M4 and M5 acute myeloid leukaemias display a high sensitivity to Bortezomib-mediated apoptosis

The present study explored the sensitivity of leukaemic blasts derived from 30 acute myeloid leukaemia (AML) patients to Bortezomib. Bortezomib induced apoptosis of primary AML blasts: 18/30 AMLs were clearly sensitive to the proapoptotic effects of Bortezomib, while the remaining cases were moderately sensitive to this molecule. The addition of tumour necrosis factor-related-apoptosis-inducing ligand, when used alone, did not induce apoptosis of AML blasts and further potentiated the cytotoxic effects of Bortezomib. The majority of AMLs sensitive to Bortezomib showed immunophenotypic features of the M4 and M5 French-American-British classification subtypes and displayed myelomonocytic features. All AMLs with mutated FLT3 were in the Bortezomib-sensitive group. Biochemical studies showed that: (i) Bortezomib activated caspase-8 and caspase-3 and decreased cellular FLICE [Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme]-inhibitory protein (c-FLIP) levels in AML blasts; (ii) high c-FLIP levels in AML blasts were associated with low Bortezomib sensitivity. Finally, analysis of the effects of Bortezomib on leukaemic cells displaying high aldehyde dehydrogenase activity suggested that this drug induced in vitro killing of leukaemic stem cells. The findings of the present study, further support the development of Bortezomib as an anti-leukaemic drug and provide simple tools to predict the sensitivity of AML cells to this drug.     

Effect of granulocyte-macrophage colony-stimulating factor and interleukin-3 on interleukin-8 production by human neutrophils and monocytes

Interleukin-8 (IL-8) is a major neutrophil chemoattractant and functional stimulant that is induced by IL-1, tumor necrosis factor alpha (TNF alpha), and lipopolysaccharide (LPS). We report that recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and rhIL-3 are also potent inducers of IL-8 messenger RNA (mRNA) accumulation and protein secretion by normal peripheral blood monocytes. Neutrophils produce IL-8 in response to GM-CSF but not to IL- 3. In contrast, recombinant human granulocyte-CSF (rhG-CSF), at concentrations as high as 100 ng/mL, does not induce IL-8 in either cell type. rhGM-CSF also induces IL-8 mRNA expression and IL-8 protein in the promonocytic cell line, U-937, whereas rhG-CSF does not. IL-8 secretion by monocytes was stimulated within 2 hours after incubation with rhGM-CSF or rhIL-3. Stimulation of neutrophils with rhGM-CSF resulted in an increase in cell-associated IL-8 at 4 hours. At 24 hours, cell-associated IL-8 levels declined, whereas secreted IL-8 levels increased. In contrast, virtually all IL-8 induced in monocytes appeared as secreted protein. Neither rhGM-CSF nor rhIL-3 induced detectable secretion of IL-1, TNF alpha, or IL-6 protein by monocytes. rhGM-CSF, and to a lesser degree rhIL-3, potently stimulated IL-8 secretion in cultures of heparinized whole blood, whereas rhG-CSF had no significant effect on IL-8 secretion. Induction of IL-8 by GM-CSF may be physiologically important in enhancing the acute inflammatory response.     

Cytotoxic effectors activated by low-dose IL-2 plus IL-12 lyse IL-2-resistant autologous acute myeloid leukaemia blasts

We have investigated the susceptibility of primary acute myeloid leukaemia (AML) samples to the lytic action of normal peripheral blood mononuclear cells (PBMC), as well as of the patients' autologous and allogeneic PBMC collected at the time of remission following stimulation with different concentrations of interleukin (IL)-2 and IL-12, both alone and in variable combinations. Primary AML blasts were resistant to IL-2-activated PBMC effectors generated from normal individuals, allogeneic and autologous patients in five, six and eight of the 10 AML samples tested, respectively. IL-12 alone proved ineffective in generating anti-leukaemic activity, whereas, in combination, the two cytokines induced anti-leukaemic killing in all five cases resistant to normal PBMC, in 4/6 samples resistant to allogeneic AML effectors and in 5/8 cases resistant to autologous effectors. In each case the lytic effect was maintained at the lowest cytokine combination (10 + 10 IU/ml) utilized. The suggestion of a synergistic effect was further strengthened by the evidence that at the lowest doses of IL-2 and IL-12 the degree of killing was greater than that promoted by each cytokine independently.
The results of this study suggest that two major limitations associated with the administration of IL-2 to AML patients, the resistance of the blasts to IL-2-generated killing and the toxicity associated with high-dose IL-2, may be overcome by the combined use of very low doses of IL-2 and IL-12. As IL-2-resistant blasts may be lysed by a low-dose combination of IL-2/IL-12, feasibility studies with this cytokine combination are worthy of exploration in vivo .     

