Key Points

  • Paradoxically higher NK-cell activity in CTCL patients is associated with increased expression of phosphorylated STAT5.

  • These highly effective NK cells are associated with poor prognosis in patients with leukemic CTCL.

Introduction

Cutaneous T-cell lymphoma (CTCL) is a type of non-Hodgkin lymphoma characterized by the expansion of malignant CD4+ T cells in the skin. There are two main subtypes of CTCL: an indolent form termed mycosis fungoides (MF), which is largely limited to the skin, and Sézary syndrome (SS), an aggressive leukemic variant of the disease which can manifest systemically.1-3 

Previous studies have demonstrated defects in cell-mediated immunity in CTCL patients, including altered cytokine profiles and impaired neutrophil function, which lead to a high incidence of recurrent bacterial and viral infections as a result of decreased Th1-mediated immunity.4-9  It has also been reported that natural killer (NK)–cell function is decreased in CTCL patients,10-14  which could contribute to an overall decrease in the innate immune response to both neoplastic cells and viral or bacterial pathogens. Previous groups have reported that NK cells from SS patients are capable of responding to activation ex vivo, indicating the potential for development of immune-based therapeutics.15 

Although MF patients often have a prolonged indolent clinical course of disease that requires localized treatment, there are few effective treatments for the successful management of patients with SS. Because of the lack of success with traditional chemotherapeutic approaches, novel immune-based therapeutics are being developed for use in a multitude of hematologic diseases, including CTCL.4,16-18  Understanding the immune microenvironment in patients with CTCL will be critical to the successful design of targeted therapies for their disease.

Previous studies by our group and by others have shown increased expression of interleukin-15 (IL-15) in malignant CD4+ T cells in CTCL patients.19  IL-15 acts through a trimeric IL-15R complex to enhance NK-cell maturation and function.20-22  Indeed, in a first-in-human phase 1 trial in patients with refractory solid cancer tumors, IL-15 treatment induced profound expansion of circulating NK cells (NCT01885897).23  Considering that IL-15 is produced by malignant cells in CTCL, we sought to study the possible effect of chronically elevated IL-15 on NK-cell function in CTCL patients. In this study, we show that NK-cell activity is significantly enhanced in CTCL, and strikingly, higher NK-cell numbers are associated with increased mortality.

Materials and methods

NK-cell numbers

NK-cell numbers were evaluated by flow cytometric analysis of peripheral blood samples drawn on the same day as the initial diagnostic complete blood cell count with differential and was performed using a 10-color technique with a gating strategy based on CD45 staining and light side scatter characteristics. NK-cell number represents the number of CD56+/CD16+/CD3 NK cells per microliter. Samples were taken from November 2007 through November 2016 from patients at The Ohio State University James Cancer Hospital who were diagnosed with biopsy-proven CTCL (Table 1).

Table 1.

