Susceptibility of Human Melanoma Cells to Autologous Natural Killer (NK) Cell Killing: HLA-Related Effector Mechanisms and Role of Unlicensed NK Cells

BACKGROUND: Despite Natural Killer (NK) cells were originally defined as effectors of spontaneous cytotoxicity against tumors, extremely limited information is so far available in humans on their capability of killing cancer cells in an autologous setting. METHODOLOGY/PRINCIPAL FINDINGS: We have established a series of primary melanoma cell lines from surgically resected specimens and here showed that human melanoma cells were highly susceptible to lysis by activated autologous NK cells. A variety of NK cell activating receptors were involved in killing: particularly, DNAM-1 and NKp46 were the most frequently involved. Since self HLA class I molecules normally play a protective role from NK cell-mediated attack, we analyzed HLA class I expression on melanomas in comparison to autologous lymphocytes. We found that melanoma cells presented specific allelic losses in 50% of the patients analyzed. In addition, CD107a degranulation assays applied to NK cells expressing a single inhibitory receptor, revealed that, even when expressed, specific HLA class I molecules are present on melanoma cell surface in amount often insufficient to inhibit NK cell cytotoxicity. Remarkably, upon activation, also the so called "unlicensed" NK cells, i.e. NK cells not expressing inhibitory receptor specific for self HLA class I molecules, acquired the capability of efficiently killing autologous melanoma cells, thus additionally contributing to the lysis by a mechanism independent of HLA class I expression on melanoma cells. CONCLUSIONS/SIGNIFICANCE: We have investigated in details the mechanisms controlling the recognition and lysis of melanoma cells by autologous NK cells. In these autologous settings, we demonstrated an efficient in vitro killing upon NK cell activation by mechanisms that may be related or not to abnormalities of HLA class I expression on melanoma cells. These findings should be taken into account in the design of novel immunotherapy approaches against melanoma

Cytolytic activity against autologous melanoma of NK cell subsets expressing no inhibitory receptors for self-HLA class I.

<p>The cytolytic activity of NK cell subsets expressing no inhibitory receptors for self-HLA class I (“HLA non-inhibited”) was compared with the counterpart expressing at least one receptor for self-HLA. <b>A:</b> Cytolytic activity was assessed by CD107a degranulation assay for total “HLA-inhibited” and total “HLA non-inhibited” NK cell subsets. A representative experiment is shown. Briefly, triple fluorescence staining with a mixture of PE-conjugated anti-KIR2DL1 mAb and a mixture of other anti-KIR (KIR2DL2/3 and KIR3DL1) and anti-NKG2A mAbs allowed to gate on the KIR⁻/NKG2A⁻ subset plus NK cell subset expressing receptors for HLA class I alleles not expressed by the related patient. On the other hand, the staining permitted also to gate on all “HLA-inhibited” subsets. Thus, as shown in panel A, CD107a expression of the whole “HLA non-inhibited” subset was analyzed and compared with that of whole “HLA-inhibited” subset, in the presence of absence of anti-HLA class I blocking mAb. In agreement with the absence of inhibitory receptors for self HLA, “HLA non-inhibited” NK cell subset did not increase their cytotyitc activity in the presence of anti-HLA class I masking mAb. A summary of all experiments performed in each patients is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008132#pone-0008132-g003" target="_blank">figure 3B(ii,iii,iv)</a> . <b>B:</b> The effectiveness of “HLA non-inhibited” NK cell subsets in killing autologous melanoma was also assessed by a standard ⁵¹chromium release assay. The whole “HLA non-inhibited” NK cell populations derived by three representative patients were sorted out by flow cytometry as double negative cells and compared with the unstained total bulk NK populations. The comparison was performed with the unstained total NK cells, rather than with HLA-inhibited NK cell subets, to avoid possible interference caused by antibody binding to functional receptors. Results shown are from three patients and data correspond to mean values±SD of experiments performed in triplicate.</p

CD107a degranulating assay on distinct NK cell subsets expressing single inhibitory receptors.

<p>CD107a degranulating ability of activated NK cell subsets from each patient was analyzed in the presence of autologous melanoma cells, either adding or not an anti-pan HLA class I blocking mAb. E/T ratio = 1.5∶1. A representative analysis of patient CA is shown in panel <b>A</b>. In order to assess the link between HLA class I expression on melanoma cells and their susceptibility to autologous NK cell-mediated lysis, the degranulation of NK cell subsets, expressing a single inhibitory receptor for distinct HLA class I molecules, was analyzed. Staining NK cells with mAbs against a specific KIR or NKG2A and with a mixture of mAbs against the other inhibitory receptors allowed to gate on the specific subset and evaluate its CD107a expression after co-culture with autologous melanoma cells. <b>B</b>: Melanoma specific deletion of a HLA class I allele in the same patient CA. HLA-Bw4 was here detected by flow cytometry on the surface of lymphocytes but not of the autologous melanoma. As a comparison, another conserved HLA-A allele is shown. In accordance with Bw4 deletion, KIR3DL1+ NK cell subset exerts a strong cytolytic activity against autologous melanoma cells, which is not significantly increased following anti-HLA class I masking.</p

Cytolitic capabilities of human NK cells against autologous melanoma.

