene transcripts for IL2, IFN-gamma, TNF-alpha, IL2R as well as TGF-beta. In tumour-involved lymph nodes of patients with SCCHN, MNC expressing genes for cytokines were found around tumour metastases but not in non-involved areas. These data suggest that tumours expressing immunogenic antigens (e.g. mucin) contain many activated MNC, while other tumours either fail to activate or suppress functions of infiltrating MNC. In SCCHN or tumour-draining lymph nodes, local down-regulation of antitumour responses might be mediated by TGF-beta produced by activated tumour-infiltrating MNC.

Cytokine gene expression in tumor-infiltrating lymphocytes (TIL) in frozen-tissue sections of 2 types of human solid tumor--ovarian adenocarcinoma and invasive breast cancer--was examined by in situ hybridization with 35S-labeled cDNA probes for human cytokines. The proportion of cells containing mRNA able to hybridize to the antisense c-DNA probes for interleukin 2 (IL-2), tumor necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma) or receptors for IL-2 (either p55 or p70) was also determined in human normal peripheral lymphoid tissues and inflammatory tissues. Few cells were positive for IL2 and TNF alpha mRNA in reactive human lymph nodes and tonsils. Inflammatory lesions, such as salpingitis or chronic active hepatitis, contained 10-20 times more cells positive for cytokine mRNA than reactive lymphoid tissue. In contrast, tumor-infiltrating lymphocytes (TIL) in the stroma of ovarian carcinomas or most ductal breast tumors only rarely expressed mRNA for TNF alpha, IL2 or IFN gamma. The intensity of mononuclear cell infiltration in these tumors correlated positively with the percentage of cells which expressed mRNA for IL-2, TNF alpha and IL-2R. In those ductal breast carcinomas which contained intracellular or intraductal mucins, up to 30% of lymphoid cells in the tumor stroma were positive for IL-2, TNF alpha, IFN gamma and IL-2R. Thus, strong evidence for local activation of mononuclear cells in situ, exemplified by the expression of genes for cytokines, was obtained only in inflammatory lesions and in mucin-producing breast carcinomas. In most carcinomas studied, few TIL expressed genes for cytokines as measured by in situ hybridization. Thus, human solid tumors appear to differ in their ability to induce gene expression for cytokines in TIL.

Successful generation of adherent lymphokine-activated killer (A-LAK) cells, highly-enriched in CD3-CD56+ antitumour effector cells, from the peripheral blood of ten patients with acute myelogenous leukaemia (AML) is described. The AML patients were either untreated or in remission. In vitro proliferation of A-LAK cells in patients with AML was generally poor, unless the cells were cocultured with irradiated concanavalin A (ConA)--prestimulated allogeneic PBL or selected lymphoblastoid cell lines (LCL) as feeder cells. Using this method, the median fold proliferation was 290 for A-LAK cells cultured with ConA-activated feeders and 291 for those grown with LCL, both significantly higher (both P less than 0.001) than the median of 2-fold expansion observed in cultures without feeders. A-LAK cultures generated in the presence of feeders consistently showed good enrichment (up to 90%) in CD3-CD56+ NK cells. Although NK activity was not significantly increased on a per cell basis in A-LAK cells grown with feeder cells, total lytic activities against both NK-sensitive target, K562, and NK-resistant target, Daudi, were significantly greater (P less than 0.02 for ConA-PBL feeders and P less than 0.005 for LCL feeders) as compared to those in paired cultures without feeders. In the presence of irradiated allogeneic feeder cells, 7/10 AML patients generated A-LAK cultures characterised by good proliferation and increased purity as well as cytotoxic activity.

