A treatise on endothelial biology and exosomes: homage to Theresa Maria Listowska Whiteside.

Exosomes are the current primary research focus of Dr. Theresa L. Whiteside. They are key mediators of intercellular communication in the head and neck, as well as other sites. Their effects in the tumor microenvironment are manifold and include suppression of immunity, promotion of angiogenesis, enabling of metastasis, as well as reprogramming of fibroblasts and mesenchymal stromal cells. The aim of this communication is to summarize Dr. Whiteside's contribution to the field of exosome research and details the interactions of exosomes with endothelial cells leading to recent findings on how to target endothelial cells using exosomes as a therapeutic approach.

Efficacy of adoptive therapy with tumor-infiltrating lymphocytes and recombinant interleukin-2 in advanced cutaneous melanoma: a systematic review and meta-analysis.

Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) has been tested in advanced melanoma patients at various centers. We conducted a systematic review and meta-analysis to assess its efficacy on previously treated advanced metastatic cutaneous melanoma. The PubMed electronic database was searched from inception to 17 December 2018 to identify studies administering TIL-ACT and recombinant interleukin-2 (IL-2) following non-myeloablative chemotherapy in previously treated metastatic melanoma patients. Objective response rate (ORR) was the primary end point. Secondary end points were complete response rate (CRR), overall survival (OS), duration of response (DOR) and toxicity. Pooled estimates were derived from fixed or random effect models, depending on the amount of heterogeneity detected. Analysis was carried out separately for high dose (HD) and low dose (LD) IL-2. Sensitivity analyses were carried out. Among 1211 records screened, 13 studies (published 1988 - 2016) were eligible for meta-analysis. Among 410 heavily pretreated patients (some with brain metastasis), 332 received HD-IL-2 and 78 LD-IL-2. The pooled overall ORR estimate was 41% [95% confidence interval (CI) 35% to 48%], and the overall CRR was 12% (95% CI 7% to 16%). For the HD-IL-2 group, the ORR was 43% (95% CI 36% to 50%), while for the LD-IL-2 it was 35% (95% CI 25% to 45%). Corresponding pooled estimates for CRR were 14% (95% CI 7% to 20%) and 7% (95% CI 1% to 12%). The majority of HD-IL-2 complete responders (27/28) remained in remission during the extent of follow-up after CR (median 40 months). Sensitivity analyses yielded similar results. Higher number of infused cells was associated with a favorable response. The ORR for HD-IL-2 compared favorably with the nivolumab/ipilimumab combination following anti-PD-1 failure. TIL-ACT therapy, especially when combined with HD-IL-2, achieves durable clinical benefit and warrants further investigation. We discuss the current position of TIL-ACT in the therapy of advanced melanoma, particularly in the era of immune checkpoint blockade therapy, and review future opportunities for improvement of this approach.

First-in-human phase I study of copanlisib (BAY 80-6946), an intravenous pan-class I phosphatidylinositol 3-kinase inhibitor, in patients with advanced solid tumors and non-Hodgkin's lymphomas.