Characteristics of patients diagnosed with biopsy-proven CTCL MF

Patient IDDiagnosisStage at diagnosisHistology/subtypeAge at diagnosis, ySexRace
CTCL, MF IA Woringer-Kolopp variant 48 White 
216 CTCL, MF IA CD30+ LPD: primary CTCL γ-δ T-cells 68 White 
27 CTCL, MF IA Hypopigmented 60 African American 
85 CTCL, MF IA Hypopigmented 38 African American 
65 CTCL, MF IA None 40 African American 
180 CTCL, MF IA None 56 African American 
CTCL, MF IA None, lyp, and CD30+ LPD 61 African American 
147 CTCL, MF IA Hypopigmented 45 Asian 
89 CTCL, MF IA Folliculotropic 48 White 
137 CTCL, MF IA Granulomatous 67 White 
28 CTCL, MF IA Hypopigmented 38 White 
38 CTCL, MF IA None 41 White 
42 CTCL, MF IA None 49 White 
52 CTCL, MF IA None 57 White 
55 CTCL, MF IA None 41 White 
61 CTCL, MF IA None 82 White 
73 CTCL, MF IA None 64 White 
82 CTCL, MF IA None 50 White 
107 CTCL, MF IA None 58 White 
111 CTCL, MF IA None 56 White 
144 CTCL, MF IA None 51 White 
145 CTCL, MF IA None 42 White 
150 CTCL, MF IA None 28 White 
152 CTCL, MF IA None 47 White 
183 CTCL, MF IA None 53 White 
200 CTCL, MF IA None 37 White 
215 CTCL, MF IA None 50 White 
226 CTCL, MF IA None 19 White 
166 CTCL, MF IA Poikilodermatous 31 White 
84 CTCL, MF IA Poikilodermatous 55 White 
188 CTCL, MF IA Woringer-Kolopp variant 31 White 
142 CTCL, MF IA Follicular 50 White 
179 CTCL, MF IA Follicular T-helper phenotype 60 White 
50 CTCL, MF IA CD8+ 58 White 
32 CTCL, MF IA Folliculotropic 37 White 
36 CTCL, MF IA Folliculotropic 61 White 
54 CTCL, MF IA Folliculotropic 81 White 
64 CTCL, MF IA Folliculotropic 25 White 
100 CTCL, MF IA Folliculotropic 62 White 
130 CTCL, MF IA Hypopigmented, CD8+ 18 White 
41 CTCL, MF IA None 84 White 
46 CTCL, MF IA None 40 White 
47 CTCL, MF IA None 78 White 
57 CTCL, MF IA None 63 White 
62 CTCL, MF IA None 57 White 
67 CTCL, MF IA None 77 White 
71 CTCL, MF IA None 45 White 
80 CTCL, MF IA None 26 White 
95 CTCL, MF IA None 60 White 
99 CTCL, MF IA None 74 White 
103 CTCL, MF IA None 82 White 
108 CTCL, MF IA None 42 White 
120 CTCL, MF IA None 62 White 
124 CTCL, MF IA None 57 White 
128 CTCL, MF IA None 54 White 
131 CTCL, MF IA None 37 White 
139 CTCL, MF IA None 26 White 
162 CTCL, MF IA None 51 White 
174 CTCL, MF IA None 76 White 
181 CTCL, MF IA None 53 White 
189 CTCL, MF IA None 44 White 
191 CTCL, MF IA None 34 White 
197 CTCL, MF IA None 70 White 
223 CTCL, MF IA None 72 White 
146 CTCL, MF IA None 48 White 
199 CTCL, MF IA None 63 White 
CTCL, MF IB None 52 African American 
CTCL, MF IB Poikilodermatous 69 African American 
203 CTCL, MF IB None 46 African American 
39 CTCL, MF IB Hypopigmented 38 African American 
56 CTCL, MF IB None 51 African American 
77 CTCL, MF IB CD8+ 58 White 
26 CTCL, MF IB Granulomatous 66 White 
87 CTCL, MF IB None 51 White 
91 CTCL, MF IB None 66 White 
136 CTCL, MF IB None 72 White 
173 CTCL, MF IB None 57 White 
194 CTCL, MF IB None 73 White 
115 CTCL, MF IB CD8+ 63 White 
209 CTCL, MF IB CD8+ 81 White 
88 CTCL, MF IB Folliculotropic 76 White 
97 CTCL, MF IB Folliculotropic 44 White 
114 CTCL, MF IB Folliculotropic 58 White 
134 CTCL, MF IB Folliculotropic 53 White 
37 CTCL, MF IB None 68 White 
58 CTCL, MF IB None 59 White 
75 CTCL, MF IB None 64 White 
78 CTCL, MF IB None 46 White 
104 CTCL, MF IB None 62 White 
112 CTCL, MF IB None 45 White 
121 CTCL, MF IB None 71 White 
122 CTCL, MF IB None 67 White 
123 CTCL, MF IB None 55 White 
220 CTCL, MF IB None 72 White 
227 CTCL, MF IB None 62 White 
217 CTCL, MF IB Follicular T-helper phenotype 36 White 
167 CTCL, MF IB None 78 White 
204 CTCL, MF IB Poikilodermatous 61 Hispanic/Latino 
132 CTCL, MF IB None 79 Hispanic/Latino 
206 CTCL, MF IIA Poikilodermatous 41 White 
33 CTCL, MF IIB Large cell transformation 50 African American 
30 CTCL, MF IIB None 66 African American 
157 CTCL, MF IIB Folliculotropic 69 African American 
168 CTCL, MF IIB Folliculotropic 53 African American 
156 CTCL, MF IIB None 80 White 
90 CTCL, MF IIB Folliculotropic 62 White 
63 CTCL, MF IIB None 88 White 
185 CTCL, MF IIB None 75 White 
153 CTCL, MF IIB Tumor 64 White 
141 CTCL, MF IIB Large cell transformation 59 White 
109 CTCL, MF IIIB Erythrodermic 55 White 
