Expression of immunomodulating genes in prostate cancer and benign prostatic tissue.

OBJECTIVE: To investigate the expression of immunomodulating genes in prostate cancer and benign prostatic tissue. STUDY DESIGN: We investigated by quantitative real-time polymerase chain reaction the expression of indoleamine 2,3-dioxygenase, arginase 1, arginase 2, inducible form of nitric oxide synthase, cyclooxygenase 2 (COX-2), programmed death ligand 1 and interleukin 10 in 36 matched pairs of samples from prostate cancer and benign prostatic tissue. RESULTS: Among the genes analyzed, arginase 2 and COX-2 showed statistically significant up-regulation and down-regulation, respectively, in malignant compared to benign prostate tissue. In addition, arginase 1 was more often present in cancer than benign samples. No significant modulation was detected for the other genes under investigation. CONCLUSION: Our data suggest that arginine metabolism may be involved in prostate cancer immune response evasion, whereas COX-2 may play a role in pathogenesis. We provide a snapshot of immunosuppressive gene expression at the transcriptional level in the prostate tumor microenvironment.

Chemokine expression in melanoma metastases associated with CD8+ T-cell recruitment.

Despite the frequent detection of circulating tumor antigen-specific T cells, either spontaneously or following active immunization or adoptive transfer, immune-mediated cancer regression occurs only in the minority of patients. One theoretical rate-limiting step is whether effector T cells successfully migrate into metastatic tumor sites. Affymetrix gene expression profiling done on a series of metastatic melanoma biopsies revealed a major segregation of samples based on the presence or absence of T-cell-associated transcripts. The presence of lymphocytes correlated with the expression of defined chemokine genes. A subset of six chemokines (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10) was confirmed by protein array and/or quantitative reverse transcription-PCR to be preferentially expressed in tumors that contained T cells. Corresponding chemokine receptors were found to be up-regulated on human CD8(+) effector T cells, and transwell migration assays confirmed the ability of each of these chemokines to promote migration of CD8(+) effector cells in vitro. Screening by chemokine protein array identified a subset of melanoma cell lines that produced a similar broad array of chemokines. These melanoma cells more effectively recruited human CD8(+) effector T cells when implanted as xenografts in nonobese diabetic/severe combined immunodeficient mice in vivo. Chemokine blockade with specific antibodies inhibited migration of CD8(+) T cells. Our results suggest that lack of critical chemokines in a subset of melanoma metastases may limit the migration of activated T cells, which in turn could limit the effectiveness of antitumor immunity.

The HLA-A2-restricted PSMA peptide LLHETDSAV is poorly immunogenic in patients with metastatic prostate cancer.

BACKGROUND: Increasing evidence suggests that prostate cancer is visible to the immune system and is potentially responsive to immunotherapeutic interventions. Previous work has identified prostate-specific membrane antigen (PSMA) as a potential antigen for T-cell recognition, and specific PSMA epitopes presented by HLA-A2 have been described. One vaccination strategy that is being pursued in the clinic utilizes peptide-pulsed peripheral blood mononuclear cells (PBMC) + IL-12. METHODS: HLA-A2(+) patients with castrate-resistant prostate cancer and normal organ function were considered. Vaccines were prepared using autologous PBMC loaded with a PSMA peptide previously reported to be immunogenic (LLHETDSAV) administered subcutaneously every 3 weeks. T-cell responses and tumor activity were assessed. RESULTS: Although the vaccine was well tolerated, no clinical responses were observed in 12 enrolled patients and no immune responses were detected based on an ex vivo IFN-gamma ELISPOT assay. To examine immunogenicity of this PSMA peptide in more detail, an in vitro priming assay was utilized with normal donor PBMC, and no detectable reactivity was observed. CONCLUSIONS: Our results suggest that the PSMA peptide LLHETDSAV is poorly immunogenic in humans and that alternative prostate cancer antigens should be pursued.

Prognostic significance of tumor iNOS and COX-2 in stage III malignant cutaneous melanoma.