Over the past 10 years, the lytic unit has become the most common means by which activity is expressed in cell-mediated cytotoxicity assays. The strengths and weaknesses of the lytic unit as a summary of cytotoxicity are discussed, and computational methods reviewed. The fundamental “assumption of proportional effect” which is implicit in the interpretation of lytic units is described and empirically tested. Based on extensive data from the assay of human natural killer (NK) activity against K562 targets, simplified computational methods are recommended. The proposed methods are easily explained, may be made robust to occasional erratic data, and permit a reasonable interpretation of lytic units even when the assumption of proportional effect breaks down.

Cancer stem cells (CSC) are thought to be responsible for tumor initiation and tumor regeneration after chemotherapy. Previously, we showed that chemotherapy of non–small cell lung cancer (NSCLC) cells lines can select for outgrowth of highly tumorigenic and metastatic CSCs. The high malignancy of lung CSCs was associated with an efficient cytokine network. In this study, we provide evidence that blocking stem cell factor (SCF)–c-kit signaling is sufficient to inhibit CSC proliferation and survival promoted by chemotherapy. CSCs were isolated from NSCLC cell lines as tumor spheres under CSC-selective conditions and their stem properties were confirmed. In contrast to other tumor cells, CSCs expressed c-kit receptors and produced SCF. Proliferation of CSCs was inhibited by SCF-neutralizing antibodies or by imatinib (Gleevec), an inhibitor of c-kit. Although cisplatin treatment eliminated the majority of tumor cells, it did not eliminate CSCs, whereas imatinib or anti-SCF antibody destroyed CSCs. Significantly, combining cisplatin with imatinib or anti-SCF antibody prevented the growth of both tumor cell subpopulations. Our findings reveal an important role for the SCF–c-kit signaling axis in self-renewal and proliferation of lung CSCs, and they suggest that SCF–c-kit signaling blockade could improve the antitumor efficacy of chemotherapy of human NSCLC. Cancer Res; 70(1); 338–46

There is increasing evidence that prolactin (PRL), a hormone/cytokine, plays a role in breast, prostate, and colorectal cancers via local production or accumulation. Elevated levels of serum PRL in ovarian and endometrial cancers have been reported, indicating a potential role for PRL in endometrial and ovarian carcinogenesis. In this study, we show that serum PRL levels are significantly elevated in women with a strong family history of ovarian cancer. We show dramatically increased expression of PRL receptor in ovarian and endometrial tumors as well as in endometrial hyperplasia, signifying the importance of PRL signaling in malignant and premalignant conditions. PRL mRNA was expressed in ovarian and endometrial tumors, indicating the presence of an autocrine loop. PRL potently induced proliferation in several ovarian and endometrial cancer cell lines. Binding of PRL to its receptor was followed by rapid phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, mitogen-activated protein kinase/ERK kinase 1, signal transducer and activator of transcription 3, CREB, ATF-2, and p53 and activation of 37 transcription factors in ovarian and endometrial carcinoma cells. PRL also activated Ras oncogene in these cells. When human immortalized normal ovarian epithelial cells were chronically exposed to PRL, a malignant transformation occurred manifested by the acquired ability of transformed cells to form clones, grow in soft agar, and form tumors in severe combined immunodeficient-beige mice. Transformation efficiency was diminished by a Ras inhibitor, providing proof that PRL-induced transformation uses the Ras pathway. In summary, we present findings that indicate an important role for PRL in ovarian and endometrial tumorigenesis. PRL may represent a risk factor for ovarian and endometrial cancers. [Cancer Res 2009;69(12):5226–33]