Effects of various recombinant human hemopoietic growth factors (rhEpo,rhG-CSF,rhGM-CSF,rhIl-3) on the growth of peripheral blood progenitor cells (BFU-E,CFU-GM)

Summary The effects of rhEpo 1, rhG-CSF, rhGM-CSF and rhIl-3 on the growth of both CFU-GM and BFU-E from normal human adult peripheral blood have been studied in plasma clot cultures. Using optimal concentrations of all growth factors, alone and in combination with all other factors, rhIl-3 showed the highest activity in regard to growth of both CFU-GM and BFU-E, whereas rhGM-CSF treatment resulted only in half-maximal colony growth compared to rhIl-3. No synergism or additive effect was seen with the combination of rhIl-3 and rhGM-CSF. Treatment with rh-G-CSF had no additional effect with optimal concentrations of rhIl-3 and/or rhGM-CSF. When suboptimal concentrations of rhGM-CSF and rhIl-3 were applied, however, they showed a marked synergism on both BFU-E and CFU-GM. RhGCSF, added to a suboptimal concentration of rhGM-CSF, resulted in a marked growth increase of CFU-GM but had no effect on BFU-E.Abbreviations BFU-E Burst forming unit-erythroid - CFU-GEM Colony forming unit-granulocytic/erythroid/monocytic/megacaryocytic - CFU-GM Colony forming unit-granulocytic/monocytic - rhEpo Recombinant human erythropoietin - rhG-CSF Recombinant human granulocyte-colony stimulating factor - rhGM-CSF Recombinant human granulocyte/monocyte-colony stimulating factor - RhIl-3 Recombinant human Il-3     

Expansion of autorecognitive cytotoxic effectors in human cancer by T cell growth factor (Interleukin 2)1

Conditions for the production of supernates from mitogen stimulated human lymphocytes with the capacity to induce proliferation in long term MLC or PHA stimulated cultures which no longer respond to PHA have been investigated. These supernates can be used to maintain lymphocytes in continuous growth with a doubling time of approximately 48 hours. Cells grown from MLC stimulated cultures show less than 80% SRBC rosetting cells, are Fc-, do not show ADCC activity and induce reduced lysis of K562 compared with freshly isolated effectors. Cultured T cells (CTC) show high lytic activity against the inducing PHA blasts but not autologous or third part targets. Similar experiments have been performed with lymphocytes from the blood, lymph node, spleen and tumour of cancer patients and CTC tested for cytotoxicity against autologous and allogeneic tumour cells and the K562 compared with freshly isolated effectors. Cultured T cells (CTC) show high lytic activity against the inducing PHA blasts but not autologous or third part targets. Similar experiments have been performed with lymphocytes from the blood, lymph node, spleen and tumour of cancer patients and CTC tested for cytotoxicity against autologous and allogeneic tumour cells and the K562 cell line. Cytotoxicity for autologous tumour was found in all samples. This was accompanied by killing of allogeneic cells in most instances but killing of K562 was only rarely demonstrable. These data would be consistent with a polyclonal expansion of cytotoxic effectors in the samples. The finding of autologous reactivity suggests the presence of autorecognitive cytotoxic T cells in cancer patients with specificity for tumour. Cloning experiments are currently in progress to investigate this possibility further.     