PURPOSE: New prognostic markers are needed for malignant melanoma. Inducible nitric oxide synthase (iNOS) and cyclooxygenase type 2 (COX-2) have been described to correlate with progression of melanoma. Moreover, activating mutations in BRAF/NRAS oncogenes are often detected in melanoma. The BRAF/NRAS mutation status and expression of COX-2 and iNOS were examined to compare their prognostic value for overall survival (OS) in stage III malignant cutaneous melanoma. EXPERIMENTAL DESIGN: The expression of iNOS and COX-2 in metastatic lymph nodes from 21 rapidly progressing (OS from date of diagnosis of stage III disease < or =14 months) and 17 slowly progressing (OS > or =60 months) stage III cutaneous melanoma patients was examined by immunohistochemistry. The presence of BRAF/NRAS mutations was analyzed using direct DNA sequencing. Chi2 exact trend test and logistic regression analysis were used for statistical analysis. RESULTS: Both iNOS (P = 0.002) and COX-2 (P = 0.048) alone significantly predicted OS. The BRAF/NRAS mutation status did not significantly differ between patient groups, although iNOS significantly (P = 0.013) correlated with BRAF mutation frequency. Furthermore, the odds ratio (OR) with respect to OS of iNOS (OR = 10.4) was higher than that of COX-2 (OR = 5.6) and was stable in the multivariate analysis of OS together with disease stage IIIB/C, ulceration, number of metastatic lymph nodes, and Breslow tumor thickness. CONCLUSION: Our data show that iNOS is an independent and stronger prognostic factor for OS in stage III malignant cutaneous melanoma than COX-2.

Diagnosis and treatment of mycoplasma-contaminated cell cultures.

Mycoplasma contamination is a serious and frequent problem in the culture laboratory. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis rests on the detection of adenosine phosphorylase secretion by infected cell lines. This appendix describes how to test for mycoplasma contamination, and also presents methods for antibiotic treatment of infected cultures.

The CD16- CD56(bright) NK cell subset is resistant to reactive oxygen species produced by activated granulocytes and has higher antioxidative capacity than the CD16+ CD56(dim) subset.

Human NK cells can be divided into CD56(dim) and CD56(bright) subsets. These two types of NK cells respond to different types of stimuli, with CD56(dim) NK cells having direct cytotoxic ability and CD56(bright) NK cells having mainly an immunoregulatory function. We show that the CD16+ CD56(dim) NK subset is characterized by sensitivity to cell death induced by activated granulocytes. We identified hydrogen peroxide (H2O2) as the major effector molecule responsible for the cytotoxic effect of granulocytes on CD56(dim) NK cells, because the ability of granulocytes to kill CD56(dim) NK cells was completely abrogated in the presence of the hydrogen peroxide scavenger catalase. When exposing NK cells to H2O2, CD56(dim) cells showed rapid mitochondrial depolarization and down-regulation of activating NKRs, eventually resulting in cell death, whereas CD56(bright) cells remained unaffected. The difference in sensitivity to H2O2 was mirrored by a difference in intracellular oxidation levels between CD56(dim) and CD56(bright) NK cells, and cell lysates from the latter subset possessed a greater ability to block H2O2-mediated oxidation. Our data may explain the preferential accumulation of CD56(bright) NK cells often seen in environments rich in reactive oxygen species, such as at sites of chronic inflammation and in tumors.

Immune resistance orchestrated by the tumor microenvironment.

It is now little disputed that most if not all cancer cells express antigens that can be recognized by specific CD8(+) T lymphocytes. However, a central question in the field of anti-tumor immunity is why such antigen-expressing tumors are not spontaneously eliminated by the immune system. While in some cases, this lack of rejection may be due to immunologic ignorance, induction of anti-tumor T-cell responses in many patients has been detected in the peripheral blood, either spontaneously or in response to vaccination, without accompanying tumor rejection. These observations argue for the importance of barriers downstream from initial T-cell priming that need to be addressed to translate immune responses into clinical tumor regression. Recent data suggest that the proper trafficking of effector T cells into the tumor microenvironment may not always occur. T cells that do effectively home to tumor metastases are often found to be dysfunctional, pointing toward immunosuppressive mechanisms in the tumor microenvironment. T-cell anergy due to insufficient B7 costimulation, extrinsic suppression by regulatory cell populations, inhibition by ligands such as programmed death ligand-1, metabolic dysregulation by enzymes such as indoleamine-2,3-dioxygenase, and the action of soluble inhibitory factors such as transforming growth factor-beta have all been clearly implicated in generating this suppressive microenvironment. Identification of these downstream processes points to new therapeutic targets that should be manipulated to facilitate the effector phase of anti-tumor immune responses in concert with vaccination or T-cell adoptive transfer.

Induction of cytotoxic granules in human memory CD8+ T cell subsets requires cell cycle progression.