BACKGROUND: To evaluate the safety, tolerability, pharmacokinetics, and maximum tolerated dose (MTD) of copanlisib, a phosphatidylinositol 3-kinase inhibitor, in patients with advanced solid tumors or non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: Phase I dose-escalation study including patients with advanced solid tumors or NHL, and a cohort of patients with type 2 diabetes mellitus. Patients received three weekly intravenous infusions of copanlisib per 28-day cycle over the dose range 0.1-1.2 mg/kg. Plasma copanlisib levels were analyzed for pharmacokinetics. Biomarker analysis included PIK3CA, KRAS, BRAF, and PTEN mutational status and PTEN immunohistochemistry. Whole-body [(18)F]-fluorodeoxyglucose positron emission tomography ((18)FDG-PET) was carried out at baseline and following the first dose to assess early pharmacodynamic effects. Plasma glucose and insulin levels were evaluated serially. RESULTS: Fifty-seven patients received treatment. The MTD was 0.8 mg/kg copanlisib. The most frequent treatment-related adverse events were nausea and transient hyperglycemia. Copanlisib exposure was dose-proportional with no accumulation; peak exposure positively correlated with transient hyperglycemia post-infusion. Sixteen of 20 patients treated at the MTD had reduced (18)FDG-PET uptake; 7 (33%) had a reduction >25%. One patient achieved a complete response (CR; endometrial carcinoma exhibiting both PIK3CA and PTEN mutations and complete PTEN loss) and two had a partial response (PR; both metastatic breast cancer). Among the nine NHL patients, all six with follicular lymphoma (FL) responded (one CR and five PRs) and one patient with diffuse large B-cell lymphoma had a PR by investigator assessment; two patients with FL who achieved CR (per post hoc independent radiologic review) were on treatment >3 years. CONCLUSION: Copanlisib, dosed intermittently on days 1, 8, and 15 of a 28-day cycle, was well tolerated and the MTD was determined to be 0.8 mg/kg. Copanlisib exhibited dose-proportional pharmacokinetics and promising anti-tumor activity, particularly in patients with NHL. CLINICALTRIALSGOV: NCT00962611; https://clinicaltrials.gov/ct2/show/NCT00962611.

Plasma biosignature and brain pathology related to persistent cognitive impairment in late-life depression.

Cognitive impairment is highly prevalent among individuals with late-life depression (LLD) and tends to persist even after successful treatment. The biological mechanisms underlying cognitive impairment in LLD are complex and likely involve abnormalities in multiple pathways, or 'cascades,' reflected in specific biomarkers. Our aim was to evaluate peripheral (blood-based) evidence for biological pathways associated with cognitive impairment in older adults with LLD. To this end, we used a data-driven comprehensive proteomic analysis (multiplex immunoassay including 242 proteins), along with measures of structural brain abnormalities (gray matter atrophy and white matter hyperintensity volume via magnetic resonance imaging), and brain amyloid-ß (Aß) deposition (PiB-positron emission tomography). We analyzed data from 80 older adults with remitted major depression (36 with mild cognitive impairment (LLD+MCI) and 44 with normal cognitive (LLD+NC)) function. LLD+MCI was associated with differential expression of 24 proteins (P<0.05 and q-value <0.30) related mainly to the regulation of immune-inflammatory activity, intracellular signaling, cell survival and protein and lipid homeostasis. Individuals with LLD+MCI also showed greater white matter hyperintensity burden compared with LLD+NC (P=0.015). We observed no differences in gray matter volume or brain Aß deposition between groups. Machine learning analysis showed that a group of three proteins (Apo AI, IL-12 and stem cell factor) yielded accuracy of 81.3%, sensitivity of 75% and specificity of 86.4% in discriminating participants with MCI from those with NC function (with an averaged cross-validation accuracy of 76.3%, sensitivity of 69.4% and specificity of 81.8% with nested cross-validation considering the model selection bias). Cognitive impairment in LLD seems to be related to greater cerebrovascular disease along with abnormalities in immune-inflammatory control, cell survival, intracellular signaling, protein and lipid homeostasis, and clotting processes. These results suggest that individuals with LLD and cognitive impairment may be more vulnerable to accelerated brain aging and shed light on possible mediators of their elevated risk for progression to dementia.

Interleukin 4 promotes the growth of tumor-infiltrating lymphocytes cytotoxic for human autologous melanoma.

Addition of IL-4 (1,000 U/ml) to either high or low concentrations of IL-2 augmented tumor-infiltrating lymphocytes (TIL) growth from human melanoma. Weekly restimulation with irradiated tumor cells in conjunction with IL-4 allowed enhanced growth of TIL. With low-dose IL-2 (10 U/ml) and IL-4, expanded TIL had little cytolytic activity against Daudi or allogeneic tumors. Further, IL-4 augmented the total lytic activity against autologous tumors in most cases. With high-dose IL-2 (1,000 U/ml), IL-4 addition decreased nonspecific killing activity against Daudi or allogeneic melanomas in many cases, and reciprocally augmented cytolytic activity against the autologous melanoma in many cases. This suggests the possible use of IL-4 in cancer therapy, especially in adoptive cellular immunotherapy using TIL or in conjunction with IL-2 administration.