213 CTCL, MF IIIB Erythrodermic 65 African American 
Patient IDDiagnosisStage at diagnosisHistology/subtypeAge at diagnosis, ySexRace
CTCL, MF IA Woringer-Kolopp variant 48 White 
216 CTCL, MF IA CD30+ LPD: primary CTCL γ-δ T-cells 68 White 
27 CTCL, MF IA Hypopigmented 60 African American 
85 CTCL, MF IA Hypopigmented 38 African American 
65 CTCL, MF IA None 40 African American 
180 CTCL, MF IA None 56 African American 
CTCL, MF IA None, lyp, and CD30+ LPD 61 African American 
147 CTCL, MF IA Hypopigmented 45 Asian 
89 CTCL, MF IA Folliculotropic 48 White 
137 CTCL, MF IA Granulomatous 67 White 
28 CTCL, MF IA Hypopigmented 38 White 
38 CTCL, MF IA None 41 White 
42 CTCL, MF IA None 49 White 
52 CTCL, MF IA None 57 White 
55 CTCL, MF IA None 41 White 
61 CTCL, MF IA None 82 White 
73 CTCL, MF IA None 64 White 
82 CTCL, MF IA None 50 White 
107 CTCL, MF IA None 58 White 
111 CTCL, MF IA None 56 White 
144 CTCL, MF IA None 51 White 
145 CTCL, MF IA None 42 White 
150 CTCL, MF IA None 28 White 
152 CTCL, MF IA None 47 White 
183 CTCL, MF IA None 53 White 
200 CTCL, MF IA None 37 White 
215 CTCL, MF IA None 50 White 
226 CTCL, MF IA None 19 White 
166 CTCL, MF IA Poikilodermatous 31 White 
84 CTCL, MF IA Poikilodermatous 55 White 
188 CTCL, MF IA Woringer-Kolopp variant 31 White 
142 CTCL, MF IA Follicular 50 White 
179 CTCL, MF IA Follicular T-helper phenotype 60 White 
50 CTCL, MF IA CD8+ 58 White 
32 CTCL, MF IA Folliculotropic 37 White 
36 CTCL, MF IA Folliculotropic 61 White 
54 CTCL, MF IA Folliculotropic 81 White 
64 CTCL, MF IA Folliculotropic 25 White 
100 CTCL, MF IA Folliculotropic 62 White 
130 CTCL, MF IA Hypopigmented, CD8+ 18 White 
41 CTCL, MF IA None 84 White 
46 CTCL, MF IA None 40 White 
47 CTCL, MF IA None 78 White 
57 CTCL, MF IA None 63 White 
62 CTCL, MF IA None 57 White 
67 CTCL, MF IA None 77 White 
71 CTCL, MF IA None 45 White 
80 CTCL, MF IA None 26 White 
95 CTCL, MF IA None 60 White 
99 CTCL, MF IA None 74 White 
103 CTCL, MF IA None 82 White 
108 CTCL, MF IA None 42 White 
120 CTCL, MF IA None 62 White 
124 CTCL, MF IA None 57 White 
128 CTCL, MF IA None 54 White 
131 CTCL, MF IA None 37 White 
139 CTCL, MF IA None 26 White 
162 CTCL, MF IA None 51 White 
174 CTCL, MF IA None 76 White 
181 CTCL, MF IA None 53 White 
189 CTCL, MF IA None 44 White 
191 CTCL, MF IA None 34 White 
197 CTCL, MF IA None 70 White 
223 CTCL, MF IA None 72 White 
146 CTCL, MF IA None 48 White 
199 CTCL, MF IA None 63 White 
CTCL, MF IB None 52 African American 
CTCL, MF IB Poikilodermatous 69 African American 
203 CTCL, MF IB None 46 African American 
39 CTCL, MF IB Hypopigmented 38 African American 
56 CTCL, MF IB None 51 African American 
77 CTCL, MF IB CD8+ 58 White 
26 CTCL, MF IB Granulomatous 66 White 
87 CTCL, MF IB None 51 White 
91 CTCL, MF IB None 66 White 
136 CTCL, MF IB None 72 White 
173 CTCL, MF IB None 57 White 
194 CTCL, MF IB None 73 White 
115 CTCL, MF IB CD8+ 63 White 
209 CTCL, MF IB CD8+ 81 White 
88 CTCL, MF IB Folliculotropic 76 White 
97 CTCL, MF IB Folliculotropic 44 White 
114 CTCL, MF IB Folliculotropic 58 White 
134 CTCL, MF IB Folliculotropic 53 White 
37 CTCL, MF IB None 68 White 
58 CTCL, MF IB None 59 White 
75 CTCL, MF IB None 64 White 
78 CTCL, MF IB None 46 White 
104 CTCL, MF IB None 62 White 
112 CTCL, MF IB None 45 White 
121 CTCL, MF IB None 71 White 
122 CTCL, MF IB None 67 White 
123 CTCL, MF IB None 55 White 
220 CTCL, MF IB None 72 White 
227 CTCL, MF IB None 62 White 
217 CTCL, MF IB Follicular T-helper phenotype 36 White 
167 CTCL, MF IB None 78 White 
204 CTCL, MF IB Poikilodermatous 61 Hispanic/Latino 
132 CTCL, MF IB None 79 Hispanic/Latino 
206 CTCL, MF IIA Poikilodermatous 41 White 
33 CTCL, MF IIB Large cell transformation 50 African American 
30 CTCL, MF IIB None 66 African American 
157 CTCL, MF IIB Folliculotropic 69 African American 
168 CTCL, MF IIB Folliculotropic 53 African American 
156 CTCL, MF IIB None 80 White 
90 CTCL, MF IIB Folliculotropic 62 White 
63 CTCL, MF IIB None 88 White 
185 CTCL, MF IIB None 75 White 
153 CTCL, MF IIB Tumor 64 White 
141 CTCL, MF IIB Large cell transformation 59 White 
109 CTCL, MF IIIB Erythrodermic 55 White 
213 CTCL, MF IIIB Erythrodermic 65 African American 