Memory CD8(+) T cell responses are thought to be more effective as a result of both a higher frequency of Ag-specific clones and more rapid execution of effector functions such as granule-mediated lysis. Murine models have indicated that memory CD8(+) T cells exhibit constitutive expression of perforin and can lyse targets directly ex vivo. However, the regulated expression of cytotoxic granules in human memory CD8(+) T cell subsets has been underexplored. Using intracellular flow cytometry, we observed that only a minor fraction of CD45RA(-)CD8(+) T cells, or of CD8(+) T cells reactive to EBV-HLA2 tetramer, expressed intracellular granzyme B (GrB). Induction of GrB-containing cytotoxic granules in both CD45RA(+) and CD45RA(-) cells was achieved by stimulation with anti-CD3/anti-CD28 mAb-coated beads, required at least 3 days, occurred after several rounds of cell division, and required cell cycle progression. The strongest GrB induction was seen in the CCR7(+) subpopulations, with poorest proliferation being observed in the CD45RA(-)CCR7(-) effector-memory pool. Our results indicate that, as with naive T cells, induction of cytotoxic granules in human Ag-experienced CD8(+) T cells requires time and cell division, arguing that the main numerical advantage of a memory T cell pool is a larger frequency of CTL precursors. The fact that granule induction can be achieved through TCR and CD28 ligation has implications for restoring lytic effector function in the context of antitumor immunity.

Immune suppression in the tumor microenvironment.

The identification of tumor-expressed antigens that can be recognized by specific T lymphocytes has made it possible both to study the properties of T cells participating in anti-tumor immune responses in patients and also to develop antigen-specific immunotherapies as a treatment modality. Interestingly, moves toward intervention have proceeded at a faster pace than have investigations toward understanding. In melanoma in particular, many clinical trials of active immunization have been performed, and many of these have shown increases in tumor antigen-specific T cells circulating in the blood. However, clinical responses have been infrequent, arguing that mechanisms of resistance downstream from initial T cell priming may be dominant in many cases. In fact, may patients show spontaneous generation of immune effector cells and/or antibodies, implying that the priming phase has occurred already in such individuals even without vaccination. Recent attention has turned toward mechanisms of immune evasion at the effector phase of the anti-tumor immune response, predominantly within the tumor microenvironment. Evidence is accumulating that T cell-intrinsic hyporesponsiveness or anergy, extrinsic suppression by regulatory cell populations, inhibitory ligands such as PD-L1, soluble factors such as TGF-beta, and the activity of nutrient-catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO), may contribute to immune escape in different settings. Murine preclinical models have shown that interfering with each of these processes can translate into T cell-mediated tumor control. Clinical studies to estimate the frequency of specific immune evasion mechanisms in individual patients, to correlate specific events with clinical outcome, and to develop strategies to counter resistance mechanisms should receive a high priority.

A randomized phase II trial of interleukin-2 in combination with four different doses of bryostatin-1 in patients with renal cell carcinoma.

PURPOSE: Bryostatin-1 is a PKC modulator with direct anti-tumor activity and immunomodulatory properties. We combined different doses of Bryostatin-1 with IL-2 to determine effects on clinical response rate and T cell phenotype in patients with advanced kidney cancer. EXPERIMENTAL DESIGN: IL-2 naïve patients were given 11 x 10(6) IU subcutaneously of IL-2 on days 1-4, 8-11, and 15-18 of every 28-day cycle. Twenty four patients were randomized to treatment cohorts of 5, 15 or 25 mcg/m2 of Bryostatin-1 on days 1, 8 and 15, starting in the second cycle. An additional nine, non-randomized patients were given 35 mcg/m2. Lymphocytes were analyzed for number, activation status, and production of IL-2, IL-4 and IFN-gamma. Response evaluation was performed every 3 cycles. RESULTS: Common grade 3 toxicities included fatigue (5), nausea/vomiting (5), myopathy (3), dyspnea (3), and syncope (3). Four patients, in the two highest dose cohorts, demonstrated evidence of tumor shrinkage, although there was only 1 objective PR. The median time to progression was 104 days (95% CI 88-120) and the median survival was 452 days (95% CI = 424-480). There was no significant boosting effect of Bryostatin-1 on lymphocytes. CONCLUSIONS: The addition of Bryostatin-1 to IL-2 was well tolerated, but the overall response rate was low (3.2%), indicating that further studies with this combination are not warranted.

Tumor progression despite massive influx of activated CD8(+) T cells in a patient with malignant melanoma ascites.