<p><b>A:</b> Susceptibility of melanoma cells to autologous NK cell-mediated lysis. Freshly isolated or IL-2 activated NK cells were used as effector cells in a specific ⁵¹chromium release assay. The effector/target (E/T) ratio used was 20∶1. Four representative patients are shown and results are expressed as means±SD derived from experiments in triplicate. <b>B:</b> Flow cytometry evaluation of total HLA class I expression (W6/32 mAb) on melanoma cells and, as a comparison, on autologous lymphocytes. <b>C:</b> Susceptibility to NK-mediated lysis of melanoma cells compared with susceptibility of HLA class I-deficient FO-1 cell line and autologous lymphocytes stimulated with PHA (PHA lymphocytes). Cytolitic activity was assessed by ⁵¹chromium release tests. In order to evaluate the inhibitory effect caused by melanoma HLA class I expression on NK cell activity, lysis of FO-1, autologous melanoma cells and PHA lymphocytes was also analysed in the presence of the anti-pan HLA class I blocking mAb (A6-136, IgM) at the indicated E/T ratios. At least three experiments were performed for each patients. Representative experiments of three patients out of six are shown. Data represent mean value±SD of experiments performed in triplicate.</p

Role of HLA class I alteration in melanoma susceptibility to lysis by autologous NK cells.

<p>Summary of CD107a degranulating assays (performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008132#pone-0008132-g002" target="_blank">figure 2A</a>) analyzed on NK cell subsets in the presence of autologous melanoma cells from six patients. Degranulation was analyzed on (i) NK cell subsets expressing the single inhibitory receptors, (ii) NK cell subsets expressing one or more inhibitory receptors (“Total HLA-inhibited”), (iii) NK cell subsets expressing no inhibitory receptors for self HLA (“Total HLA non-inhibited”) and (iv) the whole NK cell population. Data represent percentages of CD107a expression in the presence of autologous target (grey columns) or in the presence of autologous target plus anti-HLA class I mAb (black columns). Values were calculated by subtracting the spontaneous CD107a expression in the absence of the target. NK cell subsets expressing no receptors for self-HLA are depicted on a grey background. n.d. indicates that the specific NK cell subset was not detectable in a given patient. For each patient, on the right side of the figure, patient HLA class I antigens that are relevant for inhibiting NK cell activity are shown and antigens that have undergone deletions on autologous melanoma cells are marked by a slash. For HLA class I A,B,C, both molecular and serological typing were performed (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008132#pone-0008132-t001" target="_blank">Table 1</a>), while HLA-E was detected only at mRNA level. For each HLA class I antigen, matched inhibitory receptor is also specified. Results are expressed as means of 3 to 6 independent experiments. E/T ratio was 1.5∶1.</p

Melanoma surface molecules and NK cell activating receptors involved in the lysis of melanoma cells by autologous NK cells.

<p><b>A:</b> Melanoma cell expression of molecules able to trigger NK cell activity. Representative data are shown for the six melanoma cell lines whose autologous lymphocytes were also available for performing the functional experiments summarized in panel B. Melanoma cells were stained with mAbs against ligands of NKG2D and DNAM-1 and with mAbs against adhesion molecules LFA-3 and ICAM-I. <b>B:</b> Contribution to the lysis by different activating NK cell receptors. Cytotoxic activity of IL-2 activated NK cells from each patient against the autologous melanoma cells was assessed in the presence of anti-DNAM-1, -NKp30, -NKp44, -NKp46 and -NKG2D (black columns) and isotypic control (grey column) mAbs. *: p≤0.05; **: p≤0.01; n.s.: not significant. E/T ratio = 20∶1. Data shown represent mean values of the results obtained from three to six independent experiments.</p

Cronache meridionali. A. VII, n.1-12 (1960)

