Revisiting the initial diagnosis and blood staging of mycosis fungoides and Sézary syndrome with the KIR3DL2 marker.

BACKGROUND: The early diagnosis of Sézary syndrome (SS) is challenging. Loss of CD7 and CD26 expression on CD4+ T cells is the currently used criterion in the initial diagnosis and staging of patients with SS. OBJECTIVES: Our aim was to evaluate the respective value of CD26, CD7 and KIR3DL2 expression on CD4+ T cells and total lymphocytes at initial diagnosis of SS. METHODS: This prospective study included 254 patients with clinical features consistent with cutaneous T-cell lymphoma seen at our institution between March 2014 and February 2019. Peripheral blood analysis by flow cytometry was performed for each patient at the time of diagnosis and during follow-up. The diagnosis of SS was based on ISCL/EORTC criteria. RESULTS: The presence of KIR3DL2+ Sézary cells (SCs) ≥ 200 µL-1 correlated with the diagnosis of SS, with sensitivity of 88·6% and specificity of 96·3%. All 154 patients with either inflammatory skin disease or other haematological disease had KIR3DL2+ cells < 200 µL-1 , while eight of them had CD4+ CD26- T cells ≥ 1000 µL-1 . Of five patients with SS and lymphopenia, four had CD4+ CD7- T cells < 1000 µL-1 and three had CD4+ CD26- T cells < 1000 µL-1 . However, all of them had KIR3DL2+ CD4+ T cells ≥ 200 µL-1 . Among patients with available samples during evolution, all B1-staged patients with ≥ 200 µL-1 KIR3DL2+ SCs at diagnosis evolved to B2 stage within 7 months. CONCLUSIONS: KIR3DL2 expression on T cells is highly specific and helps the early diagnosis of SS, especially in those patients with lymphopenia. What's already known about this topic? In the ISCL/EORTC cutaneous T-cell lymphoma (CTCL) categorization of blood involvement (B0-B2), B2 is defined as a T-cell receptor clonal rearrangement in blood, associated with high blood-smear Sézary cell (SC) count. Flow cytometry was developed to circumvent interobserver variability of SC manual counts; however, it mostly relies on detection of cells lacking CD7 and/or CD26 expression. We previously reported the reliability of KIR3DL2 as the first positive SC marker. What does this study add? Based on our analysis of 254 patients, we propose that KIR3DL2 be added to the ISCL/EORTC criteria for initial diagnosis of Sézary syndrome (SS) and B2 staging. This marker improved sensitivity of SS B2-stage CTCL diagnosis with a specificity > 95%, especially for patients with lymphopenia. We found KIR3DL2 helped early diagnosis of SS and was more reliable than CD26 in assessing blood tumour burden during therapy. What is the translational message? SC quantification is the major means of staging at initial diagnosis and monitoring blood tumour burden in a clinical trials setting. We recommend using a threshold value of KIR3DL2+ SCs ≥ 200 µL-1 or KIR3DL2+ SCs/lymphocytes ≥ 10% in the diagnostic criteria of SS and propose a novel algorithm for CTCL B2 blood staging.

Reconstitution of regulatory T-cell subsets after allogeneic hematopoietic SCT.

Previous studies on regulatory T-cell (Treg) reconstitution after allogeneic hematopoietic SCT (HSCT) have suggested that, within the GVHD process, imbalance between effector T cells and Tregs may be more important than the absolute numbers of circulating Tregs. No study has analyzed naive vs memory Treg reconstitution in a longitudinal cohort with large numbers of patients. The reconstitution of total and subsets of Treg was prospectively analyzed by flow cytometry in 185 consecutive recipients at 3, 6, 12 and 24 months after allogeneic HSCT. The levels of total, naive and memory Tregs increased, mainly within the memory subset, but remained lower than healthy controls up to 2 years after transplantation. Reduced-intensity conditioning and peripheral blood (PBSC) as the source of stem cells were associated with better 3-month reconstitution. In multivariate analysis, PBSC, recipient age ⩽25 and no anti-thymoglobulin in the conditioning regimen were associated with a better Treg reconstitution. Naive Treg long-term reconstitution was mainly influenced by recipient age. Whereas prior acute GVHD impaired Treg reconstitution, Treg subsets (absolute numbers and frequencies relative to CD4(+) T-cell subsets) at 3, 6 and 12 months after HSCT were not associated with the occurrence of a later episode of chronic GVHD.

