Lymphocytes In Melanoma Patients Research Articles

CD4 Phenotypes Are Associated with Reduced Expansion of Tumor-Infiltrating Lymphocytes in Melanoma Patients Treated with Adoptive Cell Therapy.

Tumor-infiltrating lymphocyte (TIL) adoptive cell therapy is effective in treating malignant melanoma, but its success relies on the adequate ex vivo expansion of TIL. To assess correlates of TIL expansion, CD4+ and CD8+ TIL were analyzed by RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing of acetylated histone 3. Patients were grouped into "TIL high" and "TIL low" based on division at the median number of TIL infused. Greater numbers of TIL infused correlated with longer overall survival, and increased frequencies of CD4+ cells infused were negatively correlated with the number of TIL infused. RNA-seq analysis of CD4+ TIL showed increases in Th2/Th17/regulatory T cell-related transcripts and pathways in the TIL-low group. Analysis of a public single-cell RNA-seq dataset validated findings that increased frequencies of CD4+ cells were negatively correlated with the number of TIL infused. TIL-low patients had significantly increased frequencies of CD4+ cells expressing ETS2 and OSM and trended toward increased expression of TNFRSF18.

Activation of lymphocytes in vitro for immunotherapy of patients with melanoma

This article describes a method for activating lymphocytes isolated from the peripheral blood of melanoma patients and cultured in a medium supplemented with IL‑2 and IL‑15. It was shown that in these terms, lymphocytes have an increased proliferative and activation potential. The combination of cytokines has a positive effect on cytotoxicity, viability and the expression of activation markers (CD38, CD69, CD25, HLA-DR and NKG2D) on NK- and T-lymphocyte, and may be recommended for the culture of lymphocytes in melanoma patients for the purpose of adoptive immunotherapy.

Human pDCs Are Superior to cDC2s in Attracting Cytolytic Lymphocytes in Melanoma Patients Receiving DC Vaccination

Plasmacytoid dendritic cells (pDCs) and type 2 conventional dendritic cells (cDC2s) are currently under evaluation for use in cancer vaccines. Although both DC subsets can activate adaptive and innate lymphocytes, their capacity to recruit such cells is rarely considered. Here, we show that pDCs and cDC2s display a striking difference in chemokine secretion, which correlates with the recruitment of distinct types of immune effector cells. Activated pDCs express high levels of CXCR3 ligands and attract more CD8+ Tcells, CD56+ Tcells, and γδ Tcells invitro, compared to cDC2s. Skin from melanoma patients shows an influx of immune effector cells following intradermal vaccination with pDCs or cDC2s, with pDCs inducing the strongest influx of lymphocytes known to possess cytolytic activity. These findings suggest that combining both DC subsets could unite the preferred chemoattractive properties of pDCs with the superior Tcell priming properties of cDC2s to ultimately enhance vaccine efficacy.

A Brief Communication on Circulating PD-1-positive T-Regulatory Lymphocytes in Melanoma Patients Undergoing Adjuvant Immunotherapy With Nivolumab.

Upregulation of T-regulatory lymphocytes (Tregs) is one of numerous immune escape mechanisms of malignancies. In the present pilot study we aimed to study the effect of adjuvant nivolumab during the initiation of treatment on circulating Tregs subpopulations in patients with stage III melanoma. We subsequently recruited patients with stage III melanoma who had the indication for adjuvant anti-programmed death 1 (PD-1) treatment with nivolumab. Blood collections were performed before the initiation of nivolumab and before every 2-week therapy cycle. Flow cytometry was performed for the determination of circulating CD4CD25highCD127PD-1(PD-1Tregs) and CD4CD25highCD127CTLA-4 (CTLA-4Tregs) Treg populations. Circulating PD-1Tregs [18.1% (range, 2.9%-41.7%) vs. 4.2% (0.4%-9.8%), P=0.0001] significantly decreased after the first cycle of immunotherapy and maintained decreased during a 3-month course of treatment. By contrast, CTLA-4Tregs significantly increased after the first nivolumab dose when compared with CTLA-4Tregs before the second treatment [0.75 (0-45.5) vs. 2.1 (0.1-90.8), P=0.0002]. Blood levels of PD-1Tregs and CTLA-4Tregs remained more or less decreased and increased during a 3-month therapy with nivolumab, respectively. Data of PD-1Tregs as well as CTLA-4Tregs was not significantly associated with frequencies of immune-related adverse events (P<0.05). In conclusion, we have demonstrated that circulating PD-1Tregs of melanoma patients in stage III rapidly and continuously decline after the initiation of adjuvant treatment with the PD-1 blocking antibody nivolumab. By contrast, this decline is paralleled with an increase of CTLA-4Tregs. The expression of PD-1 and CTLA-4 on Tregs might be a potential biomarker for the efficacy of immune checkpoint blockade in melanoma.