Viral interleukin 10 (IL-10), the human herpes virus 4 cellular IL-10 homologue, induces local anergy to allogeneic and syngeneic tumors.

After the cloning of murine cytokine synthesis inhibitory factor, it was recognized that a homologous open reading frame was encoded within the Epstein-Barr virus (human herpes virus 4). This viral protein has now been termed viral interleukin 10 (vIL-10) to reflect its protein sequence homology to "cellular" IL-10 (cIL-10, either murine or human IL-10). It is now widely accepted that vIL-10 shares many functions with cIL-10, principally, the ability to enhance survival of newly infected B cells and to diminish the production of IFN-gamma and IL-2 during ongoing immune reactions. The immunomodulatory effect of locally secreted vIL-10 and murine IL-10 (mIL-10) was examined in tumor models using CL8-1 (a BL6 melanoma cell line transfected with the H-2Kb class I gene) in syngeneic animals. Although parental BL6 tumor cells grow in immunocompetent syngeneic hosts, CL8-1 are rejected. To achieve local secretion of vIL-10, we generated vIL-10 retroviral vectors. While nontransduced CL8-1 cells (1 x 10(4)) failed to grow when injected intradermally in C57BL/6 mice, CL8-1 cells (1 x 10(4)) transduced with vIL-10 formed palpable tumors and eventually killed 80% of injected animals. Suppression of tumor rejection was also noted when CL8-1 tumors with or without vIL-10 transfection were admixed with syngeneic vIL-10-transfected fibroblasts and inoculated. Since the in vitro proliferation of the tumor was not altered after transduction with the vIL-10 gene and injection of vIL-10-transduced CL8-1 does not affect the rejection of nontransduced CL8-1 inoculated at a distant site, local vIL-10 secretion appears to suppress the process of immune rejection of the target cells in a dose-dependent manner. Similar results were observed for the H-2b MCA105 sarcoma tumor model in allogeneic BALB/c mice (H-2d). Although all animals that received nontransfected MCA105 rapidly rejected these tumors, MCA105 sarcomas transfected with vIL-10 remained palpable for up to 37 d. The local immunosuppressive effect of gene-delivered vIL-10 could be neutralized by anti-human IL-10 monoclonal antibody or could be reversed by the systemic administration of IL-2 or IL-12. In marked contrast, mIL-10 transfection of CL8-1 significantly suppressed tumor growth and frequently led to the rejection of tumor. Similar results were obtained for the murine tumor cell lines MCA102.(ABSTRACT TRUNCATED AT 400 WORDS)

Peptide-pulsed dendritic cells induce antigen-specific CTL-mediated protective tumor immunity.

Cytotoxic T lymphocytes (CTLs) are a critical component of the immune response to tumors. Tumor-derived peptide antigens targeted by CTLs are being defined for several human tumors and are potential immunogens for the induction of specific antitumor immunity. Dendritic cells (DC) are potent antigen-presenting cells (APCs) capable of priming CTL responses in vivo. Here we show that major histocompatibility complex class I-presented peptide antigen pulsed onto dendritic APCs induces protective immunity to lethal challenge by a tumor transfected with the antigen gene. The immunity is antigen specific, requiring expression of the antigen gene by the tumor target, and is eliminated by in vivo depletion of CD8+ T cells. Furthermore, mice that have rejected the transfected tumor are protected from subsequent challenge with the untransfected parent tumor. These results suggest that immunization strategies using antigen-pulsed DC may be useful for inducing tumor-specific immune responses.

Interleukin (IL) 4 differentially regulates monocyte IL-1 family gene expression and synthesis in vitro and in vivo.