NK resistance of tumor cells from multiple myeloma and chronic lymphocytic leukemia patients: implication of HLA-G.

Exploiting the antitumor effect of natural killer (NK) cells has regained interest in light of data from preclinical and clinical work on the potential of alloreactive NK cells. Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) represent the two most prevalent adult hematological malignancies in the western hemisphere. To evaluate the role of NK cells in the immune surveillance and their therapeutic potential for CLL and MM, tumor cell susceptibility to NK-mediated killing was investigated. Results show relative resistance of tumor cells from CLL as well as MM (73 and 70% of the patients, respectively) to NK-mediated killing. To gain insight into molecular mechanisms of this resistance, the expression of the tolerogenic HLA-G molecule in CLL and MM and its relevance to susceptibility to NK-mediated killing were investigated. HLA-G transcript was found in tumor cells from 89% (n=19) of CLL and 100% (n=9) of MM patients examined. HLA-G1 surface expression was observed in CLL and was very low or undetectable in MM. Notably, blocking of HLA-G1 with specific antibody on CLL samples increased their susceptibility to NK-mediated killing, demonstrating that HLA-G participates in protecting CLL cells from NK-mediated killing and may thus contribute to their immune escape in vivo.

Ex vivo purging with NK-92 prior to autografting for chronic myelogenous leukemia.

Although controversial, purging of the autograft may be necessary to optimize transplant outcome, especially if better treatments become available to eliminate or control residual disease that may be left after the conditioning regimen. The intent of this study was to show that immunological purging with the cytotoxic cell line NK-92 effectively reduces the number of clonogenic cells and that the method can be performed in compliance with GMP. Owing to the easy quantification of bcr-abl transcripts, chronic myelogenous leukemia (CML) was used as a model disease for proof of principle. A detection level of 10(-7) bcr-abl+ cells and purging efficiency of four logs were achievable for the bcr-abl+ cell line, K562. Leukapheresis products collected from CML patients after stem cell mobilization were then tested. For all patients tested, residual CML cells were highly sensitive to purging by NK-92 with a purging efficacy of several logs. No adverse effect on hematopoietic progenitor cell function was noted. These results demonstrate the efficacy of NK-92 as a purging agent to decrease or eliminate malignant contamination of autologous stem cell grafts and establish proof of principle for ex vivo purging of CML autografts using cytotoxic effector cells.

Ex vivo purging of stem cell autografts using cytotoxic cells.

Autologous stem cell transplantation (SCT) is the treatment alternative offered to patients that cannot benefit from allogeneic transplantation due to lack of suitable donor or age limitations. However, the outcome of autologous SCT is largely hindered by the high relapse rate. Two major factors can account for relapse after autologous SCT: the persistence of residual malignant cells resistant to chemo/radiotherapy left either in the body or in the autograft. Therefore, the rationale for purging autografts of residual malignant cells comes from the limitations of conventional high-dose chemo/radiotherapy in achieving a complete eradication of residual tumor cells in the marrow. To date, different purging modalities have been exploited. Immunological methods of purging present the advantage of being non-cross-reactive with conventional chemotherapy. These immunologic methods include depletion using antibody targeting of the malignant cells, ex vivo activation/generation of the autologous cytotoxic cells, in particular that of natural killer/lymphokine-activated killer (NK/LAK) and cytokine-induced killer (CIK) cells, and ex vivo purging of autografts using cytotoxic cell lines. The generation of ex vivo-expanded and activated autologous cytotoxic cells (CTL or NK) has generated increasing interest for the treatment of different malignancies. Unfortunately, the isolation and expansion of these cells have proven to be technically difficult. As an alternative, the use of cytotoxic cell lines as immune effectors has been proposed. The two available human cytotoxic cell lines TALL104 and NK-92 are currently in clinical trials and a number of studies have suggested their effectiveness as an immunotherapeutic agent including for ex vivo purging of autografts.

Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92.

NK-92, a highly cytotoxic, interleukin-2 (IL-2)-dependent human natural killer (NK) cell line, has been of interest for basic and translational research. We report on a comprehensive analysis of NK-92 for factors implicated in NK cytotoxicity to elucidate factors underlying NK-92's high cytolytic activity and target range. Thus, we hope to develop a method to identify patients best suited to NK-92 immunotherapy. In addition, as a model system, we hope to increase understanding of the basis for the elevated activity exhibited by activated NK (ANK) cells. NK-92 exhibits an unusual receptor expression profile, expressing a relatively large number of activating (NKp30, NKp46, 2B4, NKGD, E, CD28) receptors. Conversely, it expresses few inhibitory receptors (NKGA/B, low levels of KIR2DL4, ILT-2), lacking most of the killer inhibitory receptors (KIRs) clonally expressed on normal NK cells. In addition, NK-92 expresses high levels of molecules involved in the perforin-granzyme cytolytic pathway as well as additional cytotoxic effector molecules including tumor necrosis factor (TNF)-superfamily members FasL, TRAIL, TWEAK, TNF-alpha, indicating the ability to kill via alternative mechanisms. NK-92 also expresses other molecules implicated immune effector cell regulation (CD80, CD86, CD40L, TRANCE) whose relevance in NK killing is unclear. This study provides initial data to develop a method to identify NK-92 susceptible cells (cells expressing ligands for NK-92 activating receptors ie CD48 for 2B4 and CD80/86 for CD28). Furthermore, this work suggests mechanisms that may contribute to ANK cell activity, including modulation of receptor expression to favor activation, up-regulation of cytotoxic effector molecules, and acquisition of new cytolytic pathways.

Characterization of genetically altered, interleukin 2-independent natural killer cell lines suitable for adoptive cellular immunotherapy.

NK-92 is a highly cytotoxic natural killer (NK) tumor cell line that possesses properties that make it an excellent candidate for adoptive cellular immunotherapy. However, the cytotoxicity of NK cells is dependent on cytokines such as interleukin 2 (IL-2). Although NK-92 cells maintain cytotoxicity for a time after withdrawal of IL-2, clinical use will probably require prolonged treatment with fully activated cells to eliminate disease effectively. The ability to support cytotoxic cells with exogenously administered IL-2 is limited by associated toxicity. Therefore, we describe the transfection of the IL-2-dependent NK-92 cell line with human IL-2 (hIL-2) cDNA by particle-mediated gene transfer to create two IL-2-independent variants, NK-92MI and NK-92 CI, and describe their characterization and comparison with parental cells. Both variants were shown to contain, express, and synthesize the hIL-2 cDNA. IL-2 synthesis was higher in NK-92MI cells compared with NK-92CI cells, with no expression in parental cells. Functionally, the cytotoxicity of all three cell lines was similar and coincubation with IL-2-independent variants did not affect hematopoietic progenitor cells. NK-92MI and NK-92CI cells were more radiosensitive than NK-92 cells, with proliferation inhibited at lower radiation doses and increased morality and decreased cytotoxicity compared with parental cells. Data presented here show that we have created by particle-mediated gene transfer two IL-2-independent variants of NK-92 that are identical to parental cells in virtually all respects, including high cytotoxic activity. The nonviral transfection of these cells makes them suitable for clinical applications. These IL-2-independent cells should allow prolonged treatment with fully active natural killer cells without the need for exogenous IL-2 support.