Adherent lymphokine-activated killer (A-LAK) cells, selected from peripheral blood lymphocytes (PBL) of normal human donors by adherence to plastic, and cultured in the presence of interleukin 2 (IL-2), are highly enriched in CD3-CD56+ natural killer (NK) cells. These IL-2-activated NK cells proliferate extensively upon further culture in conditioned medium containing IL-2. In contrast, we previously found that with PBL of some patients with advanced cancer, the same procedure often failed to yield high enrichment of NK cells or substantial expansion in the numbers of these effector cells. To obtain sufficient numbers of A-LAK cells for adoptive immunotherapy in cancer patients, an improved method for generation of human A-LAK cells with irradiated mitogen-stimulated allogeneic PBL- or Epstein-Barr virus-transformed lymphoblastoid cell lines was introduced. In paired experiments, A-LAK cultures with feeder cells showed significantly enhanced IL-2-driven proliferation of A-LAK cells obtained from normal donors or patients with metastatic melanoma, renal cell carcinoma, and other types of solid cancers. The growth-promoting effect of feeders for A-LAK cells resulted in significantly improved expansion of CD3-CD56+ (NK) effector cells in A-LAK cultures established from normal donors. Cells in these cultures also had significantly higher levels of antitumor cytotoxicity against K562 and Daudi targets than did A-LAK cells grown in the absence of feeder cells. Enrichment in CD3-CD56+ cells and antitumor activity also occurred in patient A-LAK cultures supplemented with mitogen-stimulated feeder cells, but was not statistically significant. Overall, despite improved proliferation and CD3-CD56+ cell content of A-LAK cultures established in the presence of mitogen-activated feeder cells, only 39% (21/54) of patients tested generated A-LAK cells that would be judged acceptable for large-scale therapeutic use by criteria based on fold expansion and purity of A-LAK cells. These results suggest that in comparison to normal individuals, NK cells of many patients with advanced solid tumors are defective in their ability to respond by proliferation to IL-2 even in the presence of exogenously supplied growth factors.

The addition of mitogen-prestimulated periferal blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) cultures to enriched populations of natural killer (NK) cells obtained from PBL of normal donors in the presence of rIL-2 resulted in highly significant increases in proliferation, purity, and cytolytic activity of cultured NK cells. Two sources of enriched NK cell preparations were used: (i) Adherent-lymphokine activated killer (A-LAK) cells obtained by adherence to plastic during 24 hr activation with 10(3) Cetus U/ml rIL-2; and (ii) NK cells negatively selected from PBL by removal of high-affinity rosette-forming cells and CD3+ lymphocytes. Coculture of A-LAK cells for 14 days with autologous or allogeneic Con A-activated PBL (10(6) cells/ml) or selected EBV-transformed LCL (2 x 10(5) cells/ml) as feeder cells increased fold expansion by a mean +/- SEM of 629 fold +/- 275 (P less than 0.019) and 267 fold +/- 54 (P less than 0.0001), respectively, compared to 55 +/- 20 in A-LAK cultures without feeder cells. The addition of either activated PBL or EBV lines to A-LAK cultures also led to a significant increase in the percentage of NK cells (CD3- CD56+) (84 +/- 2.4 and 84 +/- 2.6%, respectively, P less than 0.0001 for both), compared to 53 +/- 7.2% in cultures without feeders. The presence of feeder cells in cultures of A-LAK cells also led to significantly higher anti-tumor cytolytic activity compared to control cultures, as measured against NK-sensitive (K562) and NK-resistant (Daudi) target cells. Mitogen-stimulated CD4+ PBL purified by positive selection on antibody-coated flasks were better feeders than CD8+ or unseparated PBL. In the presence of feeder cells, it was possible to generate up to 6 x 10(9) activated NK cells from 2 x 10(8) fresh PBL by Day 13 of culture. Enhanced NK cell proliferation in the presence of feeder cells was not attributable to a detectable soluble factor. The improved method for generating A-LAK or activated-NK cells should facilitate cellular adoptive immunotherapy by providing sufficient numbers of highly enriched CD3- CD56+ effector cells with high anti-tumor activity.