MicroRNA Expression in Sentinel Nodes from Progressing Melanoma Patients Identifies Networks Associated with Dysfunctional Immune Response.

Sentinel node biopsy (SNB) is a main staging biomarker in melanoma and is the first lymph node to drain the tumor, thus representing the immunological site where anti-tumor immune dysfunction is established and where potential prognostic immune markers can be identified. Here we analyzed microRNA (miR) profiles in archival tumor-positive SNBs derived from melanoma patients with different outcomes and performed an integrated analysis of transcriptional data to identify deregulated immune signaling networks. Twenty-six miRs were differentially expressed in melanoma-positive SNB samples between patients with disease progression and non-progressing patients, the majority being previously reported in the regulation of immune responses. A significant variation in miR expression levels was confirmed in an independent set of SNB samples. Integrated information from genome-wide transcriptional profiles and in vitro assessment in immune cells led to the identification of miRs associated with the regulation of the TNF receptor superfamily member 8 (TNFRSF8) gene encoding the CD30 receptor, a marker increased in lymphocytes of melanoma patients with progressive disease. These findings indicate that miRs are involved in the regulation of pathways leading to immune dysfunction in the sentinel node and may provide valuable markers for developing prognostic molecular signatures for the identification of stage III melanoma patients at risk of recurrence.

Skewed Differentiation of Circulating Vγ9Vδ2 T Lymphocytes in Melanoma and Impact on Clinical Outcome

ObjectiveThe aim of this study was to evaluate over time circulating γδ T lymphocytes in melanoma patients in terms of frequency, effector functions, and relationship with clinical stage and evolution, by comparing preoperative values to those obtained at a mean follow-up of 36 months or in the event of recurrence or disease progression, and to those of healthy controls. Also, we correlated the presence of tumor-infiltrating γδ T lymphocytes with clinical evolution of melanoma.ResultsMean frequencies of circulating γδ T cells before and after melanoma removal were very similar and comparable to healthy subjects, but patients who progressed to stage III or IV showed a significantly decreased frequency of circulating Vγ9Vδ2 T cells. The distribution of Vγ9Vδ2 memory and effector subsets was similar in healthy subjects and melanoma patients at diagnosis, but circulating γδ T cells of patients after melanoma removal had a skewed terminally-differentiated effector memory phenotype. Highly suggestive of progressive differentiation toward a cytotoxic phenotype, Vγ9Vδ2T cells from patients at follow up had increased cytotoxic potential and limited cytokine production capability, while the opposite pattern was detected in Vγ9Vδ2T cells from patients before melanoma removal.ConclusionsFollow-up data also showed that tumor infiltrating γδ T cells were significantly associated with lower mortality and relapse rates, suggesting that they may serve as a prognostic biomarker, for human melanoma.

The transfer of genetically engineered lymphocytes in melanoma patients: a Phase I dose escalation study

Meeting abstracts Genetically engineered T cells have broadened the opportunity for use of T cell immunotherapy in cancer patients. The possibility of improving T cell efficacy via introduction of additional genes potentially gives them an advantage over TIL. Studies in adoptive T cell transfer

Immunomodulatory pathways regulate expression of a spliced FKBP51 isoform in lymphocytes of melanoma patients.