N. 1-2 (gennaio-febbraio 1960): Napolitano, G., Il IX congresso nazionale del P.c.i., P. 1 ; Valenza, P., Alcuni problemi dd rinnovamento del P.c.i. nel Mezzogiorno, P. 7; Grifone, P., Superamento dei contratti agrari e conquista della terra, P. 15 ; La crisi siciliana, P. 35 ; Caprara, M., Il dibattito alla Camera sulla legge per Napoli, P. 37 ; Villari, R., Una proposta per la celebrazione del centenario del '60, P. 41 ; Dalle riviste, P. 43 ; Il piano di rinascita della Sardegna, P. 47 ; Palomba, G., Intervento nel dibattito “Per lo sviluppo della piccola e media industria”, P. 58 ; Andriani, S., Intervento nel dibattito “Per lo sviluppo della piccola e media industria”, P.61 ; Assennato, M., La nuova giunta di Bari, P. 69 ; Villari, R., Denis Mack Smith: Storia d'Italia dal 1861 al 1958, p. 74 ; Ricci, P., Alberto Consiglio: Camorra, P. 77 ; Gallico, L., Giorgio Assan: La Libia e il mondo arabo, P. 79.N. 3 (marzo 1960): Laconi, R., Regioni e democrazia, P. 81 ; Colajanni, N., L'esperienza siciliana, P. 87 ; Gomez, M., Prime considerazioni sul « Piano Verde», P. 96 ; Magno, M., Coltura bieticola e prezzo dello zucchero, P. 105 ; Boffa, G., Due politiche verso i paesi sottosviluppati, P. 111 ; Fontani, A., La legge sull'utbanesimo al Senato, P. 115 ; Colombo e la Confindustria, P. 119 ; D’Antonio, M., Speculazione è parassisismo nei rapporti produzione-consumo in provincia di Napoli, P. 129 ; Misefari, E., La Calabria e la pianificazione regionale, P. 139 ; Il convegno sull'Ente di irrigazione apulo-lucano 151 ; Villari, R., Salvatore Francesco Romano: Storia dei Fasci siciliani, P. 153 ; D’Amico, M., J. J. Lador Lederer: Capitalismo mondiale e cartelli tedeschi tra le due guerre, P. 155 ; Chiaromonte, G., Franco Volpi: Le finanze comunali di un grande centro urbano, P. 159.N. 4 (aprile 1960): Alinovi, A., Inizio di una svolta?, P. 161 ; Manzocchi, B., La nazionalizzazione dell'industria elettrica, P. 165 ; Chiaromonte, G., Cassa per il Mezzogiorno e politica di sviluppo, P. 180 ; Boffa, G., La «Conferenza della fame», P. 192 ; Rabbia clericale, P. 195 Rosini, A., Il Fucino, Torlonia e la D.c., P. 211 ; Fermariello, C., Sperequazioni salariali e azione sindacale in provincia di Napoli, P. 219 ; La Torre, P., Lotte operaie e sviluppo economico in Sicilia, P. 234 ; Il IV Congresso dell'Associazione dei contadini del Mezzogiorno, P. 241 ; Il metano in Lucania, P. 247.N. 5 (maggio 1960): La difficile vetta, P. 249 ; Napolitano, G., La lotta per lo sviluppo dell'industria di Stato nel Mezzogiorno, P. 253 ; Cortese, N., La Calabria nel Risorgimento italiano, P. 265 ; Il convegno di Palma di Montechiaro, P. 281 ; La relazione del professor Pampiglione, P. 283 ; Caprara, M., Il disarmo dei due « Conti», P. 292 ; Del Rio, D., I regimi commissariali in provincia di Napoli, P. 302 ; Picciotto, G., Bilancio fallimentare dell'Opera Sila, P. 308 ; D’Antonio, M., Gunnar Myrdal: Teoria economica e paesi sottosviluppati, P. 321 ; Villari, R., Enzo Santarelli: Il socialismo anarchico in Italia, P. 325 ; Villari, R., Franco Rizzo: Francesco Saverio Nitti e il Mezzogiorno, P. 327 : Daniele, A., Impiego del tempo libero in Calabria, P. 329.N. 6 (giugno 1960): Chiaromonte, G., Il « miracolo» italiano e il Mezzogiorno, P. 333 ; Le due Italie (dalla relazione del Comitato dei ministri per il Mezzogiorno), P. 339 ; Villari, R., La liberazione del Mezzogiorno e l'unità nazionale, P. 352 ; Il Convegno delle riviste, P. 380 ; Abenante, A., Sulla crisi dell'industria molitoria, P. 388 ; La legge speciale per Napoli, P. 390 ; Russo, N., Il 10 Congresso dell'Unione siciliana cristiano-sociale, P. 393 ; Simeone, E., Il processo di Barletta, P. 