Fc receptors for IgG (CD16) have been described as the only type of immunoglobulin receptor on large granular lymphocytes (LGL). However, the ability of natural killer (NK) cells to mediate antibody-dependent cellular cytotoxicity (ADCC) in the presence of monoclonal or polyclonal IgM and the inhibition of NK activity by highly purified IgM could not be explained on the basis of FcR for IgG. In order to directly assess the expression of Fc receptors for IgM (Fc mu R), NK cells were treated with human polyclonal IgM, and its binding was visualized by a direct anti-globulin rosette assay with identification of rosette-forming LGL on Giemsa-stained smears. The data indicated that a high proportion of LGL (up to 68%) were Fc mu R-positive cells. However, this percentage varied depending on the IgM preparation (polyclonal or monoclonal), the indicator reagent used for the rosette assays, and the cell preparations studied. Two-color flow cytometry of human nonadherent lymphocyte preparations confirmed the presence of CD56+IgM+ cells, which represented from 43 to 78% of CD56+ cells. Flow cytometry was also performed using highly enriched preparations of human NK cells (the mean percentage of CD3-CD56+ cells was 84%). Up to 88% of purified NK cells bound FITC-labeled monoclonal IgM at a saturating concentration. By indirect immunofluorescence, from 34 to 62% of NK cells purified from the peripheral blood of normal donors were able to bind polyclonal IgM. Similar results were obtained with LGL from a patient with NK lymphoproliferative disease. Thus the presence of Fc mu R on a majority of human NK cells was demonstrated by different techniques, using unseparated peripheral blood mononuclear leukocytes, purified normal NK cells, and also LGL from a patient with NK lymphoproliferative disease.

The efficacy of adoptive immunotherapy for solid tumors with lymphokine-activated effector cells presumably depends on the ability of these cells to localize adequately in tumor tissues. We present here the first quantitative study of the in vivo movement of fluorescently labeled adherent lymphokine-activated killer (A-LAK) cells. These cells were injected intra-arterially along with low-dose interleukin-2 into normal (mature granulation) tissue and an implant of VX2 carcinoma grown in the rabbit ear chamber. A small proportion of A-LAK cells accumulated preferentially in the tumor microcirculation in vivo because of an increased frequency of long-term adhesive interactions with the tumor vasculature. Stasis of blood flow in the tumor vasculature was observed 1 to 2 days after injection. Subsequent necrosis of the tumors was observed, along with diffuse infiltrates of lymphocytes, monocytes, and granulocytes in the interstitial space within the tumor. Development of necrosis despite low ratios of effector cells to target cells suggests that in addition to direct cytotoxicity, the response to adoptive immunotherapy is mediated via the tumor vasculature. This novel mechanism for adoptive immunotherapy must be taken into account in the development of improved strategies for cancer treatment.

In a nude mouse model of human squamous-cell carcinoma of the head and neck (SCCHN), locoregional therapy with interleukin 2 and human lymphokine-activated killer (LAK) cells resulted in a significant inhibition of growth of 3-day established tumors. The same model was used for therapy of 7-day established tumors with highly enriched populations of human adherent (A)-LAK (CD3- CD56+) cells and IL-2. Peritumoral transfer of 10 x 10(6) A-LAK cells, whose in vitro cytotoxicity against a SCCHN cell line (PCI-I) was not significantly different from that of LAK cells, resulted in complete regression of all 3-day or 7-day human SCCHN in nude mice. An initial inflammatory-type reaction, which appeared within hours of the first peritumoral cell transfer, was accompanied by infiltration initially by granulocytes and plasma cells, and later by mononuclear cells into the tumor stroma. A-LAK cells labelled with a fluorescent dye prior to injection appeared in the tumor stroma within 24 hr and were localized around or in the basal epithelial tumor layer by 48 hr. Histologic sections revealed an increasing epithelial disorganization and progressively decreasing basal epithelial layer, which were proportional to the increasing number of A-LAK cells transferred. Within 4 weeks, the tumors were reduced to amorphous keratinic remnants surrounded by the connective tissue containing abundant mononuclear cells. Local administration of human A-LAK cells and IL-2 to SCCHN tumors growing in nude mice led to accelerated tumor differentiation, keratinization and regression.