Interleukin (IL) 4 is a multifunctional T cell-derived cytokine that inhibits cytokine production and certain effector functions in human monocytes, while enhancing others. We show that IL-4 may contribute to the downregulation and resolution of an inflammatory response by selectively promoting expression of the IL-1 receptor antagonist (IL-1ra) that blocks the action of IL-1. IL-1ra specifically binds to the IL-1 receptor without initiating signal transduction. Peripheral blood monocytes obtained from cancer patients, before and immediately after a regimen of IL-4 immunotherapy, were examined for IL-1ra gene expression. After IL-4 therapy, monocytes from the patients showed a marked increase in IL-1ra mRNA. This selective induction of IL-1ra mRNA in circulating monocytes was reflected by significantly enhanced serum levels of IL-1ra (p < 0.01) during IL-4 therapy, which declined after IL-4 treatment. In vitro analysis of IL-4 regulation of monocytes from normal individuals revealed a dose-dependent induction of IL-1ra mRNA within 2-4 h after stimulation without a concomitant effect on the expression of IL-1 mRNA. Increased IL-1ra mRNA was not due to RNA stabilization, but occurred at the level of transcription. In the presence of LPS, IL-4 not only augmented IL-1ra levels, but markedly inhibited LPS-induced IL-1 mRNA expression. The selective upregulation of IL-1ra by resting or activated monocytes, coupled with inhibition of IL-1 production by activated monocytes, as we demonstrate both in vitro and in vivo, suggests that IL-4 may prove clinically useful as a systemic antiinflammatory agent.

Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines.

Antigen presentation by host dendritic cells (DC) is critical for the initiation of adaptive immune responses. We have previously demonstrated in immunogenic murine tumor models that bone marrow (BM)-derived DC pulsed ex vivo with synthetic tumor-associated peptides, naturally expressed by tumor cells, serve as effective antitumor vaccines, protecting animals against an otherwise lethal tumor challenge (Mayordomo, J.I., T. Zorina, W.J. Storkus, C. Celluzzi, L.D. Falo, C.J. Melief, T. Ildstad, W.M. Kast, A.B. DeLeo, and M.T. Lotze. 1995. Nature Med. 1:1297-1302). However, T cell-defined epitopes have not been identified for most human cancers. To explore the utility of this approach in the treatment of tumors expressing as yet uncharacterized epitopes, syngeneic granulocyte/macrophage colony-stimulating factor-stimulated and BM-derived DC, pulsed with unfractionated acid-eluted tumor peptides (Storkus, W.J., H.J. Zeh III, R.D. Salter, and M.T. Lotze. 1993. J. Immunother. 14:94-103) were used to treat mice bearing spontaneous, established tumors. The adoptive transfer of 5 x 10(5) tumor peptide-pulsed DC dramatically suppressed the growth of weakly immunogenic tumors in day 4 to day 8 established MCA205 (H-2b) and TS/A (H-2d) tumor models, when applied in three biweekly intravenous injections. Using the immunogenic C3 (H-2b) tumor model in B6 mice, tumor peptide-pulsed DC therapy resulted in the erradication of established d14 tumors and long-term survival in 100% of treated animals. The DC-driven antitumor immune response was primarily cell mediated since the transfer of spleen cells, but not sera, from immunized mice efficiently protected sublethally irradiated naive mice against a subsequent tumor challenge. Furthermore, depletion of either CD4+ or CD8+ T cells from tumor-bearing mice before therapy totally suppressed the therapeutic efficacy of DC pulsed with tumor-derived peptides. Costimulation of the host cell-mediated antitumor immunity was critical since inoculation of the chimeric fusion protein CTLA4-Ig virtually abrogated the therapeutic effects of peptide-pulsed DC in vivo. The analysis of the cytokine pattern in the draining lymph nodes and spleens of tumor-bearing mice immunized with DC pulsed with tumor-eluted peptides revealed a marked upregulation of interleukin (IL) 4 and interferon (IFN) gamma production, as compared with mice immunized with DC alone or DC pulsed with irrelevant peptides. DC-induced antitumor effects were completely blocked by coadministration of neutralizing monoclonal antibody directed against T helper cell 1-associated cytokines (such as IL-12, tumor necrosis factor alpha, IFN-gamma), and eventually, but not initially, blocked by anti-mIL-4 mAb. Based on these results, we believe that DC pulsed with acid-eluted peptides derived from autologous tumors represents a novel approach to the treatment of established, weakly immunogenic tumors, and serves as a basis for designing clinical trials in cancer patients.

