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. 2009 Mar;126(3):423-35.
doi: 10.1111/j.1365-2567.2008.02910.x. Epub 2008 Sep 5.

Characterization of the recognition and functional heterogeneity exhibited by cytokine-induced killer cell subsets against acute myeloid leukaemia target cell

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Characterization of the recognition and functional heterogeneity exhibited by cytokine-induced killer cell subsets against acute myeloid leukaemia target cell

Yeh C Linn et al. Immunology. 2009 Mar.

Abstract

The polyclonal cytokine-induced killer (CIK) cells exhibit potent cytotoxicity against a variety of tumour cells including autologous and allogeneic acute myeloid leukaemic (AML) targets. At maturity, three lymphocyte subsets: CD3(-) CD56(+), CD3(+) CD56(-) and CD3(+) CD56(+), constitute the bulk of the CIK cell culture. The CD3(-) CD56(+) subset behaves like classical natural killer (NK) cells where cytotoxicity is potentiated by blocking the human leucocyte antigen Class I molecules in the AML targets. Both the CD3(+) CD56(+) and CD3(+) CD56(-) subsets, though known to kill autologous and allogeneic targets to a comparable degree and therefore non-major histocompatibility complex (MHC)-restricted, nevertheless require the presence of the MHC molecule on the target, which interacts with their CD3-T-cell receptor complex. Although CIK cells are often termed 'NK-like' T cells, we have demonstrated that the well-characterized NK receptors KIR, NKG2C/E, NKG2D and DNAM-1 are not involved in the process of AML recognition for the CD3(+) CD56(-) and CD3(+) CD56(+) subsets. The CD3(+) CD56(+) and CD3(+) CD56(-) subsets express a polyclonal and comparable TCRVbeta repertoire in a Gaussian distribution. The CD3(+) CD56(+) subset kills AML targets more efficiently than its CD3(+) CD56(-) counterpart because of the presence of a higher proportion of CD8(+) cells. The CD3(+) CD56(+) subset comprise more terminally differentiated late effector T cells that bear the CD27(+) CD28(-) or CD27(-) CD28(-) phenotype, with a higher granzyme A content. In comparison, the phenotype of the CD3(+) CD56(-) subset is consistent with early effector T cells that are CD27(+) CD28(+) and CD62L(+), known to be less cytotoxic but possess greater proliferative potential.