FKBP51 (gene FKBP5) is an immunophilin capable of immunosuppression expressed in melanoma and lymphocytes. We found increased levels of a spliced FKBP5 variant in the PBMCs of 124 patients with melanoma. This variant encodes for an unknown isoform (FKBP51s). We hypothesized that FKBP51s resulted from tumour interaction with immune cells, through PDL-1/PD-1. To address this issue, we performed melanoma/PBMC cocultures. Furthermore, the immunohistochemistry of 76 melanoma specimens served to investigate whether FKBP51s stained tumour infiltrating lymphocytes. Our results showed that PBMCs expressed FKBP51s when cocultured with melanoma. Tumour PDL-1 knockdown or anti-PD-1 reduced FKBP51s expression in cocultured PBMCs. IHC showed a strong FKBP51s signal in tumour infiltrating lymphocytes, and lymphocytes of the invasion front of the tumour, along with melanoma PDL-1 expression. When overexpressed in melanoma, FKBP51s facilitated PDL-1 expression on the cell surface. In conclusion, our study shows that FKBP51s marks the PBMCs of patients with melanoma and is exploited by the tumour to immunomodulate through PDL-1.

Deciphering Treg accumulation in melanoma.

e22170 Background: A high density of regulatory T-cells (Tregs) at melanoma tumor sites correlates with poor prognosis due to immunosuppressive effects on the host immune system. While the concentration of circulating Tregs in patients with melanoma is not significantly different from that of healthy controls, Tregs tend to accumulate at tumor sites. We hypothesized that the influx of Tregs observed in melanoma tumors may be explained by enhanced tumor homing. Methods: To quantify the relative expression of chemokine receptors CCL4 and CCL8, and skin homing receptor CLA, blood was drawn from five metastatic melanoma patients and five healthy controls. Lymphocytes were ficolled and negatively sorted for CD3 expression. T-cells were subjected to 7-color FACS staining where Tregs were defined as CD4+CD25+CD127lowFoxP3+ and were simultaneously assessed for expression of CCR4, CCR8, and CLA. To further understand chemokine-receptor signaling involved in Treg homing in melanoma we analyzed the number of CCL22 producing cells in four human metastatic melanoma tissues and four healthy skin tissues obtained during surgery. Dermal CCL22+ cells were detected by indirect immunohistochemistry and quantified to a depth of three times the thickness of the epidermis. Results: Lymphocytes of melanoma patients were found to exhibit enhanced expression of CCR4 (mean of 65.9 versus 51.2%, p&lt;0.01) and CCR8 (3.0 versus 1.5%, p&lt;0.05), but not CLA (21.9 versus 23.9%) as compared with healthy controls. The mean fluorescence intensities of CCR4 and CCR8 were likewise elevated by 63 and 49% over controls (p&lt;0.05 and p&lt;0.01), respectively. A markedly increased number of CCL22 secreting cells were found in tumor samples (mean of 1063.1 versus 207.4, p= 0.02) as compared with healthy controls. Conclusions: These data strongly suggest that enhanced homing of CCR4+ Tregs towards the ligand CCL22 is likely involved in accumulating Tregs at melanoma tumor sites, ultimately impeding effective anti-tumor immune responses. Targeting chemokine-receptor signaling may thus be an effective intervention strategy in melanoma.

1452 POSTER Efficacy of DNA Repair Mechanisms in Blood Lymphocytes of Melanoma Patients as Prognostic Factor of Chemotherapy

1452 POSTER Efficacy of DNA Repair Mechanisms in Blood Lymphocytes of Melanoma Patients as Prognostic Factor of Chemotherapy

Absence of amplification of CD4+CD25high regulatory T cells during in vitro expansion of tumor‐infiltrating lymphocytes in melanoma patients