Mass spectrometric identification of a naturally processed melanoma peptide recognized by CD8+ cytotoxic T lymphocytes.

We and others have previously reported that melanoma-specific, cytotoxic T lymphocytes (CTL) define a minimum of six class I-presented peptide epitopes common to most HLA-A2+ melanomas. Here we show that three of these peptide epitopes are coordinately recognized by a CTL clone obtained by limiting dilution from the peripheral blood of an HLA-A2+ melanoma patient. Tandem mass spectrometry was used to characterize and sequence one of these three naturally processed melanoma peptides. One of the potential forms of the deduced peptide sequence (XXTVXXGVX, X = I or L) matches positions 32-40 of the recently identified melanoma gene MART-1/Melan-A. This peptide (p939; ILTVILGVL) binds to HLA-A2 with an intermediate-to-low affinity and is capable of sensitizing the HLA-A2+ T2 cell line to lysis by CTL lines and clones derived from five different melanoma patients. A relative high frequency of anti-p939-specific effector cells appear to be present in situ in HLA-A2+ melanoma patients, since p939 is also recognized by freshly isolated tumor infiltrating lymphocytes. p939 represents a good candidate for the development of peptide-based immunotherapies for the treatment of patients with melanoma.

Therapy of murine tumors with p53 wild-type and mutant sequence peptide-based vaccines.

The BALB/c Meth A sarcoma carries a p53 missense mutation at codon 234, which occurs in a peptide, termed 234CM, capable of being presented to cytotoxic T lymphocytes (CTL) by H-2Kd molecules (Noguchi, Y., E.C. Richards, Y.-T. Chen, and L.J. Old. 1994. Proc. Natl. Acad. Sci. USA. 91:3171-3175). Immunization of BALB/c mice with bone marrow-derived dendritic cells (DC), generated in the presence of granulocyte macrophage colony-stimulating factor and interleukin 4, and prepulsed with the Meth A p53 mutant peptide, induced CTL that specifically recognized peptide-pulsed P815 cells, as well as Meth A cells naturally expressing this epitope. Immunization with this vaccine also protected naive mice from a subsequent tumor challenge, and it inhibited tumor growth in mice bearing day 7 subcutaneous Meth A tumors. We additionally determined that immunization of BALB/c mice with DC pulsed with the p53 peptide containing the wild-type residue at position 234, 234CW, induced peptide-specific CTL that reacted against several methylcholanthrene-induced BALB/c sarcomas, including CMS4 sarcoma, and rejection of CMS4 sarcoma in vaccination and therapy (day 7) protocols. These results support the efficacy of DC-based, p53-derived peptide vaccines for the immunotherapy of cancer. The translational potential of this strategy is enhanced by previous reports showing that DC can readily be generated from human peripheral blood lymphocytes.

Human intestinal Vdelta1+ lymphocytes recognize tumor cells of epithelial origin.