F, female; LPD, lymphoproliferative disorder; lyp, lymphomatoid papulosis; M, male.

NK-cell isolation and cytotoxicity

NK cells were isolated from fresh peripheral blood samples by negative enrichment (STEMCELL Technologies) followed by sorting on a BD Aria II analyzer. No phenotypic alterations were noted between the presorted and postsorted NK cells. Purified NK cells were co-cultured with chromium-labeled K562 target cells for 4 hours in a standard chromium release cytotoxicity assay.24  K562 cells were obtained from American Type Culture Collection (ATCC) and were kept in culture less than 1 month. Cells were routinely tested for mycoplasma per routine protocol. Tables 2 and 3 provide clinical information on the patients who participated in NK-cell functional studies.

Table 2.

Characteristics of patients with biopsy-proven CTCL SS

Patient IDDiagnosisStage at diagnosisAge at diagnosis, ySexRaceClonality on initial flow cytometric analysis
69 CTCL, SS IVA1 51 White 84% CD3+/CD26 T-cells; 82% CD4+/CD26; 75% CD7+/CD26 
76 CTCL, SS III 82 White 22% CD3+/CD4+/CD7 T-cells 
86 CTCL, SS IVA1 77 White 92% CD3+/CD4+/CD26; 22% CD3+/CD4+/CD7 
96 CTCL, SS IVA1 67 White 82% CD3+/CD26/CD2 
135 CTCL, SS IVA1 59 White 92% CD3+/CD26 
164 CTCL, SS IVA1 60 White 63% CD3+/CD4+/CD7+/CD26; 34% CD3+/CD4/CD8/CD7/CD26 
187 CTCL, SS IVA1 64 African American 60% CD3+/CD26; 50% CD3 biphasic/CD7 
224 CTCL, SS IVA1 82 White 98% CD2+/CD4+/CD5+/CD3/CD7/CD8/CD26 
227 CTCL, SS IVA1 56 White 93% CD4+/CD26 
Patient IDDiagnosisStage at diagnosisAge at diagnosis, ySexRaceClonality on initial flow cytometric analysis
69 CTCL, SS IVA1 51 White 84% CD3+/CD26 T-cells; 82% CD4+/CD26; 75% CD7+/CD26 
76 CTCL, SS III 82 White 22% CD3+/CD4+/CD7 T-cells 
86 CTCL, SS IVA1 77 White 92% CD3+/CD4+/CD26; 22% CD3+/CD4+/CD7 
96 CTCL, SS IVA1 67 White 82% CD3+/CD26/CD2 
135 CTCL, SS IVA1 59 White 92% CD3+/CD26 
164 CTCL, SS IVA1 60 White 63% CD3+/CD4+/CD7+/CD26; 34% CD3+/CD4/CD8/CD7/CD26 
187 CTCL, SS IVA1 64 African American 60% CD3+/CD26; 50% CD3 biphasic/CD7 
224 CTCL, SS IVA1 82 White 98% CD2+/CD4+/CD5+/CD3/CD7/CD8/CD26 
227 CTCL, SS IVA1 56 White 93% CD4+/CD26 
Table 3.

Characteristics of patients with CTCL, treatment, and experimental assay performed

