Helena Harlin

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.

Immune resistance orchestrated by the tumor microenvironment

Summary:  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‐β 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.

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.

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-γ 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.

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-κB, 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-κBα 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-κB. 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.

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.

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.

Clinical responses following nonmyeloablative allogeneic stem cell transplantation for renal cell carcinoma are associated with expansion of CD8+ IFN-γ-producing T cells

Summary:Nonmyeloablative allogeneic stem cell transplantation (NST) is thought to be an immunologic therapy in which donor T cells mediate a graft-versus-tumor effect. We recently reported the clinical outcome of a phase II trial of NST in metastatic renal cell carcinoma (RCC). However, the immune response correlates of clinical activity remain unknown. We now describe the analysis of T-cell subsets and T-cell cytokine-producing potential for those patients evaluable for immune monitoring. The incidence of graft-versus-host disease (GVHD) correlated with clinical outcome, with all responders exhibiting chronic GVHD. Following initial tapering of immunosuppression, an increase in the total numbers of CD8+ T cells but not CD4+ T cells was observed among responders compared to nonresponders. In addition, a greater ratio of CD8+ to CD4+ T cells producing IFN-γ and IL-2 was seen in clinical responders at the time when clinical responses were first detected (day 180 after transplantation). Our results support the hypothesis that the antitumor effects of NST may be mediated by IFN-γ-producing CD8+ T cells, and indicate that isolation of putative tumor antigen-specific T cells, ideally, should be pursued around day +180.

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‐3β 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.

CTLA‐4 engagement regulates NF‐κB 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‐κB, a transcription factor implicated in promoting T cell survival and cytokine production. However, whether NF‐κB 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‐κB activity was increased in T cells from CTLA‐4–/– mice. To determine the importance of NF‐κB inhibition upon CTLA‐4 engagement in vivo, CTLA‐4–/– mice were interbred with mice expressing a transdominant IκBα mutant under the control of the Lck promoter. The resulting mice had reduced spontaneous NF‐κB 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‐κB 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‐κB activity in T cells prevents specific aspects of the CTLA‐4–/– phenotype, suggesting that inhibition of NF‐κB activation is one of the key biochemical events regulated by CTLA‐4 ligation in vivo.