gammadelta T cells can be grouped into discrete subsets based upon their expression of T cell receptor (TCR) variable (V) region families, their tissue distribution, and their specificity. Vdelta2+ T cells constitute the majority of gammadelta T cells in peripheral blood whereas Vdelta1+T cells reside preferentially in skin epithelium and in the intestine. gammadelta T cells are envisioned as first line host defense mechanisms capable of providing a source of immune effector T cells and immunomodulating cytokines such as interleukin (IL) 4 or interferon (IFN) gamma. We describe here the fine specificity of three distinct gammadelta+ tumor-infiltrating lymphocytes (TIL) obtained from patients with primary or metastatic colorectal cancer, that could be readily expanded in vitro in the presence of IL-1beta and IL-7. Irrespective of donor, these individual gammadelta T cells exhibited a similar pattern of reactivity defined by recognition of autologous and allogeneic colorectal cancer cells, renal cell cancer, pancreatic cancer, and a freshly isolated explant from human intestine as measured by cytolytic T cell responses and by IFN-gamma release. In contrast, tumors of alternate histologies were not lysed, including lung cancer, squamous cell cancer, as well as the natural/lymphocyte-activated killer cell-sensitive hematopoietic cell lines T2, C1R, or Daudi. The cell line K562 was only poorly lysed when compared with colorectal cancer targets. Target cell reactivity mediated by Vdelta1+ T cells was partially blocked with Abs directed against the TCR, the beta2 or beta7 integrin chains, or fibronectin receptor. Marker analysis using flow cytometry revealed that all three gammadelta T cell lines exhibit a similar phenotype. Analysis of the gammadelta TCR junctional suggested exclusive usage of the Vdelta1/Ddelta3/Jdelta1 TCR segments with extensive (< or = 29 bp) N/P region diversity. T cell recognition of target cells did not appear to be a major histocompatibility complex restricted or to be correlated with target cell expression of heat-shock proteins. Based on the ability of some epithelial tumors, including colorectal, pancreatic, and renal cell cancers to effectively cold target inhibit the lysis of colorectal cancer cell lines by these Vdelta1+ T cell lines, we suggest that intestinal Vdelta1+ T cell lines, we suggest that intestinal Vdelta1+ T cells are capable of recognizing cell surface Ag(s) shared by tumors of epithelial origin.

Coordinate NF-kappaB and STAT1 activation promotes development of myeloid type 1 dendritic cells.

NF-kappaB and STAT1 are critically involved in the initiation of the inflammatory cascade. Using semi-automated imaging cytometry and fluorescent antibodies, we screened several factors for their ability to induce nuclear translocation of RelA/NF-kappaB and STAT1 in subsets of monocyte-derived dendritic cells (DC). Detailed kinetics and dose-response studies are presented for IL-1-, LPS-, CD40L-, IFN-gamma- and IFN-alpha-stimulated responses. The results are consistent with the notion that simultaneous activation of both STAT1 and NF-kappaB pathways at the initiation of differentiation culture is required for efficient priming of IL-12 production by DC. Maturation of DC led to characteristic NF-kappaB and STAT1 distribution and response patterns. During the resting stage, DC differentiated under the presence of IFN-gamma showed sustained STAT activation and remained responsive to LPS. By contrast, PGE2-supplemented DC could be characterized by negligible responses to LPS and IFN-gamma and a remarkable NF-kappaB response to CD40L. STAT1 pathway was suppressed in PGE2-supplemented cells. We conclude that the magnitude and temporal kinetics of the nuclear shift of STAT1 and NF-kappaB in myeloid DC are associated with IL-12p70 production and are dependent on the nature of the stimulating factors and the polarization state of cells.

Tumor escape from immune recognition: lethal recurrent melanoma in a patient associated with downregulation of the peptide transporter protein TAP-1 and loss of expression of the immunodominant MART-1/Melan-A antigen.

In the last few years, mutiple protein target antigens for immunorecognition by T cells have been identified on human melanoma. How melanoma lesions escape from functional antigen-specific immune recognition remains poorly understood. We have identified the concomitant loss of the immunodominant T cell-defined MART-1/Melan-A antigen and downregulation of the TAP-1 gene in a recurrent metastatic melanoma that was resected in 1993. This phenotype was not observed for an earlier autologous melanoma lesion resected in 1987. The "antigen loss" could be restored in the variant tumor cell line by simultaneously providing both the MART-1/Melan-A gene (by retroviral transfer) and the TAP-1 gene (by a bioballistic approach) resulting in tumor cell sensitivity to MART-1/Melan-A-specific cytotoxic T lymphocytes. This suggests that tumor escape from immune surveillance may have occurred in vivo as a sequential result of (a) antigen loss, and (b) downregulation of the peptide-transporter protein TAP-1 expression by this patient's tumor over a 6-yr period from 1987 to 1993. These results suggest that the characterization of the T cell response to melanoma in individual patients and definition of the immunologically relevant genetic defects in tumors may be required to select the most effective therapeutic strategies for a given patient.

The dendritic cell and human cancer vaccines.