Phenotypic and functional characteristics of tumor-infiltrating lymphocytes (TIL) obtained from human primary and metastatic liver tumors were studied. Lymphocytes isolated from 18 tumors and autologous (A) peripheral blood (6 cases) were phenotyped by 2-color flow cytometry and cloned in a limiting dilution system, which allows virtually all normal T lymphocytes to proliferate; 70-80% of fresh TIL were T cells (i. e., CD3+), and the ratio of CD4+/CD8+ cells was 1.2 in both primary and metastatic liver tumors. TIL contained significantly more CD56+ (NKHI+) cells, half of which were CD3+ CD56+, CD3+ CD25+ cells and CD3+ HLA-DR+ cells, than A-PBL. The frequencies of proliferating T-cell precursors (PTL-p) and cytolytic T-lymphocyte precursors (CTL-p) reactive with K562, allogeneic tumor cells and autologous tumor cells, were determined. Mean PTL-p frequencies for TIL from hepatocellular carcinomas, cholangiocarcinomas and metastatic liver tumors were 0.52 (0.22-0.83), 0.10 (0.05-0.16) and 0.16 (0.01-0.30), respectively. The frequency of CTL-p with natural-killer-like activity was lower in TIL than in A-PBL. The frequency of CTL-p for autologous tumor cells in fresh TIL isolated from primary liver tumors was 0.02-0.13 and 12/81 clones were reactive against autologous tumor. In contrast, only 1/66 TIL clones obtained from colon carcinomas metastatic to liver showed autotumor reactivity. No clones reactive with autologous tumor were obtained from peripheral blood of patients with liver cancer. These data indicate that substantial differences in anti-tumor functions of TIL between primary and metastatic liver tumors exist, which can be detected at a clonal level.

The isolation of mononuclear cells from human liver tissues was achieved by a simple method consisting of enzymatic and mechanical dissociation, density gradient centrifugation, and adherence to plastic. The method was optimized to obtain a nearly complete recovery of different lymphoid subpopulations. The mononuclear cells recovered from "normal" liver tissues consisted of 33 +/- 9% (mean +/- SD) small lymphocytes, 44 +/- 6% large granular lymphocytes, 9 +/- 2% monocytes/macrophages, 9 +/- 3% granulocytes, and 5 +/- 2% other cells as determined by microscopic analysis of May-Grünwald-Giemsa-stained cytocentrifuge smears. Phenotypic analysis of the liver-infiltrating lymphocytes (LIL) by two-color flow cytometry showed that CD3-CD56+ NK cells represented 43 +/- 6% (mean +/- SD), CD3+CD56- T cells, 30 +/- 9%, and CD19+ B cells 3 +/- 1%. The predominant phenotype of the liver-infiltrating NK cells was CD3-CD56+CD16- in contrast to that of circulating NK cells, which are largely CD3-CD56+CD16+. The alpha/beta TCR+ T cells were 42 +/- 14% and gamma/delta T cells were infrequent (4 +/- 4%). The proportions of the three major lymphoid populations (T, NK, and B cells) recovered from liver tissues of 10 patients with different liver diseases were altered. Tissue sections from the same specimens were stained by the immunoperoxidase method to compare the in situ cellular composition with that determined by flow cytometry. LIL recovered from normal (control) and virally infected (non-A, non-B hepatitis) liver tissues had high NK activity (up to 1,000 LU/10(7) cells) as measured against K-562 targets in 4-hr 51Cr-release assays. NK activity was significantly lower (P less than 0.05) in LIL recovered from other liver diseases. LIL had spontaneous cytotoxicity against NK-resistant Daudi targets which was significantly higher (P less than 0.05) in control and virally infected than in other liver tissues. Our results indicated that human LIL recovered from normal and diseased liver tissues contained a high proportion of functionally active NK cells and that NK and lymphokine-activated killer activities but not the percentages of CD56+ cells were decreased in end-stage primary biliary cirrhosis and primary sclerosing cholangitis. In contrast, the proportions of NK cells and NK activity remain high in non-A, non-B hepatitis.