NK cell-mediated killing of AML blasts: role of histamine,monocytes and reactive oxygen metabolites

Abstract: Blasts recovered from patients with acute myelogenous leukaemia (AML) were lysed by heterologous natural killer (NK) cells treated with NK cell-activating cytokines such as interleukin-2 (IL-2) or interferon-α (IFN-α). The cytokine-induced killing of AML blasts was inhibited by monocytes, recovered from peripheral blood by counterflow centrifugal elutriation. Histamine, at concentrations exceeding 0.1 μM, abrogated the monocyte-induced inhibition of NK cells; thereby, histamine and IL-2 or histamine and IFN-α synergistically induced NK cell-mediated destruction of AML blasts. The effect of histamine was completely blocked by the histamine H2-receptor (H2R) antagonist ranitidine but not by its chemical control AH20399AA. Catalase, a scavenger of reactive oxygen metabolites (ROM), reversed the monocyte-induced inhibition of NK cell-mediated killing of blast cells, indicating that the inhibitory signal was mediated by products of the respiratory burst of monocytes. It is concluded that (i) monocytes inhibit anti-leukemic properties of NK cells, (ii) the inhibition is conveyed by monocyte-derived ROM, and (iii) histamine reverses the inhibitory signal and, thereby, synergizes with NK cell-activating cytokines to induce killing of AML blasts.     

Upregulation of lipocortin 1 inhibits tumour necrosis factor-induced apoptosis in human leukaemic cells: a possible mechanism of resistance to immune surveillance

The signal transduction pathway through which tumour necrosis factor (TNF) induces apoptosis in leukaemic cells may involve activation of cytosolic phospholipase A(2) (cPLA(2)). The steroids dexamethasone (Dex) and 1,25(OH)(2) D(3) both render U937 leukaemic cells resistant to TNF-induced apoptosis. In this study, we found that Dex inhibited both spontaneous and TNF-induced activation of cPLA(2). Dex had no direct effect on cellular cPLA(2) levels, but facilitated cPLA(2) degradation upon subsequent stimulation of cells with TNF. In addition, Dex increased synthesis of the endogenous cPLA(2) inhibitor lipocortin 1 (LC1). An antisense oligonucleotide to LC1 could completely abrogate Dex-induced resistance to the cytotoxic action of TNF. Constitutive LC1 levels were relatively higher in myeloid leukaemic blasts showing resistance to TNF than TNF-sensitive myeloid leukaemic cell lines. Our data suggest that Dex confers the resistance of U937 cells to TNF-induced apoptosis by upregulating intracellular levels of LC1 and by facilitating a negative-feedback loop, which is activated upon stimulation with TNF. High constitutive levels of LC1 in leukaemic blasts may protect them against immune-mediated killing.     

Telomerase activity in acute myelogenous leukaemia: clinical and biological implications

We examined telomerase activity in myeloid leukaemic cell lines, normal haemopoietic cells, and leukaemic blasts from acute myelogenous leukaemia (AML) patients. Normal bone marrow mononuclear (BMNC) cells expressed low telomerase activity. Higher telomerase activity was detected in 10 myeloid leukaemic cell lines compared to normal BMNC cells. Treatment with 1,25(OH)₂D₃, and vitamin D₃ analogues, EB1089 and KH1060, reduced telomerase activity in vitamin D₃-sensitive HL-60 cells, whereas vitamin D₃ insensitive K562 cells did not change its activity. This down-regulation of telomerase activity by EB1089 was associated with induction of p21 protein. The rank order of telomerase activity was leukaemic CD34⁻ cells > leukaemic CD34⁺ cells > normal CD34⁻ cells > normal CD34⁺ cells. Telomerase activity was positive in all of the AML patients tested; however, heterogeneity of telomerase activity was found amongst this group. Therefore we compared telomerase activity with clinical response. Unexpectedly, we found that a higher rate of complete remission was noted in AML patients with higher telomerase activity. No association between telomerase activity and biological parameters including percentage of S-phase, cytotoxicity to cytosine arabinoside and percentage of CD34⁺ cells in AML blasts was found. These results suggest that telomerase activity in AML patients is detected with high frequency, but is heterogenous. Expression level of telomerase activity may have a clinical implication in AML patients regarding clinical response.     