The exact role of CD4+CD25(high) regulatory T cells (Treg) in adoptive immunotherapy of melanoma is still to be determined, and an association between an expansion of Treg cells and the expansion of therapeutic tumor-infiltrating lymphocytes (TIL) remains unelucidated. In this context, the aim of our study was to determine whether functional Treg cells were detectable and amplified among in vitro-expanded TIL from 10 metastatic melanoma lymph nodes (LNs). In this study, we investigated the expression of forkhead/winged helix transcription factor 3 (Foxp3) in melanoma-invaded LNs and determined proportion and functionality of Treg cells among TIL extracted from these 10 metastatic melanoma LNs at different steps of their in vitro expansion. We found that metastatic melanoma LNs expressed very heterogeneous levels of Foxp3 and that CD4+CD25(high) Treg cells extracted from these LNs were detectable at each step of the in vitro culture of TIL but decreasing during the culture. In addition, functional assays demonstrated that these CD4+CD25(high) T cells were capable of suppressing autologous CD8+ and CD4+CD25- T cell proliferation. These cells were indeed Treg cells as they expressed Foxp3. In conclusion, our work suggests that CD4+CD25(high) Foxp3 expressing T cells are not expanded during in vitro amplification of TIL obtained from melanoma-invaded LNs.

Analysis of Pmel17/gp100 expression in primary human tissue specimens: implications for melanoma immuno- and gene-therapy.

Pmel17/gp100-encoded tumor-associated antigens are recognized by cytotoxic T lymphocytes in melanoma patients and may represent attractive target antigens for immuno- and gene-therapeutic strategies. An important prerequisite for identification and monitoring of melanoma patients that could potentially benefit from Pmel17/gp100-based immuno- and gene-therapies is the detailed knowledge of Pmel17/gp100 expression in vivo. Immunophenotyping is considerably hampered by the different immunoreactivities of Pmel17/gp100-reactive antibodies. Therefore, we analyzed an extended series of different primary normal and malignant human tumor specimens for Pmel17/gp100 expression at the mRNA level. Transcripts were detectable in all malignant melanoma tissue specimens representing all stages of tumor progression, with significant levels even in early and amelanotic melanoma lesions. In contrast, normal melanocytes exhibited significantly less Pmel17/gp100 mRNA in vivo, as determined by comparative in situ hybridization. Tissue specimens from the retina and substantia nigra also contained Pmel17/gp100 mRNA, whereas other normal and malignant human tissues were negative. As determined by comparative in situ hybridisation and HMB-45 immunostaining, even tumor tissue lacking Pmel17/gp100 immunoreactivity contained Pmel17/gp100 transcripts. Our results indicate a melanocytic-cell-lineage-restricted expression of Pmel17/gp100 with significant transcript levels in all stages of melanoma progression, including early and amelanotic melanoma lesions, and a significantly differential expression between melanoma cells and normal melanocytes in vivo. Owing to its higher sensitivity, phenotyping of individual tumor specimens by mRNA expression analysis seems to be more valuable than phenotyping by immunostaining.

UV-induced N-ras mutations are T-cell targets in human melanoma

Human cutaneous melanoma is heterogeneous with respect to the genetic aberrations involved and the genes altered are potential targets for the immune system. The incidence of cutaneous melanoma is known to be linked to UV peak exposure, and the N-ras oncogene is clearly one of the genes involved in the UV carcinogenesis in melanoma. It is mutated in a significant proportion of melanomas and therefore may serve as a target for T cells. Here, we report that an human leukocyte antigen-A2 binding peptide CLLDILDTAGL, encompassing the frequently found 61-Leu mutation in N-ras, induces cytotoxic T lymphocytes from healthy donor blood that lyse 61-Leu N-ras transfected melanoma cells. Furthermore, we have found an association between the presence of N-ras mutations and clinical response to immunotherapy with interleukin-2 plus interferon in a group of stage IV melanoma patients. Although the overall survival of these patients was not affected by the N-ras status of their melanomas, these studies suggest that mutated N-ras may provide a target for cytotoxic T lymphocytes in melanoma patients.

Dendritic cells generated from peripheral blood transfected with human tyrosinase induce specific T cell activation.