Over the past ten years, the identification of the critical role that dendritic cells (DCs) play in stimulating a specific immune response has led to their use in cancer and HIV therapy. Interesting responses have been reported but the most effective approach and the duration of these responses are still unclear. The quality of DCs, the means by which tumor antigens are delivered to DCs and the problems associated with monitoring the immune response have made individual studies difficult to compare. Much work is still needed to determine the role that DC-based cancer vaccines will have, the most effective way to deliver DCs to patients and the most relevant antigens to provide to DCs.

FLT3: receptor and ligand. Biology and potential clinical application.

Flt3 ligand (FL) is a recently identified cytokine having a central role in the proliferation, survival and differentiation of early murine and human hematopoietic precursor/stem cells. FL acts synergistically in vitro with a number of other hematopoietic growth factors such as IL-3, IL-6, IL-11, IL-12, KIT Ligand and GM-CSF. Recently, it has been shown the in vivo administration of FL results in a significant alteration of hematopoiesis in murine bone marrow (BM), spleen, peripheral blood, liver and lymph nodes. In addition, treatment with FL resulted in a significant accumulation of functionally active dendritic cells within murine lymphoid tissues. The possible applications of FL in dendritic cell-based immunotherapies are discussed.

Lymphokine-activated killer (LAK) cell phenomenon. IV. Lysis by LAK cell clones of fresh human tumor cells from autologous and multiple allogeneic tumors.

Activated killer cells are generated by the incubation of peripheral blood mononuclear leukocytes (PBL) in the lymphokine interleukin 2 (IL-2). Unseparated populations of these lymphokine-activated killer (LAK) cells lyse a variety of fresh noncultured human tumor targets, but they do not kill normal PBL. This study analyzed the generation and lytic specificity of LAK cell clones. Of 49 (84%) clones isolated by limiting-dilution techniques from a whole population of LAK cells, 41 manifested significant LAK cell activity. LAK cell clones had varied cell surface phenotypes. Clones with high and intermediate LAK cell activity were Leu 2+3-4+7-DR+Tac+ and Leu 2-3+4+7-DR+Tac+, respectively. Single LAK cell clones lysed multiple fresh human tumor targets including autologous sarcoma, 5 allogeneic sarcomas, and a colon cancer in addition to the cultured cell line K562. Autologous PBL were not lysed. Tumor targets were each lysed by multiple LAK cell clones. Sixteen subclones were derived from 5 of these LAK cell clones. These subclones had 99% or greater probability of being derived from a single cell. These subclones also exhibited lysis of multiple tumor targets. These findings suggest the existence of a shared determinant, expressed by multiple human tumors, which is recognized in common by multiple LAK cell clones.

Prospective randomized trial of high-dose interleukin-2 alone or in conjunction with lymphokine-activated killer cells for the treatment of patients with advanced cancer.

BACKGROUND: Treatment using interleukin-2 (IL-2) alone or in conjunction with lymphokine-activated killer (LAK) cells has been shown to mediate disease regression in selected patients with advanced cancer. PURPOSE: This prospective randomized trial was designed to determine whether the administration of LAK cells in conjunction with high-dose IL-2 alters response and survival rates, compared with those for IL-2 alone, in patients with advanced cancer. METHODS: The 181 patients who had metastatic cancer that had failed to respond to standard therapy or who had disease for which no effective therapy existed received treatment with high-dose IL-2 alone or with LAK cells plus IL-2. Both treatment groups were to receive the same dose of IL-2 administered according to the same schedule. IL-2 doses were omitted depending on the tolerance of the patient. Of the 181 patients, 97 had renal cell cancer and 54 had melanoma. RESULTS: Median potential follow-up was 63.2 months. There were 10 complete responses among the 85 assessable patients who received IL-2 plus LAK cells, compared with four among the 79 who received IL-2 alone. There were 14 and 12 partial responses, respectively. Complete response continues in seven patients at 50-66 months. The 36-month actuarial survival with IL-2 plus LAK cells was 31%, compared with 17% with IL-2 alone (two-sided P value [P2] = .089). A trend toward improved survival was seen for patients with melanoma who received IL-2 plus LAK cells, compared with those who received IL-2 alone (24-month survival: 32% versus 15%; 48-month survival: 18% versus 4%; P2 = .064 [corrected]). None of 26 patients with melanoma who received IL-2 alone are alive; five of 28 who received IL-2 plus LAK cells are alive, and three continue in complete response. No difference in survival was seen in patients with renal cell cancer in the two treatment groups. There were six treatment-related deaths (3.3%); three were due to myocardial infarction. Other toxic effects resolved by discontinuation of IL-2. Many toxic effects were related to increased vascular permeability induced by IL-2. CONCLUSIONS: Some patients with metastatic cancer have prolonged remission when they are treated with high-dose IL-2 alone or in conjunction with LAK cells. Our results suggest a trend toward increased survival when IL-2 is given with LAK cells in patients with melanoma, but no trend was observed for patients with renal cell cancer. IMPLICATIONS: As these studies continue, efforts are underway to develop improved immunotherapies using tumor-infiltrating lymphocytes (TIL) and gene-modified TIL.