T-cell acute lymphoblastic leukemia with natural killer cell phenotype

To determine the type and proportion of cases within that type of acute lymphoblastic leukemia (ALL) that has a natural killer (NK) cell phenotype, we examined leukemic blasts from 31 children with ALL (14 with T-ALL, 17 with non-T-ALL) for expression of antigens detected by NK-specific monoclonal antibodies Leu 11b, Leu 7, and 1G2 (an antibody we have developed that cross-reacts with Leu 7). None of the patients had leukemic blasts that reacted with Leu 11b. However, leukemic blasts from four T-ALL patients were 1G2+ and/or Leu 7+. Blasts from two of these had spontaneous lytic activity against standard NK target cell line K562; blasts from one killed K562 only when incubated with interferon; blasts from the other had no lytic activity against K562 but did manifest antibody-dependent cell-mediated cytotoxicity against antibody-coated cells from NK-resistant cell line SB. Blasts from all four Leu 7+ patients had L2 morphology. In one, the leukemic blasts had azurophilic cytoplasmic granules similar to those found in NK-enriched normal populations of large granular lymphocytes. These findings suggest that a significant proportion of T-cell acute lymphoblastic leukemias may be malignancies of NK cell origin.     

Suppressive effect of granulocyte-macrophage colony-stimulating factor on the generation of natural killer cells in vitro.

We investigated the effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombinant human granulocyte-CSF (rhG-CSF) on the generation of natural killer (NK) cells in vitro. NK cells were cultured from selected human bone marrow cells obtained after the elimination of mature T and NK cells. rhGM-CSF significantly suppressed the generation of CD56+ cells and NK activity (P less than .01) in a dose-dependent manner. The generation of large granular lymphocytes (LGL) was also suppressed in the presence of rhGM-CSF (P less than .01). In contrast, rhG-CSF had no effect on LGL (P greater than .05). Both rhGM-CSF and rhG-CSF had no influence on the CD56+ cell count in the peripheral blood. These results suggest that rhGM-CSF suppresses the in vitro generation of NK cells.     

Generation of cytokine-induced killer cells from leukaemic samples with in vitro cytotoxicity against autologous and allogeneic leukaemic blasts

Cytokine-induced killer (CIK) cells are CD3(+)CD56(+) non-major histocompatibility complex (MHC)-restricted immune effector cells. The present report demonstrates that it was possible to expand CIK cells obtained at diagnosis from patients with acute leukaemia. The percentage of CD3(+)CD56(+) CIK cells generated following culture ranged between 7.6% and 65% (median of 35.3%) and these cells were able to kill the human natural killer target K562 cells. Although the same effector cells were able to lyse autologous acute myeloid leukaemia (AML) target cells, they were not able to lyse autologous acute lymphoblastic leukaemia target cells. Pre-absorption of the CIK effector cells by K562 cells did not completely abrogate the cytotoxicity of CIK cells against autologous blasts in 9 out of 12 samples tested. Moreover, it was observed that the cytotoxicity generated by the CIK effector cells against allogeneic leukaemic blasts was similar to that against autologous blasts. The present study suggests the potential application of CIK cells in the immunotherapy of AML, either in minimal disease state, as donor lymphocyte infusion in relapse post allogeneic transplant, or in cases of chemotherapy refractory leukaemia.     

Recovery of drug sensitivity by MS-209, a new multidrug resistance-reversing agent,on acute myelogenous leukaemic blasts and K562 cells resistant to adriamycin cell line

Abstract: The efficacy of MS-209, a quinoline derivative synthesized as a new multidrug resistance (MDR)-reversing agent, was studied on blast cells from 33 acute myelogenous leukaemia (AML) patients and on the human myelogenous leukaemia K562 cell line resistant to adriamycin (K562/ADM). By the addition of MS-209, the intracellular daunorubicin (DNR) contents which had been found to be low in P-gp-positive AML blasts and in K562/ADM were significantly enhanced to the level of P-gp-negative blasts and that of sensitive K562. The intracellular rhodamine (Rh123) contents also increased in P-gp-positive blasts and K562/ADM cells with MS-209. A leukaemic blast colony assay also demonstrated the effect of MS-209, i.e. a high D₁₀ value for DNR of P-gp-positive blasts was reduced to the D₁₀ level similar to that observed in P-gp-negative blasts by the addition of MS-209. The greater DNR sensitivity reversing effect of MS-209 was observed in blasts with higher P-gp positivity. These findings suggest the potential usefulness of MS-209 in overcoming MDR in AML patients, especially those with high P-gp expression. This study clarified the relationship between the clinical outcome of the patients and the P-gp positivity, intracellular DNR content and DNR drug sensitivity of leukaemic progenitors.     

Effect of cisplatin treatment of human monocyte cell line U937 on the induction of tumoricidal activity.