Although melanoma tumors usually express antigens that can be recognized by T cells, immune-mediated tumor rejection is rare. In many cases this is despite the presence of high frequencies of circulating tumor antigen-specific T cells, suggesting that tumor resistance downstream from T cell priming represents a critical barrier. Analyzing T cells directly from the melanoma tumor microenvironment, as well as the nature of the microenvironment itself, is central for understanding the key downstream mechanisms of tumor escape. In the current report we have studied tumor-associated lymphocytes from a patient with metastatic melanoma and large volume malignant ascites. The ascites fluid showed abundant tumor cells that expressed common melanoma antigens and retained expression of class I MHC and antigen processing machinery. The ascites fluid contained the chemokines CCL10, CCL15, and CCL18 which was associated with a large influx of activated T cells, including CD8(+) T cells recognizing HLA-A2 tetramer complexes with peptides from Melan-A and NA17-A. However, several functional defects of these tumor antigen-specific T cells were seen, including poor production of IFN-gamma in response to peptide-pulsed APC or autologous tumor cells, and lack of expression of perforin. Although these defects were T cell intrinsic, we also observed abundant CD4(+)CD25(+)FoxP3(+) T cells, as well as transcripts for FoxP3, IL-10, PD-L1/B7-H1, and indoleamine-2,3-dioxygenase (IDO). Our observations suggest that, despite recruitment of large numbers of activated CD8(+) T cells into the tumor microenvironment, T cell hyporesponsiveness and additional negative regulatory mechanisms can limit the effector phase of the anti-tumor immune response.

Diagnosis and treatment of Mycoplasma-contaminated cell cultures.

Mycoplasma contamination is a serious and frequent problem in the culture laboratory. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis rests on the detection of adenosine phosphorylase secretion by infected cell lines. This appendix describes how to test for mycoplasma contamination, and also presents methods for antibiotic treatment of infected cultures.

Dose-ranging pharmacodynamic study of tipifarnib (R115777) in patients with relapsed and refractory hematologic malignancies.

PURPOSE: Tipifarnib, an orally bioavailable inhibitor of farnesyl transferase, has activity in hematologic malignancies, but the dose required to achieve the proposed biologic end point, inhibition of farnesylation, is unknown. PATIENTS AND METHODS: The impact on post-translational farnesylation was assessed in 42 patients with refractory hematologic malignancies and bone marrow involvement. Tipifarnib was taken orally for 21 days of a 28-day cycle. For cycle 1, patients were randomly assigned to one of four dose levels: 100 mg bid, 200 mg bid, 300 mg bid, and 600 mg bid. In cycle 1, peripheral blood and bone marrow mononuclear cells were analyzed for inhibition of HDJ2 prenylation by Western blot analysis at baseline and on day 21. RESULTS: Twenty-three patients were assessable for analysis of HDJ2 prenylation before and after therapy. Inhibition of farnesylation was noted at all dose levels, although the highest level of inhibition was noted at the 300-mg-bid dose. The inhibition of farnesylation in the peripheral blood correlated with the inhibition in the bone marrow (r = 0.62). Of the 26 patients assessable for clinical activity after cycle 1, three patients had a significant decrease in total blasts count (acute myeloid leukemia in two patients, and chronic myelogenous leukemia in one patient). The inhibition of farnesylation was greater in the three responders than the nonresponders (P = .03). CONCLUSION: Farnesylation as measured by HDJ2 analysis was inhibited at all dose levels administered. Clinical activity may correlate with the degree of farnesylation inhibition, rather than dose of tipifarnib, and escalation beyond 300 mg bid might not result in additional clinical activity.

Disparate functions of immature and mature human myeloid dendritic cells: implications for dendritic cell-based vaccines.

Although antigen-loaded dendritic cells (DC) are being investigated as antitumor vaccines, which DC differentiation state is most effective is not clear. Three DC functions that may be critical for immunization potential are expression of CD80/86, cytokine production following CD40 engagement, and migration to chemokine receptor 7-binding chemokines. We therefore examined highly purified human monocyte-derived immature and mature DC for these properties from normal donors and cancer patients. Although high expression of CD80/86 and migration to 6Ckine + macrophage-inflammatory protein-3beta were properties of mature DC, cytokine production following CD40 ligation was superior by immature DC. Loss of cytokine secretion occurred with multiple maturation conditions, was not apparently reversible, and was also seen with lipopolysaccharide stimulation in correlation with down-regulated Toll-like receptor expression. Our results suggest that the functions thought to contribute to optimal T cell priming are not coexpressed by the same DC population and that immature and mature DC likely possess distinct CD40-mediated signaling events.

Immunization with Melan-A peptide-pulsed peripheral blood mononuclear cells plus recombinant human interleukin-12 induces clinical activity and T-cell responses in advanced melanoma.