Immunoregulatory effects of human macrophages on natural killer (NK) activity were studied. Monocytes were isolated by adherence to plastic, after leukapheresis of normal blood donors, and cultured for 1 to 14 days. In vitro-differentiated (5-7 days) human macrophages consistently and significantly (P less than 0.01) augmented NK activity of fresh autologous or allogeneic PBMNC. During culture, these macrophages also developed increased antitumor cytostatic activity. The optimal time for both the expression of cytostatic activity and up-regulation of NK activity was 5-7 days in culture. In contrast, 12- to 14-day macrophages significantly suppressed NK activity and had less cytostatic activity. Macrophages in culture demonstrated shifts in Leu-M3+HLA-DR+ phenotype from the mean of 60% +/- 11 (SD) in fresh monocytes to 90% +/- 5 between Days 5 and 7 in culture and then down to 10% +/- 5 in 14-day cultures. The activity of NK (CD56+CD3-) cells, purified by Percoll gradient centrifugation and flow cytometry, was up-regulated directly by in vitro-differentiated macrophages at low macrophage to NK cell ratios, and this up-regulation was not dependent on T lymphocytes or other accessory cells. The modulation of NK activity by differentiated macrophages was not MHC-restricted and depended on the viability and cellular integrity of macrophages. Sonicated macrophages could no longer up-regulate NK activity. This study shows that antitumor effects mediated by human in vitro differentiated LeuM3+HLA-DR+ macrophages may simultaneously involve more than one mechanism, namely direct cytostasis of tumor cells and activation of NK cells.

Current evidence indicates that natural killer (NK) cells, which are large granular lymphocytes that mediate non-major histocompatibility complex (MHC)-restricted cytotoxicity and antibody-dependent cytotoxicity and that are involved in multiple regulatory, developmental, and immunologic processes, are important in health. Immunodeficiency states presenting with low NK activity are often associated with malignancies, chronic viral infections, or autoimmune diseases. Monitoring of NK function appears to be indicated as an aid to diagnosis, prognosis, and follow-up after therapy. Reliable performance of NK assays in a clinical laboratory requires that uniform criteria be established and followed for the acceptability of results. Statistical analysis of daily variability can be of great assistance in identifying and tracking sources of error, but routine statistical adjustments are not generally advisable. The quality control program described here provides a degree of assurance that this cytotoxicity assay can be dependable whether performed at one time point or serially. The successful implementation of this program requires laboratory resources, biostatistical support, and interpretative skills, all of which are available in a modern clinical laboratory.

The scale and scope of the cancer problem in the United States today is much greater than four decades ago when the formal war against the disease was first announced. Patterns of the disease are not fully explained by known risk factors. Much progress has been made in understanding the molecular basis of carcinogenesis, particularly the near consensus (realization) that virtually all cancers arise from an accumulation of genetic mutations and the more recent recognition of the role of inflammation and the tissue microenvironment, in particular for hormone-dependant cancers. However, most genetic mutations that contribute to cancer are not inherited, and thus must be attributable to accumulation of somatic mutations and epigenetic changes, from as yet poorly understood environmental factors, that certainly cannot be explained entirely by tobacco, use and arise over the course of a lifetime. Much of the national effort to control cancer has focused on detecting and treating the disease—not on seeking approaches to prevent cases from arising. Given this reality, we present a cross-disciplinary framework for establishing comprehensive research and policy centers focused on environmental oncology to be based at selected academic cancer centers across the country. The principal goal of such centers is to improve the ability to prevent cancer, by developing effective interventions based on insights obtained from epidemiology, including molecular epidemiology and basic scientific research on genomic, metabolomic, and other biomarkers of exposure, susceptibility, and disease. As the needed scientific evidence for environmental factors contributing to cancer is revealed, these academic centers will develop specific interventions and/or policy recommendations regarding ways to lower the burden of cancer, based on existing information about cancer hazards in the personal, occupational, and general environment. Ultimately the centers will improve the ability to identify and control the underlying causes of the occurrence of cancer and its progression.