U937, a human monocyte-like, cell line was checked for cytotoxic activity against tumor target cells. Untreated U937 cells showed little cytotoxicity against tumor cells. Granulocyte-macrophage colony stimulating factor (GM-CSF) and LPS significantly activated the U937 cells to tumoricidal state. Treatment of U937 cells with cisplatin did not enhance the tumoricidal activity. Similarly, interferon gamma (IFN-Y) and macrophage colony stimulating factor (M-CSF) could also not activate either the tumoricidal activity of U937 cells. Pretreatment of U937 cells with GM-CSF for 24 h and then the treatment with cisplatin significantly augmented the tumoricidal activity as compared to that of GM-CSF alone.     

Human natural killer cells, activated lymphocyte killer cells, and monocytes possess similar cytotoxic mechanisms.

The relationship between the killing mechanisms of human natural killer (NK) cells, mitogen- and mixed-lymphocyte-culture-induced activated lymphocyte killer (ALK) cells, and monocytes was investigated with a monoclonal antibody. The IgG2 antibody 9.1C3 was prepared from mice immunized with purified human large granular lymphocytes and selected from clones that inhibited NK cell killing. The 9.1C3 antibody bound to all mononuclear cells but not to granulocytes or K562 cells, and it selectively blocked killing of K562 targets by both NK and ALK cells without affecting the binding of effector to target cells. The antibody blocked killing when present from time zero and it still inhibited partially even when added 1 hr after initiation of the lytic reaction. Killing of Epstein-Barr virus-transformed B lymphoblasts by classical cytotoxic T lymphocytes was not inhibited. Of interest, 9.1C3 did block the killing of K562 target cells by cultured peripheral blood monocytes. Other monoclonal antibodies that bound to monocytes did not block killing, and a nonspecific effect of the antibody on monocytes was excluded. These data suggest that NK cells, ALK cells, and monocytes can kill tumor cell targets by using similar lytic mechanisms.     

Administration of recombinant human granulocyte-macrophage colony- stimulating factor after chemotherapy regulates the expression and secretion of monocyte tumor necrosis factor (TNF) and TNF receptors p55 and p75

Monocyte expression and secretion of tumor necrosis factor (TNF) and TNF receptors (TNF-R) p55 and p75 was studied in patients receiving granulocyte-macrophage colony-stimulating factor (GM-CSF) after intensive chemotherapy. TNF expression and secretion of biologically active TNF was increased at regeneration compared with that of patients who had received chemotherapy alone. This effect persisted for several weeks after cessation of growth factor therapy. GM-CSF restored the responsiveness of monocytes to bacterial lipopolysaccharide (LPS), which appeared to be diminished after chemotherapy alone. Expression and secretion of TNF-R p55 and p75 by monocytes was augmented by GM-CSF therapy in association with the increase in TNF protein. We propose that GM-CSF administration after chemotherapy restores the normal responsiveness of monocytes to a secondary stimulus such as LPS and primes monocytes to respond to LPS with increased expression and secretion of TNF and TNF-R.     

Human CD80/IL2 lentivirus-transduced acute myeloid leukaemia (AML) cells promote natural killer (NK) cell activation and cytolytic activity: implications for a phase I clinical study

Immunotherapeutic strategies may promote T and/or natural killer (NK) cell cytotoxicity. NK cells have the potential to exert a powerful anti-leukaemia effect, as demonstrated by studies of allogeneic transplantation. We have previously shown that CD80/interleukin 2 (IL2) lentivirus (LV)-transduced AML cells stimulate in-vitro T cell activation. The present study demonstrated that allogeneic and autologous culture of peripheral blood mononuclear cells with CD80/IL2-expressing AML cells also promoted NK cell cytotoxicity. Expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA-DR significantly increased following allogeneic culture and a consistent increased expression of NKp30, NKp44, NKp46, NKG2D, NKG2C and CD69, and up-regulation of the cytolytic marker CD107a was detected following autologous culture with LV-CD80/IL2 AML cells. Furthermore, increased NK cell lysis of K562 and primary AML blasts was detected. The lytic activity increased by twofold against K562 (from 46·6% to 90·4%) and allogeneic AML cells (from 11·8% to 20·1%) following in-vitro stimulation by CD80/IL2-expressing AML cells. More importantly for potential therapeutic applications, lysis of primary AML cells by autologous NK cells increased by more than 40-fold (from 0·4% to 22·5%). These studies demonstrated that vaccination of patients with CD80/IL2-transduced AML cells could provide a powerful strategy for T/NK cell-mediated stimulation of anti-leukaemic immunological responses.     