Separation and functional studies of the human lymphokine-activated killer cell.

Cell separation studies were undertaken in an attempt to purify the lymphokine-activated killer (LAK) precursor cell. Null cells, prepared by the sequential depletion of monocytes, T- and B-lymphocytes from human peripheral blood mononuclear cells, were found to be potent mediators of LAK activity. Such preparations were Leu-11+ but Leu-4- and displayed high levels of natural killer activity. Incubation of these cells with recombinant interleukin 2 (IL-2) for periods in excess of 24 h induced LAK lysis of fresh tumor targets which were resistant to lysis by unstimulated null effectors. In contrast, lymphocytes which formed high affinity rosettes with sheep RBC (E+ lymphocytes) were poor mediators of both natural killer and LAK activity. Interleukin 2 stimulated null cells, retained a Leu-11+, Leu-4- phenotype, and expressed only low levels of receptors for IL-2 and transferrin. An increase in the number of binding sites, on null cells but not on T-cells, for Vicia villosa lectin with IL-2 stimulation was noted. Following IL-2 stimulation, null and T-cells were able to conjugate to K562 and fresh tumor but not to autologous lymphoblast targets.

Immunohistochemical correlates of response to recombinant interleukin-2-based immunotherapy in humans.

We have evaluated immunohistochemical characteristics of tumors and the infiltrating cells in patients treated with various immunotherapy regimens. Forty-eight patients with advanced malignancies were treated with high dose i.v. recombinant interleukin-2 alone or in combination with cyclophosphamide, recombinant tumor necrosis factor, recombinant interferon-alpha, antimelanoma antibody 9.2.27, adoptively transferred tumor infiltrating lymphocytes, or lymphokine-activated killer cells. Thirty-four patients with metastatic melanoma and two patients with breast carcinoma underwent excision of one or more s.c. metastases either before, during, or after treatment. Twelve patients with metastatic renal cell carcinoma underwent pretreatment nephrectomy and these tumors were also studied. Tumor cells were evaluated for class I (HLA-A,B,C) and II (HLA-DR) antigen expression and the mononuclear infiltrate was characterized using an avidin-biotin immunoperoxidase technique. All melanomas were class I antigen positive. Fifty-three % of biopsied metastatic melanoma lesions, 58% of primary renal cell carcinomas, and neither of the two breast carcinomas expressed class II antigen prior to therapy. The pretreatment expression of class II antigens by a tumor was not predictive of a clinical response to recombinant interleukin 2-based therapy. After treatment, however, seven of seven biopsied regressing individual metastases intensely expressed DR antigen on over fifty percent of the cells while only three of ten nonresponding lesions did so. Regressing lesions were permeated with macrophages and both CD4 and CD8 T-cell subsets. There were no CD1 or NKH-1 positive infiltrating cells detected in any lesion. The response to recombinant interleukin 2-based immunotherapy is associated with T-cell as well as macrophage infiltration. DR antigen expression by tumor cells and T-cell infiltrate appear in individual lesions to be associated with this response.