Natural killer cells were first described in the early 1970s on a functional basis according to their ability to lyse tumor cells in the absence of prior stimulation. Since their discovery, NK cells have been shown to have other important functions that led to the use of NK cells as a form of adoptive immunotherapy. Over the next 5-10 years, with the advances in the field of NK cells we will undoubtedly see the use of allogeneic and autologous NK cells at the forefront of cancer immunotherapy.

The emerging field of cancer prevention through chemoprevention agents and cancer vaccines offers significant promise for reducing suffering and death from cancer. However, that promise may not be kept unless major barriers to progress are lowered or eliminated. Among the most significant barriers are the relatively small investment from government and industry in research and development of cancer preventive agents; a predominant emphasis of translational cancer research on therapeutic interventions for metastatic or advanced cancer; complexities of prevention trial design; a relatively uncharted Food and Drug Administration (FDA) approval process for preventive agents; insufficient public and patient understanding of the importance and potential for cancer preventive measures, with consequent unpredictable public and patient willingness to take preventive agents; an uncertain reimbursement from payors; and limitations in patent law, liability protection, and data package exclusivity that undermine the opportunity for recouping investment. Viewed individually or collectively, each of these barriers serves as a substantial deterrent to intellectual and financial investment by all sectors of the cancer community. In an effort to ultimately overcome these barriers, a Cancer Prevention Research Summit was assembled June 12–13, 2006 in Bethesda, Maryland, organized by C-Change with support from the AACR. The Summit brought together some 120 leaders from private, public, and not-for-profit entities, including cancer researchers and clinicians; federal health officials; regulatory agency representatives; pharmaceutical, biotech, and food industry leaders; patent attorneys; economists; public and private provider group executives; and advocates. Participants engaged in a detailed process to more carefully define the major barriers, identify potential solutions, and formulate initial priorities and recommendations for action. At the conclusion of this dialogue among experts, the following recommended actions were outlined: define policy solutions to patent, intellectual property, and liability law barriers; create an advisory document about the approval process for cancer chemopreventive agents and vaccines for the FDA; develop new design models for cancer chemopreventive clinical trials; outline the business case for chemopreventive agents and vaccines for federal research agencies, payors and investors; and implement a communications strategy to increase public awareness about the importance of chemoprevention and cancer preventive vaccines. (Cancer Res 2006; 66(24): 11540-9)

Chronic fatigue syndrome (CFS) is a serious health concern affecting over 800,000 Americans of all ages, races, socioeconomic groups and genders. The etiology and pathophysiology of CFS are unknown, yet studies have suggested an involvement of the immune system. A symposium was organized in October 2001 to explore the possibility of an association between immune dysfunction and CFS, with special emphasis on the interactions between immune dysfunction and other abnormalities noted in the neuroendocrine and autonomic nervous systems of individuals with CFS. This paper represents the consensus of the panel of experts who participated in this meeting. Data suggest that persons with CFS manifest changes in immune responses that fall outside normative ranges, but current research does not provide definitive evidence on whether these immune abnormalities are a cause or result of the illness. It has become clear that CFS cannot be understood based on single measurements of immune, endocrine, cardiovascular, or autonomic nervous system dysfunction. This panel encourages a new emphasis on multidisciplinary research into CFS.