Induction of specific CTL by MAGE-3/CEA peptide-pulsed dendritic cells from HLA-A2/A24<Superscript>+</Superscript> gastrointestinal cancer patients

PURPOSE: In this study, we aimed to investigate whether MAGE3/CEA peptide-pulsed dendritic cells could induce specific cytotoxic T lymphocytes (CTL). METHODS: In this pilot study, we selected 25 patients expressing MAGE-3-HLA-A2/A24 or CEA-HLA-A24. Patients' dendritic cells (DCs) were expanded in vitro in the presence of recombined-human granular macrophage colony stimulating factor (rhGM-CSF) and recombined-human interleukin 4 (rhIL-4), pulsed with MAGE-3/CEA (HLA-A2/A24) peptide. The cytolytic cells' activity, induced by peptide-pulsed DCs and unpurified T cells as effector cells, and with Mel526, 803, Raji, and K562 as target cells, were measured using LDH-releasing assay. RESULTS: DCs were obtained by in vitro expansion in all cases although DC harvest rates varied among different patients (7.1+/-3.2%). Compared with T-IL-2 (IL-2-induced T cells), T-DC-P - which resulted from T-IL-2 co-cultured with DCs pulsed by MAGE3 or CEA peptides - exhibited an increase in cytolytic activity against Mel526 (expressing MAGE-3-HLA-A2) and 803 (expressing CEA-HLA-A24) cell lines by about 25-30% ( P<0.01). In contrast, there was no significant difference between the activity against Raji and K562 cells, which are negative for both peptides. CONCLUSIONS: This study showed that combined usage of rhGM-CSF and rhIL-4 in vitro could expand DCs, and that the DCs pulsed with specific peptides could induce MAGE- and CEA-specific CTL responses. The DC-based vaccine may provide an important method for the immunological treatment of gastrointestinal cancers.     

Studies in acute leukemia. I. Antibody-dependent and spontaneous cellular cytotoxicity by leukemic blasts from patients with acute nonlymphoid leukemia.

Leukemic blasts from patients with acute nonlymphoid leukemia were examined for the presence of Ig, receptors for IgGFc, and for their capacity to mediate antibody-dependent cellular cytotoxicity (ADCC) against chicken red blood cells (RBC) coated with IgG and spontaneous cell-mediated cytotoxicity (SCMC) against cells of K562 cell line. Leukemic blasts from acute myeloblastic leukemia (AML) patients lacked both Fc receptors and Ig on their surface, had no SCMC activity and majority, but not all of them, lacked ADCC activity. Leukemic blasts from patients with acute monocytic leukemia (AMOL) had Fc receptors, and 50% had IgG on their surface. IgG was cytophilic and appeared not to be directed against cell-surface antigens. This antibody did not interfere with the ADCC activity of leukemic cells. Leukemic blasts from majority of patients with AMOL mediated ADCC, but had no SCMC activity. An association between ADCC and presence of Fc receptor was observed.     

Full chimaeric CAR.CIK from patients engrafted after allogeneic haematopoietic cell transplant: Feasibility,anti-leukaemic potential and alloreactivity across major human leukocyte antigen barriers

Cytokine-induced killer lymphocytes (CIK) are a promising alternative to conventional donor lymphocyte infusion (DLI), following allogeneic haematopoietic cell transplantation (HCT), due to their intrinsic anti-tumour activity and reduced risk of graft-versus-host disease (GVHD). We explored the feasibility, anti-leukaemic activity and alloreactive risk of CIK generated from full-donor chimaeric (fc) patients and genetically redirected by a chimeric antigen receptor (CAR) (fcCAR.CIK) against the leukaemic target CD44v6. fcCAR.CIK were successfully ex-vivo expanded from leukaemic patients in complete remission after HCT confirming their intense preclinical anti-leukaemic activity without enhancing the alloreactivity across human leukocyte antigen (HLA) barriers. Our study provides translational bases to support clinical studies with fcCAR.CIK, a sort of biological bridge between the autologous and allogeneic sources, as alternative DLI following HCT.