Patient IDAssaySexRaceCTCL typeStage at sample procurementTreatment at time of collection
MF1 Flow cytometry White MF IA Imiquimod 
MF2 Flow cytometry White MF IA Desoximetasone 
MF3 Flow cytometry White MF IA Clobetasol 
U16-2023 Cytotoxicity assay White MF IB PUVA, triamcinolone, imiquimod 
U16-2101 Cytotoxicity assay White MF IB Bexarotene 
U16-2102 Cytotoxicity assay White MF IB Bexarotene 
U16-2225 Cytotoxicity assay White MF IIB Gemcitabine/doxorubicin 
U16-2228 Cytotoxicity assay White MF IIB Bexarotene 
U16-2237 Cytotoxicity assay African American MF IA Topical steroids 
U17-0092 Cytotoxicity assay White MF IB Bexarotene 
U17-0093 Cytotoxicity assay White MF IB Topical steroids 
U17-0094 Cytotoxicity assay White MF IA nbUVB 
U17-0095 Cytotoxicity assay/RNA sequencing White MF IA Bexarotene 
U17-0253 Cytotoxicity assay/RNA sequencing White MF IA Topical bexarotene 
U17-0254 Cytotoxicity assay/RNA sequencing White MF IB nbUVB 
U17-0314 Flow cytometry African American SS IVB Romidepsin 
U17-0315 Flow cytometry White MF 1A Topical steroids 
U17-0316 Flow cytometry White MF IB Valchlor gel 
U17-0341 Flow cytometry White MF 1A Oral or topical bexarotene 
U17-0342 Flow cytometry White MF 1A Mechlorethamine gel, clobetasol 
U17-0343 Flow cytometry White MF 1A Mechlorethamine gel, TAC 
U17-0350 Flow cytometry African American MF IA Topical steroids 
U17-0352 Flow cytometry White MF IA Oral bexarotene 
U17-0353 Flow cytometry White MF 1B Interferon, nbUVB, TAC, clobetasol 
U17-0543 RNA sequencing White SS 1VA Romidepsin, IPH 4102 
U17-0593 RNA sequencing White MF 1B Gemcitabine/ doxorubicin 
U17-0594 RNA sequencing White SS IVA1 Topical steroids 
U17-0595 RNA sequencing White MF IA TAC 
U17-1613 Phospho-specific flow cytometry White MF IIB Brentuximab 
U17-1614 Phospho-specific flow cytometry White SS 1VA2 Bendamustine + brentuximab vedotin 
U17-1615 Phospho-specific flow cytometry White MF IA nbUVB 
U17-1616 Phospho-specific flow cytometry White MF 1B TAC, nbUVB 
U17-1654 Phospho-specific flow cytometry White MF 1A Clobetasol 
U17-1657 Phospho-specific flow cytometry White MF IA TAC 
U17-1658 Phospho-specific flow cytometry White MF 1A TAC, topical imiquimod 
U17-1649 Phospho-specific flow cytometry White MF 1B Romidepsin 
U17-1651 Phospho-specific flow cytometry White MF 1B nbUVB, TAC, interferon, oral bexarotene 
U17-1652 Phospho-specific flow cytometry White MF IA Topical bexarotene 
U17-1653 Phospho-specific flow cytometry White MF IA TAC 
U17-1819 Phospho-specific flow cytometry White MF IA Romidepsin 
U17-1936 Phospho-specific flow cytometry White SS IVA Bexarotene and interferon 
U17-1937 Phospho-specific flow cytometry White SS IIIA None 
U17-1938 Phospho-specific flow cytometry White MF 1A None 
Patient IDAssaySexRaceCTCL typeStage at sample procurementTreatment at time of collection
MF1 Flow cytometry White MF IA Imiquimod 
MF2 Flow cytometry White MF IA Desoximetasone 
MF3 Flow cytometry White MF IA Clobetasol 
U16-2023 Cytotoxicity assay White MF IB PUVA, triamcinolone, imiquimod 
U16-2101 Cytotoxicity assay White MF IB Bexarotene 
U16-2102 Cytotoxicity assay White MF IB Bexarotene 
U16-2225 Cytotoxicity assay White MF IIB Gemcitabine/doxorubicin 
U16-2228 Cytotoxicity assay White MF IIB Bexarotene 
U16-2237 Cytotoxicity assay African American MF IA Topical steroids 
U17-0092 Cytotoxicity assay White MF IB Bexarotene 
U17-0093 Cytotoxicity assay White MF IB Topical steroids 
U17-0094 Cytotoxicity assay White MF IA nbUVB 
U17-0095 Cytotoxicity assay/RNA sequencing White MF IA Bexarotene 
U17-0253 Cytotoxicity assay/RNA sequencing White MF IA Topical bexarotene 
U17-0254 Cytotoxicity assay/RNA sequencing White MF IB nbUVB 
U17-0314 Flow cytometry African American SS IVB Romidepsin 
U17-0315 Flow cytometry White MF 1A Topical steroids 
U17-0316 Flow cytometry White MF IB Valchlor gel 
U17-0341 Flow cytometry White MF 1A Oral or topical bexarotene 
U17-0342 Flow cytometry White MF 1A Mechlorethamine gel, clobetasol 
U17-0343 Flow cytometry White MF 1A Mechlorethamine gel, TAC 
U17-0350 Flow cytometry African American MF IA Topical steroids 
U17-0352 Flow cytometry White MF IA Oral bexarotene 
U17-0353 Flow cytometry White MF 1B Interferon, nbUVB, TAC, clobetasol 
U17-0543 RNA sequencing White SS 1VA Romidepsin, IPH 4102 
U17-0593 RNA sequencing White MF 1B Gemcitabine/ doxorubicin 
U17-0594 RNA sequencing White SS IVA1 Topical steroids 
U17-0595 RNA sequencing White MF IA TAC 
U17-1613 Phospho-specific flow cytometry White MF IIB Brentuximab 
U17-1614 Phospho-specific flow cytometry White SS 1VA2 Bendamustine + brentuximab vedotin 
U17-1615 Phospho-specific flow cytometry White MF IA nbUVB 
U17-1616 Phospho-specific flow cytometry White MF 1B TAC, nbUVB 
U17-1654 Phospho-specific flow cytometry White MF 1A Clobetasol 
U17-1657 Phospho-specific flow cytometry White MF IA TAC 
U17-1658 Phospho-specific flow cytometry White MF 1A TAC, topical imiquimod 
U17-1649 Phospho-specific flow cytometry White MF 1B Romidepsin 
U17-1651 Phospho-specific flow cytometry White MF 1B nbUVB, TAC, interferon, oral bexarotene 
U17-1652 Phospho-specific flow cytometry White MF IA Topical bexarotene 
U17-1653 Phospho-specific flow cytometry White MF IA TAC 
U17-1819 Phospho-specific flow cytometry White MF IA Romidepsin 
U17-1936 Phospho-specific flow cytometry White SS IVA Bexarotene and interferon 
U17-1937 Phospho-specific flow cytometry White SS IIIA None 
U17-1938 Phospho-specific flow cytometry White MF 1A None 

nbUVB narrow-band ultraviolet B light therapy; PUVA, psoralen and ultraviolet A (light therapy); Tac, triamcinolone cream.

RNA sequencing analysis

NK cells (CD56+/lineage) were isolated as described above. Total RNA was isolated from cell samples using standard methods (Active Motif). To generate heat maps of the most differential genes, additional comparative metrics were calculated such as fold-change between averages of fragments per kilobase of transcript per million mapped reads (FPKM) values between groups of interests, and analysis of variance P values comparing 2 or more groups. Genes with no values in all samples or a value in only 1 of the 9 samples were removed.

Phosphorylated STAT staining and analysis

Fresh peripheral blood samples were obtained from CTCL patients and age-matched normal donors. Phosphorylated signal of transducer and activator of transcription 3 (pSTAT3) and STAT5 (pSTAT5) were evaluated by direct whole blood antibody labeling (BD Biosciences). Median fluorescence intensity was calculated for each STAT protein.

Results

The absolute number of NK cells in peripheral blood was evaluated in CTCL patients and compared with that in normal donors (n = 51). There was no statistical difference in absolute number of NK cells when all patients with CTCL were included (Figure 1A); however, SS patients had on average 57.4% fewer NK cells compared with normal donors (supplemental Figure 1). We then evaluated the association between absolute NK-cell counts and overall survival. NK-cell counts were significantly associated with overall survival (P = .041; Figure 1B). To evaluate NK-cell function, NK cells were purified from fresh peripheral blood (Figure 1C) and evaluated for cytotoxic function against K562 target cells.24  CTCL patients had significantly higher levels of NK-cell cytotoxicity compared with normal donors (Figure 1D). Although these findings differ from those in previous reports, earlier work did not use NK cells isolated from fresh peripheral blood,10-12  evaluate frozen samples, or use cytokine stimulation.14 

Figure 1.