PURPOSE: Preclinical studies showed that immunization with peripheral blood mononuclear cells (PBMC) loaded with tumor antigen peptides plus interleukin-12 (IL-12) induced CD8+ T-cell responses and tumor rejection. We recently determined that recombinant human (rh) IL-12 at 30 to 100 ng/kg is effective as a vaccine adjuvant in patients. A phase II study of immunization with Melan-A peptide-pulsed PBMC + rhIL-12 was conducted in 20 patients with advanced melanoma. PATIENTS AND METHODS: Patients were HLA-A2-positive and had documented Melan-A expression. Immunization was performed every 3 weeks with clinical re-evaluation every three cycles. Immune responses were measured by ELISpot assay before and after treatment and through the first three cycles, and were correlated with clinical outcome. RESULTS: Most patients had received prior therapy and had visceral metastases. Nonetheless, two patients achieved a complete response, five patients achieved a minor or mixed response, and four patients had stable disease. The median survival was 12.25 months for all patients and was not yet reached for those with a normal lactate dehydrogenase. There were no grade 3 or 4 toxicities. Measurement of specific CD8+ T-cell responses by direct ex vivo ELISpot revealed a significant increase in interferon gamma-producing T cells against Melan-A (P =.015) after vaccination, but not against an Epstein-Barr virus control peptide (P =.86). There was a correlation between the magnitude of the increase in Melan-A-specific cells and clinical response (P =.046). CONCLUSION: This immunization approach may be more straightforward than dendritic cell strategies and seems to have clinical activity that can be correlated to a biologic end point.

CTLA-4 engagement regulates NF-kappaB activation in vivo.

CTLA-4 engagement inhibits TCR-dependent functions and CTLA-4(-/-) mice develop a lymphoproliferative disorder leading to early lethality. In vitro, ligation of CTLA-4 reduces TCR-mediated activation of NF-kappaB, a transcription factor implicated in promoting T cell survival and cytokine production. However, whether NF-kappaB inhibition downstream of CTLA-4 is necessary for down-regulation of T cell responses is not known. We hypothesized that signaling pathways that are antagonized when CTLA-4 is engaged should be augmented when CTLA-4 is absent and found thatspontaneous NF-kappaB activity was increased in T cells from CTLA-4(-/-) mice. To determine the importance of NF-kappaB inhibition upon CTLA-4 engagement in vivo, CTLA-4(-/-) mice were interbred with mice expressing a transdominant IkappaBalpha mutant under the control of the Lck promoter. The resulting mice had reduced spontaneous NF-kappaB activity in T cells,delayed mortality, and reduced leukocytic accumulation in spleen, lymph nodes, and exocrine pancreas as compared with CTLA-4(-/-) littermates. However, impaired NF-kappaB activation in T cells did not prevent the up-regulation of activation markers on T cells or the acquisition of effector cytokine production. Thus, impaired NF-kappaB activity in T cells prevents specific aspects of the CTLA-4(-/-) phenotype, suggesting that inhibition of NF-kappaB activation is one of the key biochemical events regulated by CTLA-4 ligation in vivo.

TCR-independent CD30 signaling selectively induces IL-13 production via a TNF receptor-associated factor/p38 mitogen-activated protein kinase-dependent mechanism.

Initiation of T lymphocyte responses to most Ags requires concurrent stimulation through the TCR and costimulatory receptors such as CD28. Following initial activation, secondary receptors are up-regulated that can costimulate T cells in concert with TCR engagement. One such receptor is the TNFR family member CD30. In this study, we report that unlike CD28, ligation of CD30 on normal effector T cells induces IL-13 production in the absence of concurrent TCR engagement. TCR-independent CD30-mediated IL-13 release correlated with activation of c-Jun N-terminal kinase, p38 mitogen-activated protein kinase (MAPK), and NF-kappaB, and was completely inhibited by the expression of a TNFR-associated factor 2 (TRAF2) dominant-negative transgene (TRAF2.DN-Tg), but not by that of an I-kappaBalpha dominant-negative transgene. In parallel, expression of the TRAF2.DN-Tg selectively prevented the induction of c-Jun N-terminal kinase and p38 MAPK, but not that of NF-kappaB. Furthermore, IL-13 production was reduced in a dose-dependent manner by the p38 MAPK inhibitor SB203580. Together, these results suggest that TCR-independent CD30-mediated production of IL-13 is triggered by association of CD30 with TRAF family members and subsequent activation of p38 MAPK. Inasmuch as IL-13 can promote airway inflammation and cancer progression, production of IL-13 in a TCR-independent manner has important pathological implications in vivo.