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Figures

Figure 1
Figure 1
(a) Cytolysis of K562 target cells by cytokine-induced killer (CIK) cells was directly correlated with the proportion of CD3 CD56+ natural killer cell subset in the bulk CIK culture. A total of 27 cytotoxicity assays were performed and each dot represents the result of one reading of a CIK cell culture against K562 target at an effector to target (E : T) ratio of 10 : 1. Coefficient of correlation = 0·64. (b) Neither purified CD3+ CD56+ nor CD3+ CD56 cell subsets killed K562 target cells. Five separate experiments are shown for both cell subsets against K562 target cells at E : T ratio of 40 : 1, P = 0·224. (c) Cytotoxicity of the CD3 CD56+ cell subset (♦) against acute myeloid leukaemia target cells was enhanced when human leucocyte antigen class I molecules on the target cells were blocked (◊), to a degree similar to allogeneic killing (formula image). Figure represents one of the two comparison sets done, P < 0·05 for both.
Figure 2
Figure 2
(a) Cytotoxicity of purified CD3+ CD56+ (▀) and CD3+ CD56 cells (•) against acute myeloid leukaemia (AML) targets were abrogated when the human leucocyte antigen (HLA) class I molecules on the target cells were blocked by the W6/32 monoclonal antibody (mAb; □,○). Figure shows one representative assay. Eight comparison sets were performed for the CD3+ CD56+ subset, P < 0·05 in seven out of eight sets. Eight comparison sets were performed for the CD3+ CD56 subset, P < 0·05 for all eight sets. Histograms show isotype control (left) and HLA class I expression on blasts without (middle) and with W6/32 blocking (right) respectively, of one representative assay. (b) Cytotoxicity of the CD3+ CD56+ cells (▀) and CD3+ CD56 cells (•) was enhanced when HLA class I molecules on the AML target cells were up-regulated by preincubation with interferon-γ (IFN-γ; □ and ○, respectively). Figure shows one representative assay of two comparison sets carried out for both CD3+ CD56+ and CD3+ CD56 subsets, P < 0·05 for both. Histograms show isotype control (left) and HLA class I expression on blasts without (middle) and with preincubation with IFN-γ (right) respectively, of one representative assay. (c) Cytotoxicity of the CD3+ CD56+ cells (▀) and CD3+ CD56 cells (•) was inhibited when the CD3/T-cell receptor (TCR) complexes on the effector cells were blocked by a TCR-blocking mAb T10B9. The figure shows one representative assay. Four comparison sets were made for the CD3+ CD56+ subset, P < 0·05 in three out of four sets. Four comparison sets were made for the CD3+ CD56 subset, P < 0·05 for all. Histograms show isotype control (left) and TCR expression on cytokine-induced killer cells without (middle) and with T10B9 blocking (right) respectively, of one representative assay. (d) Cytotoxicity of the CD3+ CD56+ cells (▀) and CD3+ CD56 cells (•) was enhanced when the CD3/TCR complexes on the effector cells were either stimulated with OKT3 (Δ) or with a stimulating anti-TCR mAb (□). Two comparison sets were carried out for the CD3+ CD56+ subset and three comparison sets for the CD3+ CD56 subset, P < 0.05 for all. (e) Mean fluorescent intensity of degranulation marker CD107a over time after ligation with OKT3. There were three samples at serial time-points: 0 min (□), 30 min (formula image), 60 min (formula image), 120 min (formula image), 240 min (▀).
Figure 3
Figure 3
(a) One representative cytotoxicity assay of CD3+ CD56+ subset against acute myeloid leukaemia (AML) target cells (▀) in the presence of anti-CD158a and anti-CD158b monoclonal antibody (mAb; Δ), and with anti-CD94 mAb (□). Three comparison sets comparing the blocking with anti-CD158a/anti-CD158b and anti-CD94 were done for the CD3+ CD56+ and CD3+ CD56 subsets respectively, P = NS for all. (b) Real-time polymerase chain reaction analyses of the relative expression level of NKG2C and NKG2E transcripts after normalization to β-actin. Analysis was done on CD3+ CD56+ subsets from two independent AML samples (AML#1 and AML#2) and two independent acute lymphoblastic leukaemia (ALL) samples (ALL#1 and ALL#2), before and after coincubation with their respective autologous targets. Relative expression level of CD3+ CD56+ subset at baseline (open column) is compared with that after 4 hr of incubation with the respective AML or ALL targets (grey column). (c) Histogram showing one representative fluorescence-activated cell sorting (FACS) analysis of the expression of NKG2D receptor in CD3+ CD56+ cells after cytokine-induced killer cell (CIK) cell culture. One representative cytotoxicity assay of CD3+ CD56+ cells against autologous AML target cells in the absence (▀) and presence (□) of blocking anti-NKG2D mAb (right panel). There were five comparison sets, P = NS for all. (d) Histogram showing one representative FACS analysis of the expression of DNAM-1 receptor in CD3+ CD56+ cells after CIK cell culture. One representative cytotoxicity assay of CD3+ CD56+ cells against autologous AML target cells in the absence (▀) and presence (□) of blocking anti-DNAM-1 mAb (right panel). Three comparison sets were done, P = NS for all.
Figure 4
Figure 4
(a) Cytotoxicity of the bulk cytokine-induced killer (CIK) cell culture against acute myeloid leukaemia (AML) target cells was correlated with the increase in the percentage of CD3+ CD56+ cells upon culture. The percentage of CD3+ CD56+ cells in the bulk CIK cell culture was shown in brackets; D denotes days in culture for the CIK cells. (b) Cytotoxicity of the CD3+ CD56+ subset (▀) against autologous AML target cells was superior to that of the CD3+ CD56 cell subset (□), at all effector to target ratios. *P < 0·05 using a two-tailed paired Student’s t-test performed on 20 independent sets of cytotoxicity assays. (c) Frequency histogram showing intracellular granzyme A expression intensity. Mean fluorescence intensity 4·2 for isotype control, 11.2 for CD3+ CD56 subset and 28·7 for CD3+ CD56+ subset.
Figure 5
Figure 5
(a) T-cell receptor Vβ (TCRVβ) spectratyping of T lymphocytes obtained from the marrow sample of patient with acute myeloid leukaemia (AML) at diagnosis. (b) TCR repertoire of cytokine-induced killer (CIK) T cells at maturity. Fifty-six neg denotes CD3+ CD56 cells; 56 pos denotes CD3+ CD56+ cells. (c) Flow cytometric analyses of the composition of the different TCRVβ subfamilies for the CD3+ CD56 (upper panel) and CD3+ CD56+ cell subsets (middle panel). The table summarizes the % of each of the TCRVβ subfamilies within the two T-cell subsets (lower panel).
Figure 6
Figure 6
(a) Comparison of the % of CD8+ and CD4+ cells in the bulk cytokine-induced killer (CIK) cultures of six independent acute myeloid leukaemia (AML) patients at diagnosis (D0) and after CIK cell culture (Maturity). (b) Flow cytometric analyses of the % of CD8+ and CD4+ cells in the CD3+ CD56+ cell subset. % of CD8+ cells = 72% [19·75/(19·75 + 7·75)] and % of CD4+ cells = 32% [10·48/(10·48 + 19·89)]. (c) Comparison of the level of cytotoxicity for the four different purified cell subsets: CD8+ CD56+(♦), CD8+ CD56 (⋄), CD4+ CD56+(▴) and CD4+ CD56 (Δ) against the same AML target cell.
Figure 7
Figure 7
(a) Flow cytometric analyses for the presence of memory T-cell phenotype in the leukaemic marrow sample obtained at diagnosis. (b) Proportion of early CD27+ CD28+ (□), intermediate CD27+ CD28 (formula image) and late CD27 CD28 TE-cell subsets (formula image) present in the starting population (D0), within the CD3+ CD56+ (56+) and CD3+ CD56 (56) subsets in the matured cytokine-induced killer (CIK) cell culture. Cultures #1 to #4 represent four separate CIK cell cultures of four patients with acute myeloid leukaemia (AML; bar chart). The dot plot shows the fluorescence-activated cell sorting (FACS) profiles of a representative culture, sample #2. (c) Level of CD62L expression on T cells before culture (D0), at the peak of expression (D15–23), and at maturity of CIK cell culture (D25–30) for six separate CIK cell cultures of six AML patients. Expression of CD62L at maturity was higher in the CD3+ CD56 subset than in CD3+ CD56+ subset (upper panel). The lower panel shows the FACS profiles of a representative culture sample. *Indicates statistical significance at P < 0·05 by two-way paired Student’s t-test. (d) Level of CD127 expression on T cells before culture (D0), at the peak of expression (D15–23), and at maturity of CIK cell culture (D25–30) for six separate CIK cell cultures of six AML patients. CD127 was dimly expressed at variable levels in the CD3+ CD56 subset at maturity but absent in CD3+ CD56+ subset (upper panel). The lower panel shows the FACS profiles of a representative culture sample. *Indicates statistical significance at P < 0·05 by two-way paired Student’s t-test.

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