NK-cell number and correlation with CTCL patient survival. (A) Absolute NK-cell numbers were calculated in normal donors (n = 51; mean ± standard error of the mean [SEM], 0.2442 ± 0.02 ) and CTCL patients (n = 121; 0.208 ± 0.01; P = .08). (B) Kaplan-Meier curves for overall survival at possible absolute NK-cell counts in CTCL patients (n = 121). (C) NK cells, CD56+/lineage (CD3/CD14/CD20), were isolated from freshly obtained peripheral blood samples from CTCL patients and normal control donors. (D) Purified NK cells were co-cultured with K562 leukemic targets in a standard chromium release assay at indicated ratios. Data are presented as mean ± SEM. *P ≤ .05; **P ≤ .01; ***P ≤ .001; unpaired 2-tailed Student t test. FSC, forward scatter; ns, not significant; SSC, side scatter.

Figure 1.

NK-cell number and correlation with CTCL patient survival. (A) Absolute NK-cell numbers were calculated in normal donors (n = 51; mean ± standard error of the mean [SEM], 0.2442 ± 0.02 ) and CTCL patients (n = 121; 0.208 ± 0.01; P = .08). (B) Kaplan-Meier curves for overall survival at possible absolute NK-cell counts in CTCL patients (n = 121). (C) NK cells, CD56+/lineage (CD3/CD14/CD20), were isolated from freshly obtained peripheral blood samples from CTCL patients and normal control donors. (D) Purified NK cells were co-cultured with K562 leukemic targets in a standard chromium release assay at indicated ratios. Data are presented as mean ± SEM. *P ≤ .05; **P ≤ .01; ***P ≤ .001; unpaired 2-tailed Student t test. FSC, forward scatter; ns, not significant; SSC, side scatter.

A comprehensive surface immunophenotypic analysis of NK cells revealed no significant differences (supplemental Figure 2), so we hypothesized that there could be alterations in pathway activation in CTCL patients. Whole transcriptome analysis was completed to evaluate NK-cell activation. A heat map generated by hierarchical clustering of the differentially expressed genes shows the unique patterns of gene expression in NK cells from CTCL patients and healthy donors (Figure 2A). NK cells from CTCL patients had increased expression of cytolytic mediators, including perforin, granzyme A, granzyme B, Fas, and tumor necrosis factor-alpha–related apoptosis-inducing ligand (TRAIL) (Figure 2B). Furthermore, we observed significantly increased cytokine (interferon-γ [IFN-γ]) production in patient NK cells. Using ingenuity pathway analysis, we determined differential expression of key pathways, including IL-15, IL-15/IL-2 receptors, IFN-γ, and several surface activating molecules such as CD2, CD40, FCER1G, CD80, KLR, SELL, and CD244. Furthermore, NF-kB and ICOS were inhibited in our data set (Figure 2C; supplemental Figures 3 and 4).

Figure 2.

Differential RNA expression analysis in CTCL patients. NK cells were isolated from CTCL patients and normal donors and evaluated by RNA sequencing. (A) Heat map of differentially regulated transcripts among normal donors (n = 3) and CTCL MF (n = 5) and CTCL SS (n = 2) patients. (B) Mediators of cytolytic activity and NK-cell activation were evaluated in all CTCL patients compared with normal controls. Perforin mean ± SEM of transcript in healthy donor vs CTCL patients for NK cells (91.39 ± 9.18 [n = 3] vs 485.7 ± 43.15 [n = 7]; P = .0004), granzyme A (GZMA) (58.15 ± 6.605 [n = 3] vs 218.7 ± 29.78 [n = 7]; P = .0094), granzyme B (GZMB) (143 ± 23.79 [n = 3] vs 431 ± 68.13 [n = 7]; P = .03), Fas (1.413 ± 0.01849 [n = 3] vs 3.967 ± 0.4395 [n = 7]; P = .0063), tumor necrosis factor-alpha–related apoptosis-inducing ligand (TRAIL) (3.756 ± 0.4925 [n = 3] vs 7.634 ± 1.033 [n = 7]; P = .0477), and IFN-G (2.861 ± 0.5007 [n = 3] vs 7.325 ± 1.621 [n = 7]; P = .1218). (C) An ingenuity pathway analysis upstream functional analysis was performed in an enriched data set from the NK cells. Red indicates upregulation in CTCL patients compared with normal donors; green indicates reduced expression. (D) RNA expression of receptor components required for both IL-2 and IL-15 signaling were evaluated in CTCL patients and compared with that in normal donors (IL-15Rα: mean ± SEM of relative RNA in normal vs CTCL, 0.8677 ± 0.1018 [n = 3] vs 1.772 ± 0.2055 [n = 7]; P = .03; IL-15Rγ, 57.02 ± 7.525 [n = 3] vs 154.7 ± 12.14 [n = 7]; P = .001. IL-15Rβ was also elevated (128 ± 23.68 [n = 3] vs 223.2 ± 25.43 [n = 7]; P = .056). (E) Protein expression of pSTAT3 and pSTAT5 on NK cells was determined in freshly obtained whole blood samples from CTCL patients and matched with that of normal donors by flow cytometry. Graph indicates mean fluorescence intensity (MFI) of normal (gray bars [n = 7]) compared with CTCL patients (green bars [n = 14]); pSTAT3: normal, 2417 ± 154.1 (n = 7) vs CTCL, 2734 ± 199; P = .32; pSTAT5: normal, 1253 ± 50.23 (n = 8) vs CTCL, 1405 ± 64.35; P = .028. (F) Schematic of interaction between malignant CD4+ T cells in CTCL patients producing IL-15, which binds to the upregulated IL-15 receptor complex on NK cells and enhances downstream activating pathways in NK cells. Data are presented as mean ± SEM. *P ≤ .05; **P ≤ .01; ***P ≤ .001; unpaired 2-tailed Student t test. FPKM, fragments per kilobase million.