Colorectal cancer is one of the most common fatal malignancies in the United States, with an incidence second only to lung cancer. The liver is the most common site of colorectal metastases and frequently the only affected organ once the primary tumor has been surgically removed. The only potentially curative treatment for metastatic colorectal cancer in the liver is surgery, although most patients are not eligible for resection. We have therefore, evaluated the therapeutic efficacy of dendritic cells (DCs) engineered to express IL-12 in a liver metastasis model. Direct administration of DCs into the portal vein significantly inhibited the growth of established MC38 colon carcinoma in the liver in C57BL/6 mice. This effect was accompanied by an intratumoral accumulation of CD4+, CD8+, and NLDC-145+ immune effector cells, and also resulted in a systemic immune response as determined by enhanced production of IFN-gamma by T lymphocytes isolated from both spleen and draining lymph nodes. Evaluation of homing of Cy3-labeled DCs following the portal vein injection confirmed their distribution in the liver and lymphoid tissue. Thus, a local delivery of DCs transduced with the IL-12 gene can not only inhibit colorectal tumor growth in vivo but also mount systemic antitumor immune responses. This approach is likely to improve the outcome of immunotherapy for metastatic colorectal cancer since high numbers of tumor-associated DCs positively correlate with a more favorable prognosis. Simultaneous local gene therapy with IL-12 will further improve clinical efficacy without placing the patient at risk for systemic toxicity.

Natural killer (NK) cells are spontaneously cytotoxic immune effector cells with the ability to selectively destroy tumor cells without harming normal cells. To perform this function, NK cells utilize two main cytotoxicity pathways, the well known perforin/granzyme-mediated secretory/necrotic killing and the recently defined TNF family ligand-mediated non-secretory/apoptotic killing. The former mechanism is manifested mainly against a few cultured leukemia cell targets, while the latter mediates killing against a large variety of tumor cell targets. Therefore, the biological role and significance of these mechanisms might be different. The NK cell-mediated necrotic killing has been reliably and selectively measured in humans by the standard 4-h 51Cr release assay (CRA) against K562 myeloid leukemia cell targets. However, no standardized high throughput assay is available for testing the NK cell-mediated apoptotic killing. Here, we introduce the modified MCA as a convenient method for measuring perforin/granzyme-independent NK cell-mediated apoptotic killing. The assay is performed in microwells of Terasaki tissue culture microtest plates, using adherent tumor cell targets, which are selectively susceptible to non-secretory/apoptotic killing and resistant to secretory/necrotic killing mediated by NK cells. Target cells are plated in microwells and incubated overnight to adhere to the plastic surface and to regenerate cell surface-bound TNF family receptors. Following this adherence, target cells are co-incubated with freshly isolated human peripheral blood mononuclear leukocytes (PBMNL) or purified subpopulations of immune cells for 24 h in various effector/target (E/T) ratios. During this incubation, dead target cells become non-adherent and are removed by washing the wells. Remaining adherent (viable) target cells are fixed, stained and optically counted. A notable dose-dependent (peak at 200:1 E/T ratio), time-dependent (peak at 24 h of incubation) and donor-dependent killing of tumor cells was consistently and reproducibly induced by PBMNL of normal donors. Using purified subpopulations of immune cells, it was demonstrated that among PBMNL, CD3(-)CD56(+)CD16(+) mature NK cells are the only mediators of tumor cell killing in MCA, as well as in CRA. Comparative studies of NK activity detected by MCA and CRA, performed with PBMNL from normal individuals and breast cancer patients, showed no significant correlation between the cytotoxicities measured in the two assays. In addition, while NK activity measured in CRA was normal in most breast cancer patients, NK activity assessed in MCA was decreased in a large majority of the patients. Thus, MCA is a sensitive NK assay, which is biologically different from CRA, and may be clinically relevant. MCA has also a higher throughput, and is more practical and economical than CRA.

A 61-year-old woman with extensive cutaneous vasculitis and hairy cell leukemia treated with cladribine is described. She achieved a dramatic hematological and dermatological response which has lasted more than 6 years. © 2000 Elsevier Science Ltd. All rights reserved.