TNF-alpha induction of CD44-mediated leukocyte adhesion by sulfation.

Regulation of cell adhesion is important for immune system function. CD44 is a tightly regulated cell adhesion molecule present on leukocytes and implicated in their attachment to endothelium during an inflammatory immune response. The proinflammatory cytokine tumor necrosis factor-alpha, but not interferon-gamma, was found to convert CD44 from its inactive, nonbinding form to its active form by inducing the sulfation of CD44. This posttranslational modification was required for CD44-mediated binding to the extracellular matrix component hyaluronan and to vascular endothelial cells. Sulfation is thus a potential means of regulating CD44-mediated leukocyte adhesion at inflammatory sites.

Induction of sensitivity to NK-mediated cytotoxicity by TNF-alpha treatment: possible role of ICAM-3 and CD44.

SR-91 is a natural killer (NK)-resistant leukemic cell line expressing a low level of ICAM-1. Pre-treatment of SR-91 cells with TNF-alpha or IFN-gamma, increased both ICAM-1 (CD54) expression on SR-91 cells and binding to the human NK cell line NK-92. However, only TNF-alpha-treated SR-91 cells became sensitive to killing by NK-92 cells. The increased binding induced by both cytokines and the TNF-alpha-induced sensitivity of SR-91 cells to NK-92 cell killing were abrogated by anti-LFA-1 mAb as well as by a combination of antibodies against the three ligands of LFA-1 (CD11a/CD18), ICAM-1 (CD54), ICAM-2 (CD102) and ICAM-3 (CD50). This indicated that LFA-1 interaction with the three ICAMs on SR-91 cells is essential for effector-target cell binding (which is a prerequisite for subsequent target cell lysis), but is insufficient to render the SR-91 cells sensitive to killing by NK-92 cells. TNF-alpha, but not IFN-gamma also induced the activation of LFA-1, CD44 and beta1 integrins on SR-91 cells. Based on these observations we propose that the differential effect of TNF-alpha and IFN-gamma could be related to the activation of certain adhesion molecules on the surface of SR-91 cells by TNF-alpha that, upon interaction with their counter-receptors on NK-92 cells, lead to the activation of the NK-92 cells.

A cytotoxic NK-cell line (NK-92) for ex vivo purging of leukemia from blood.

Myeloablative chemo-/radiotherapy supported by transplantation of autologous bone marrow or blood progenitor cells for acute leukemia, lymphoma, or myeloma continues to be associated with a high relapse rate because of the infusion of malignant stem cells and the lack of an in vivo graft-vs.-leukemia (GVL) effect. Although various methods of purging are established for marrow, purging procedures for blood progenitor cell preparations have not been widely used primarily because of the technical challenges to process a higher number of cells. As a broadly applicable method for immunological purging, we tested whether highly cytotoxic cells from a natural killer (NK) cell line characterized previously (NK-92) could be used for immunological purging of blood preparations. The NK-92 cell line, which was established from a patient with non-Hodgkin's lymphoma, can lyse in vitro a broad range of leukemia, lymphoma, and myeloma cell lines even at very low effector:target (E:T) ratios; this lysis is superior to cytotoxicity obtained from normal peripheral blood mononuclear cells (PBMCs) stimulated for 4 days with interleukin (IL)-2. In an attempt to quantitate the purging achievable with NK-92 cells, normal PBMCs were spiked with 10% K562 cells that had been transfected with the neo(r) marker gene (K562-neo(r). Various numbers of NK-92 cells were then added to the cell mixtures, which were incubated for 4 or 48 hours at 37 degrees C with or without IL-2 (500 U/mL). In order to prevent their proliferation, NK-92 cells were irradiated with 1000 cGy (cesium source). This radiation dose was determined to suppress proliferation of NK-92 cells, but at the same time maintain full cytotoxic activity. After co-culture, the cells were plated in methylcellulose containing 0.8 mg/mL G418. The number of surviving K562-neo(r) colonies was counted under the microscope 7 days later and the results were considered a quantitative readout for the purging efficacy of NK-92 cells. No neomycin-resistant K562 colonies could be detected up to a ratio of NK-92:K562-neo(r) cells of 5:1 (effective NK-92:PBMC ratio of 0.5:1). The presence or absence of IL-2 during the culture period did not affect the results. At this ratio of NK-92:PBMC, the growth of normal clonogenic hematopoietic progenitor cells was not compromised as determined by a standard methylcellulose assay. Considering that K562 is a rapidly proliferating cell line and that the input number of K562 cells (10%) tested here was high, the data suggest that the cytotoxic NK-92 clone (after irradiation to prevent proliferation) could be used effectively for immunological ex vivo purging without compromising hematopoietic cell function.

Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells.

The cell line described here was established for a 50-year-old male patient with rapidly progressive non-Hodgkin's lymphoma whose marrow was diffusely infiltrated with large granular lymphocytes (LGL). Immunophenotyping of marrow blasts and peripheral lymphocytes was positive for CD56, CD2 and CD7, and negative for CD3. Cytotoxicity of peripheral blood mononuclear cells at an effector: target (E:T) cell ratio of 50:1 was 79% against K562 cells and 48% against Daudi cells. To establish the line, cells from the peripheral blood were placed into enriched alpha medium containing 12.5% fetal calf serum, 12.5% horse serum, 10(-4) M beta-mercaptoethanol and 10(-6) M hydrocortisone. Growth of the line (termed NK-92) is dependent on the presence of recombinant IL-2 and a dose as low as 10 U/ml is sufficient to maintain proliferation. Conversely, cells die within 72 h when deprived of IL-2; IL-7 and IL-12 do not maintain long-term growth, although IL-7 induces short-term proliferation measured by 3H-thymidine incorporation. None of the other cytokines tested (IL-1 alpha, IL-6, TNF-alpha, IFN-alpha, IFN-gamma) supported growth of NK-92 cells which have the following characteristics: surface marker positive for CD2, CD7, CD11a, CD28, CD45, CD54, CD56bright; surface marker negative for CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, CD34, HLA-DR. DNA analysis showed germline configuration for T-cell receptor beta and gamma genes. CD25 (p55 IL-2 receptor) is expressed on about 50% of all cells when tested at 100 U/ml of IL-2 and its expression correlates inversely with the IL-2 concentration. The p75 IL-2 receptor is expressed on about half of the cells at low density irrespective of the IL-2 concentration. NK-92 cells kill both K562 and Daudi cells very effectively in a 4 h51-chromium release assay (84 and 86% respectively, at an E:T cell ratio of 5:1). The cell line described here thus displays characteristics of activated NK-cells and could be a valuable tool to study their biology.

Establishment and characterization of a human leukemic cell line (SR-91) with features suggestive of early hematopoietic progenitor cell origin.

The characterization of a cell line (designated SR-91) from a patient with clinical and morphological diagnosis of acute lymphoblastic leukemia (CD3-, CD2+, CD7+, germline TCR genes) who relapsed early after allogeneic bone marrow transplantation, is reported. The line was established from blood cells obtained at diagnosis and placed in suspension culture with medium conditioned by 5637 cells. SR-91 cells are negative for lymphoid surface markers (CD3-, CD2-, CD7-) but positive for markers indicative of myeloid progenitor cells, such as CD33 and CD34. It is likely that the conditioned medium has induced myeloid differentiation from a lymphohematopoietic progenitor cell. After establishment, cells proliferated in response to GM-CSF stimulation but they are not factor-dependent and do not produce GM-CSF. No proliferative response to IL-1, IL-2, IL-3, IL-4, IL-6 or M-CSF was observed. Cells were completely resistant to anti-proliferative effects of tumor necrosis factor-alpha and interferon-alpha or -gamma, and showed no lysis after incubation with freshly isolated natural killer cells or IL-2-activated natural killer cells.