Figure 2.

Differential RNA expression analysis in CTCL patients. NK cells were isolated from CTCL patients and normal donors and evaluated by RNA sequencing. (A) Heat map of differentially regulated transcripts among normal donors (n = 3) and CTCL MF (n = 5) and CTCL SS (n = 2) patients. (B) Mediators of cytolytic activity and NK-cell activation were evaluated in all CTCL patients compared with normal controls. Perforin mean ± SEM of transcript in healthy donor vs CTCL patients for NK cells (91.39 ± 9.18 [n = 3] vs 485.7 ± 43.15 [n = 7]; P = .0004), granzyme A (GZMA) (58.15 ± 6.605 [n = 3] vs 218.7 ± 29.78 [n = 7]; P = .0094), granzyme B (GZMB) (143 ± 23.79 [n = 3] vs 431 ± 68.13 [n = 7]; P = .03), Fas (1.413 ± 0.01849 [n = 3] vs 3.967 ± 0.4395 [n = 7]; P = .0063), tumor necrosis factor-alpha–related apoptosis-inducing ligand (TRAIL) (3.756 ± 0.4925 [n = 3] vs 7.634 ± 1.033 [n = 7]; P = .0477), and IFN-G (2.861 ± 0.5007 [n = 3] vs 7.325 ± 1.621 [n = 7]; P = .1218). (C) An ingenuity pathway analysis upstream functional analysis was performed in an enriched data set from the NK cells. Red indicates upregulation in CTCL patients compared with normal donors; green indicates reduced expression. (D) RNA expression of receptor components required for both IL-2 and IL-15 signaling were evaluated in CTCL patients and compared with that in normal donors (IL-15Rα: mean ± SEM of relative RNA in normal vs CTCL, 0.8677 ± 0.1018 [n = 3] vs 1.772 ± 0.2055 [n = 7]; P = .03; IL-15Rγ, 57.02 ± 7.525 [n = 3] vs 154.7 ± 12.14 [n = 7]; P = .001. IL-15Rβ was also elevated (128 ± 23.68 [n = 3] vs 223.2 ± 25.43 [n = 7]; P = .056). (E) Protein expression of pSTAT3 and pSTAT5 on NK cells was determined in freshly obtained whole blood samples from CTCL patients and matched with that of normal donors by flow cytometry. Graph indicates mean fluorescence intensity (MFI) of normal (gray bars [n = 7]) compared with CTCL patients (green bars [n = 14]); pSTAT3: normal, 2417 ± 154.1 (n = 7) vs CTCL, 2734 ± 199; P = .32; pSTAT5: normal, 1253 ± 50.23 (n = 8) vs CTCL, 1405 ± 64.35; P = .028. (F) Schematic of interaction between malignant CD4+ T cells in CTCL patients producing IL-15, which binds to the upregulated IL-15 receptor complex on NK cells and enhances downstream activating pathways in NK cells. Data are presented as mean ± SEM. *P ≤ .05; **P ≤ .01; ***P ≤ .001; unpaired 2-tailed Student t test. FPKM, fragments per kilobase million.

Our group previously demonstrated that CD4+ T cells from CTCL patients exhibit elevated levels of IL-15, a key cytokine that mediates NK-cell activation and homeostasis.19  There was significant upregulation of several IL-15 receptor subunits on NK cells (Figure 2D). To further confirm activation of the IL-15 pathway, we evaluated STAT signaling in fresh peripheral blood NK cells by flow cytometry, because IL-15 signals through both STAT3 and STAT5.25  There was a nonsignificant trend toward increased pSTAT3 expression and a significant upregulation of pSTAT5 in CTCL patients compared with normal donors (Figure 2E). Overall, we propose a mechanism by which malignant CD4+ T cells produce IL-15, which binds to the highly expressed IL-15 receptor complex on NK cells from CTCL patients. NK-cell activation is reflected in enhanced cytotoxicity, STAT5 phosphorylation, and upregulation of downstream effectors (Figure 2F).