Peptides of melanosomal proteins have recently been shown to be recognized in an HLA-restricted mode by specific cytolytic T lymphocytes in melanoma patients. Dendritic antigen-presenting cells (DC) are considered to be the most effective stimulators of T cell responses, and the use of these cells has therefore been proposed to generate therapeutic responses to tumor antigens in cancer patients. We, therefore, generated DC from peripheral blood of normal donors in the presence of granulocyte/macrophage colony-stimulating factor and interleukin-4. Flow cytometric analysis of the cells during a 2-week culture revealed a loss of CD14 and CD34 expression, a concomittent increase of CD1a, CD11a,b and c, CD44, CD45, CD54, HLA-class I and II, and intermediate levels of CD26, CD80 and CD86. Cultured DC stimulated proliferation of allogeneic T cells and induced a marked, up to 20-fold, stimulation of T cell proliferation after pulsing with tetanus toxoid. To achieve independence of already-identified antigenic peptides presented in HLA class I-restricted fashion, which limits the general applicability of such peptides for vaccination of melanoma patients, we tested whether DC are transfectable with eukaryotic expression plasmids. DC transfected with two reporter genes (CAT, beta-galactosidase) using a liposome-based transfection technique, exhibited only low levels of enzymatically active proteins, but were able to degrade rapidly intracellular proteins and to process peptides efficiently. Chloramphenicol acetyltransferase as well as tyrosinase mRNA were detectable after transfection by reverse-transcriptase-polymerase chain reaction, and enzyme activities became measurable. Furthermore, DC transfected with the tyrosinase gene were able to induce specific T cell activation in vitro, indicating appropriate peptide processing and presentation in DC after transfection. These data suggest new approaches to future tumor vaccination strategies.

The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas.

Lymphocytes of melanoma patients can be restimulated in vitro with autologous tumor cells to generate antitumor cytolytic T lymphocytes (CTL). Previous reports have indicated that, when such CTL are obtained from HLA-A2 melanoma patients, they often display broad reactivity on A2 melanoma cell lines. Such antitumor CTL clones, which appeared to recognize the same antigen, were isolated from two patients. We report here the cloning of a cDNA that directs the expression of the antigen recognized by these CTL. This cDNA corresponds to the transcript of the tyrosinase gene. The gene was found to be active in all tested melanoma samples and in most melanoma cell lines. Among normal cells, only melanocytes appear to express the gene. The tyrosinase antigen presented by HLA-A2 may therefore constitute a useful target for specific immunotherapy of melanoma. But possible adverse effects of antityrosinase immunization, such as the destruction of normal melanocytes and its consequences, will have to be examined before clinical pilot studies can be undertaken.

Analysis of the expression of N-glycolylneuraminic acid-containing gangliosides in cells and tissues using two human monoclonal antibodies.

The specificities of two human monoclonal antibodies (2-39M and 32-27M), produced by hybridomas derived from the lymphocytes of melanoma patients (Yamaguchi, H., Furukawa, K., Fortunato, S. R., Livingston, P. O., Lloyd, K. O., Oettgen, H. F., and Old, L. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2416-2420) have been elucidated. Using a large panel of glycolipids, it has been shown that the two monoclonal antibodies (mAbs) identified a number of N-glycolylneuraminic acid (NeuGc)-containing gangliosides. mAb 2-39M reacted with (NeuGc)GM3, (NeuGc)sialylparagloboside, and (NeuGc)sialylhexaglycosylceramide; no reactivity was observed with gangliosides containing only N-acetylneuraminic acid (NeuAc) or with disialogangliosides. These reactive species have the NeuGc alpha 2----3Gal- sequence in common. mAb 32-27M reacted strongly with (NeuGc)2 GD3 and (NeuGc)2disialylparagloboside, and moderately with (NeuAc-NeuGc-)GD3 and (NeuAc-NeuGc-)disialylparagloboside. The reactive species have sialic acid alpha 2----8NeuGc alpha 2----3Gal- sequences in common. These two antibodies were used to demonstrate the species-related presence of different NeuGc-containing gangliosides in various animal erythrocytes by thin layer chromatography immunostaining. No reactivity of either mAb was observed with gangliosides isolated from fresh human colon cancer, melanoma specimens, or some normal tissues, including brain. On the other hand, it was shown that mAb 32-27M reacted with gangliosides isolated from human melanoma and astrocytoma cells grown in fetal bovine serum but not from those grown in synthetic medium. Within the sensitivities of the methods used, these data, and related chemical analyses, do not support the presence of NeuGc-containing gangliosides in human tumors.