397 ; Costantino, D., Problemi del Reggino, P. 399 ; Di Carlo, D., La qualificazione professionale della donna, P. 402 ; Di Benedetto, S., Piccole e medie imprese e sviluppo economico, P. 419 ; Fontani, A., Milano, Corea - Inchiesta sugli immigrati (a cura di F . Alasia e D. Montaldi), P. 432 ; Chiaromonte, G., Pasquale Saraceno: Iniziativa privata e azione pubblica nei piani di sviluppo economico, P. 434 ; Valenzi, M., J. D. Bernal: Mondo senza guerra, P. 437 ; Gruppi, L., Pietro Secchia: Capitalismo e classe operaia nel centro laniero d'Italia, P. 439N. 7-8 (luglio-agosto 1960): Alinovi, A., Per uno sviluppo democratico della società nazionale, P. 441 ; Caprara, M., Il processo di Torre del Greco, P. 447 ; Barbadoro, I., Metano, industria di Stato e sviluppo economico, P. 453 ; Fermariello, C., Napoli contro il governo Tambroni, P. 464 ; Pro La Torre: Dallo sciopero di Palermo alle manifestazioni dell' 8 luglio, P. 469 ; Pistillo, M., Il movimento dei braccianti pugliesi, P. 477 ; Villani, R., Per la morte di Federico Chabod, P. 483 ; Tamburrino, L., Alcuni dati sul sottosalario a Napoli, P. 489 ; Masi, G., Osservazioni sulla ripresa delle quotizzazioni demaniali nel Mezzogiorno (1861), P. 493 ; Cinanni, P., Bilancio e prospettive della riforma agraria in Calabria, P. 503 ; D’ Antonio, M., Eugenio Peggio, Mario Mazzarino, Valentino Parlato, Industrializzazione e sottosviluppo, P. 524 ; Daniele, A., La Calabre, P. 526.N. 9 (settembre 1960): Napolitano, G., Una nuova politica per il Mezzogiorno, p. 529 ; Pavolini, L., La Fiera del Levante e il M.e.c., P. 535 ; Berti, G., Una inchiesta parlamentare sulla « mafia» di Agrigento, P. 539 ; Fontani, A., Un bilancio negativo, P. 557 ; Pace, G., Lo spopolamento della Calabria, P. 561 ; Scutari, D., L'emigrazione dalla provincia di Potenza, P. 569 ; Voutto, E., L'esodo dai comuni irpini, P. 575 ; Lepre, A., Un documento di storia agraria del Mezzogiorno, P. 581 ; Lombardi, V., Il lavoro femminile nell'economia napoletana, P. 585 ; Gomez, M., Un progetto di legge sui fitti agrari, P. 597 ; Pinna, P., I pescatori sardi contro i balzelli feudali, P. 598 ; Il convegno sull'artigianato meridionale, P. 606.N. 10-11 (ottobre-novembre 1960): l risultati elettorali nel Mezzogiorno continentale, P. 609 ; Il Convegno internazionale sullo sviluppo delle regioni arretrate, P. 618 ; Cortese, N., Napoli e l'Unità italiana nel 1860, P. 646 ; Pinna, P., Sul banditismo in Sardegna, P. 658 ; Esposto, A., Le elezioni dei rappresentanti degli assegnatari nell'E.r.a.s., P. 662 ; Tamburrino, L., Le lotte sindacali a Napoli e la politica di sviluppo, P. 666 ; Boffa, G., Vecchio e nuovo colonialismo in Africa, P. 672 ; D’Antonio, M., Una inchiesta sugli studenti universitari meridionali, P. 67 ; Villari, R., Giovanni Amendola: La democrazia italiana contro il fascismo, P. G89 ; Giudice, G., Luigi Incoronato: Il governatore (G. Giudice), P. 691 ; Pedio, T., Giuseppe Bufalari: La masseria - Giuseppe G. Loschiavo: Il mare di pietra, P. 695.N. 12 (dicembre 1960): Una scelta politica decisiva, P. 697 ; D’Antonio, M., Mezzogiorno e Comunità Europea, P. 713 ; Tamborrino, L., Un convegno sul sottosalario, P. 721; Villa, R., Come hanno votato le donne in una frazione di Casalnuovo, P. 728 ; Villano, P., Napoli 1860: Il ritorno degli esuli, P. 729 ; Di Marino, G., l risultati elettorali del 6 novembre nelle campagne meridionali, P. 746 ; Chiaromonte, G., Azione meridionalistica ed alternativa democratica m provincia di Napoli, P. 755 ; Silipo, L., Su alcune caratteristiche del voto calabrese, P. 762 ; Cardia, U., Verso le elezioni regionali sarde, P. 766 Le elezioni universitarie a Napoli, P. 770