Discussion

Although our initial goal with this work was to define the functional capacity of freshly isolated NK cells in CTCL patients, our comprehensive analysis of both NK-cell function and expression profiles uncovered intriguing results. One of the most interesting findings of this study was the significant association between NK-cell number and CTCL patient survival. To the best of our knowledge, this is the first description of higher NK-cell numbers in a malignancy being associated with decreased short-term survivability. The cause of this significant clinical relationship is unknown. Few recent studies have described the tumor-promoting potential of NK cells through their ability to upregulate certain oncogenic pathways such as VEGF-A,25  mediated in part by reduced STAT5 activity. We described increased pSTAT5 and did not identify upregulation of VEGF-A or other immunosuppressive factors. It is possible that there are other yet undiscovered mechanisms of tumor promotion by NK cells, and these may lead to the failure of NK cells to control tumor progression despite elevated cytotoxic activity. It is also possible that there may be additional immunosuppressive mechanisms that are present in the microenvironment of CTCL patients. For example, previous studies by our group and others have demonstrated alterations in NK-cell signaling because of the presence of suppressive myeloid cells such as myeloid-derived suppressor cells and tumor-associated macrophages in cancer patients,26  and both populations have been reported among CTCL patients.27-29 

Additional checkpoint inhibitors, such as PD-1, CTLA-4, TIGIT, and TIM-3 may also play a role in decreasing NK-cell function in patients, because previous studies demonstrated that these inhibitors may be increased in the presence of IL-15.30-32  Transcript analysis of PD-1 did show a moderate increase in the NK cells isolated from CTCL patients (supplemental Figure 5), and because K562 cells are known to express low levels of the PD-1 ligand (PD-L1), this inhibitory effect might not be observed in vitro.33  Analysis of inhibitory ligand expression on CTCL cells in circulation in SS patients or in skin biopsies from MF patients suggests that novel potential therapeutic targets could be focused on removing this barrier to immune cell activation.

It is known that γ cytokines such as IL-7 and IL-15 are important for CTCL progression.19,34,35  Indeed, as disease progresses, neoplastic CD4+ T cells express higher levels of IL-15.19  Furthermore, IL-15 overexpression alone can induce CTCL in a murine model of the disease.19  IL-15 is also known to stimulate NK-cell proliferation and cytotoxicity via phosphorylation of STAT5.13,36-38  Thus, we proposed that IL-15 derived from the neoplastic CD4+ T cells in CTCL may contribute to increased NK-cell cytotoxicity observed in patient samples. Indeed, transcriptome analysis reveals upregulation of IL-15–induced signaling pathways, and protein levels of pSTAT5 were significantly elevated in patients with CTCL, further confirming this relationship between increased NK-cell activation and cytotoxicity in CTCL patients. However, recent reports by multiple groups have demonstrated that continuous exposure to IL-15 can lead to a decrease in NK-cell proliferation, cell cycle arrest, and NK-cell exhaustion, suggesting that chronic exposure to IL-15 could also lead to immune-cell exhaustion in CTCL patients.39,40  It is also possible that although NK cells from peripheral blood exhibit higher levels of cytotoxicity, those localized in skin lesions where localized levels of IL-15 may be higher have additional defects that would render them ineffective at lysing the CD4+ malignant T cells.

The reason for this counterintuitive finding is not known; however, we speculate that although NK cells are maintained in a hyperactive state in CTCL patients, malignant cell recognition is impaired. It is also possible that the ability of NK cells to form an effective immune synapse and polarize actin and cytolytic granules is altered in the microenvironment of CTCL patients. In support of this theory, a key mediator of NK-cell polarization, phosphatase and tensin homolog (PTEN), is significantly overexpressed in NK cells from CTCL patients in the RNA sequencing analysis (data not shown). We have previously demonstrated a role for PTEN in the organization of the components of the immunologic synapse and the appropriate convergence of cytolytic granules.41  It is also possible that the process of NK-cell recognition of malignant CD4+ T cells is altered in CTCL patients. Previous studies by Bouaziz et al15  suggest that NK cells are potentially able to be activated to kill autologous CTCL cells, which suggests that malignant CD4+ T cells are susceptible to NK-cell killing, but additional mechanisms of inhibition such as the ones discussed above prevent this in CTCL patients. Although further investigation of multiple facets of NK-cell recognition is warranted, it is clear that the overall immunosuppressive microenvironment in CTCL patients contributes to insufficiency of patient NK cells to effectively control CTCL progression.

The full-text version of this article contains a data supplement.

Acknowledgments

Support for this study was provided by the American Association for Cancer Research (17-20-46-MUND) (B.M.-B.), Spatz Foundation (A.M.), American Skin Association (A.M.), Cutaneous Lymphoma Foundation (A.M.), Pelotonia (A.M.), DeStefano Lymphoma Research funds (A.M.), and the National Institutes of Health, National Cancer Institute (CA016058). The authors gratefully acknowledge The Ohio State University Comprehensive Cancer Center Leukemia Tissue Bank Shared Resource (P30CA016058) for patient samples.

Authorship

Contribution: B.M.-B., L.C., H.C.M., Y.Y., G.L., N.D., and A.M. assisted with experimental design and implementation; E.M. and D.K. assisted with statistical modeling and bioinformatics analysis; P.P., B.W., and S.H. assisted with patient identification and sample acquisition; N.C., D.A.L., and A.M. assisted with ingenuity pathway analysis; B.M.-B., R.K., and A.M. assisted with writing and analyzing experiments; and A.G.F., P.P., and M.A.C. provided critical design and manuscript assistance.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Anjali Mishra, Comprehensive Cancer Center, Division of Dermatology, Department of Internal Medicine, The Ohio State University, 886 Biomedical Research Tower, 460 W. 12th Ave, Columbus, OH 43210; e-mail: anjali.mishra@osumc.edu; and Bethany Mundy-Bosse, Comprehensive Cancer Center, Division of Hematology, Department of Internal Medicine, The Ohio State University, 882 Biomedical Research Tower, 460 W. 12th Ave, Columbus, OH 43210; e-mail: bethany.mundy@osumc.edu.

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