In vivo activation of lymphocytes in melanoma patients receiving escalating doses of recombinant interleukin 2.

A phase-I study of the recombinant, non-mutagenized interleukin 2 (rIL2, BioleukinTM) was performed in 12 melanoma patients (Pts). From 100 to 800 micrograms/m2 of rIL2 were administered by i.v. bolus injection, TID for 4-8 days. Side-effects included fever, malaise, low serum K+ and Ca++ values, electrocardiographic abnormalities, leukopenia and thrombocytopenia. No major organ toxicity and no significant fluid retention were observed at the administered doses. Treatment induced a rapid depletion of peripheral blood lymphocytes (PBL) with a rebound (2-6 times the pre-treatment values), 24-48 hr after rIL2 discontinuation. PBL obtained between the 5th treatment day and the 2nd post-treatment day showed: (a) enhanced proliferation (II/12 Pts) with stimulation indexes of 6-52; (b) increased cytotoxicity against autologous tumor cells (2/2 Pts), allogeneic melanomas (5/7 Pts), the Daudi (5/6 Pts) and K562 cell lines (7/12 Pts); and (c) increased expression of IL2 receptors (8/12 Pts) and of DR antigens (6/12 Pts). Lymphocytes collected 1-2 days after treatment and activated in vitro with rIL2 showed a more rapid development of tumor cytotoxicity, with an earlier loss of activity. Spontaneous proliferation, autologous or allogeneic tumor cytotoxicity and expression of IL2 receptors obtained after in vivo treatment with rIL2 were significantly weaker than those induced during in vitro stimulation. No major objective responses were detected in these patients.

Infusion of autologous alloactivated lymphocytes in melanoma patients: toxicity and immunologic monitoring.

Previous work has shown that infusion of autologous helper-enriched, alloactivated lymphocytes in melanoma patients may induce, in addition to other mild signs of toxicity, a transient but sharp elevation of blood pressure. To avoid such a disturbing symptom, the in vitro protocol of peripheral blood lymphocyte activation has been modified. In the present study we show that such a modification has led to a lower toxicity of autologous lymphocyte infusion in 4 melanoma patients; in particular, hypertension was no longer observed. In addition, an immunologic monitoring was carried out in these patients. In 1 of 4 patients the treatment enhanced the in vitro cytotoxic activity of peripheral blood lymphocytes against autologous tumor cells. Other parameters such as NK activity and T4/T8 ratio did not show significant trends. The possible implications of these findings for clinical trials of adoptive immunotherapy with lymphocytes are discussed.

Inhibition of human melanoma growth in nude mice by autologous, alloactivated peripheral blood lymphocytes.

Peripheral blood lymphocytes of melanoma patients were stimulated in vitro by a pool of allogeneic lymphocytes and shown to be cytotoxic against autologous melanoma cells. To evaluate the in vivo antitumor activity of the cytotoxic alloactivated autologous peripheral blood lymphocytes, tumor neutralization (Winn) assay was carried out by injecting such lymphocytes admixed with autologous melanoma cells in athymic BALB/c nude mice. In 3 of 6 cases, complete inhibition of tumor growth was obtained at lymphocytes to tumor cells ratio of 10:1 and in one case also of 5:1. In all cases the appearance of tumors was delayed and the growth rate was significantly reduced in a dose-dependent fashion as compared to control mice injected with tumor cells alone. We conclude that in vitro alloactivated peripheral blood lymphocytes can inhibit and/or impair the growth of autologous melanoma cells in nude mice.

Prolonged increase of sister-chromatid exchanges in lymphocytes of melanoma patients after CCNU treatment

Prolonged increase of sister-chromatid exchanges in lymphocytes of melanoma patients after CCNU treatment