Expression of HLA class II determinants by normal and chronic myeloid leukemia progenitors

It has been suggested that the expression of some HLA class II antigens, derived from three loci (DR, DP, DQ) is important in the regulation of both the immune response and the response of haemopoietic progenitors to regulation factors, such as acidic isoferritins (AIF), as well as in the interaction between T lymphocytes and erythroid progenitors (BFU-E). Changes in the expression of class II antigens have been reported on the surface of granulo-monocyte progenitors in chronic myeloid leukemia (CML) and correlated to the abnormal proliferation of such cells. In this study, monoclonal antibodies against DR and DQ monomorphic determinants were used to investigate the expression of these antigens on the surface of normal and CML bone marrow and peripheral blood BFU-E by means of complement mediated cytotoxicity. It was found that most normal and leukemic BFU-E express DR antigens. Antigens density tends to be greater on marrow as opposed to peripheral precursors. In addition, leukemic BFU-E are more sensitive to cytolytic treatment than their normal counterparts. Normal BFU-E do not express detectable amounts of DQ antigens, whereas these are present on a proportion of leukemic BFU-E.     

Effect of prolactin on class II HLA antigen expression by MCF7 cell line

Effects of prolactin on Class II HLA Ag expression have been identified for the first time in a human breast cancer cell line maintained in long-term tissue culture (MCF7) and were reported in this work as follows. Quantification methods for assaying Class II HLA Ag expression modulated by prolactin were established. Class II HLA Ags were internally labelled with [35S] methionine, extracted with Nonidet P-40, immunoprecipitated specifically with anti-Class II HLA MoAbs, isolated on protein A-Sepharose and quantified by chromatofocusing. For low doses of prolactin added to a final concentration (0.015 to 0.350 micrograms/ml culture medium), no change in Class II HLA Ags expressed by MCF7 cells was observed, when compared with controls, the percent of Class II HLA Ags assayed by chromatofocusing was then 4.03 +/- 0.57. For high doses of prolactin added to the final concentration (1.50 micrograms to 3.00 micrograms/ml medium), the percent of Class II HLA Ags increased to 6.05 +/- 0.72. When prolactin was added to the culture medium of MCF7 human breast cancer cell line, increased Class II HLA Ag expression by membrane cells was noted. Prolactin induction of Class II HLA Ag expression by human breast cancer cell lines should prove very useful to study the biology of prolactin in the tumorogenesis of the human breast.     

Lack of HLA class II antigen expression in microsatellite unstable colorectal carcinomas is caused by mutations in HLA class II regulatory genes

Colorectal cancers (CRCs) develop on the basis of a deficient DNA mismatch repair (MMR) system in about 15% of cases. MMR‐deficient CRC lesions show high‐level microsatellite instability (MSI‐H) and accumulate numerous mutations located at coding microsatellite loci that lead to the generation of immunogenic neopeptides. Consequently, the host's antitumoral immune response is of high importance for the course of the disease in MSI‐H CRC patients. Accordingly, immune evasion mediated by impairment of HLA class I antigen presentation is frequently observed in these cancers. In this study, we aimed at a systematic analysis of alterations affecting HLA class II antigen expression in MSI‐H CRC. HLA class II antigens are expressed by only two‐thirds of MSI‐H CRCs. The mechanisms underlying the lack of HLA class II antigens in a subset of MSI‐H CRCs remain unknown. We here screened HLA class II regulatory genes for the presence of coding microsatellites and identified mutations of the essential regulator genes RFX5 in 9 (26.9%) out of 34 and CIITA in 1 (2.9%) out of 34 MSI‐H CRCs. RFX5 mutations were related to lack of or faint HLA class II antigen expression (p = 0.006, Fisher's exact test). Transfection with wild‐type RFX5 was sufficient to restore interferon gamma‐inducible HLA class II antigen expression in the RFX5‐mutant cell line HDC108. We conclude that somatic mutations of the RFX5 gene represent a novel mechanism of loss of HLA class II antigen expression in tumor cells, potentially contributing to immune evasion in MSI‐H CRCs.     

Comparison of Class II HLA antigen expression in normal and carcinomatous human breast cells

Class II HLA antigen expression in breast carcinoma and normal breast gland cells was compared using a method more accurate than immunofluorescence. This new method involves labeling membrane proteins with 131I and the anti-Class II HLA monoclonal antibody with 125I. The isolation and purification of the doubly labeled (125I-131I) immune complex was performed by affinity chromatography and chromatofocusing successively. When the specific activity of glycoproteins is known, the amount of glycoproteins which bind specifically to the anti-Class II HLA monoclonal antibody can be deduced. In breast carcinoma cells, 1.5 to 2% of the purified glycoproteins bind specifically to the monoclonal antibody, whereas less than 0.3% of normal breast gland cells binds. In contrast, leukemic cells, of which 80 to 90% possess Class II HLA antigens, 2 to 3% of Class II HLA glycoproteins bind specifically with the anti-Class II HLA monoclonal antibody.     

Effect of 4-hydroperoxycyclophosphamide on leukemic and normal human myeloid progenitor cells

The sensitivity of myeloid progenitor cells from normal subjects (N-CFU-GM) and from leukemic patients in complete remission (LR-CFU-GM) to 4-hydroperoxycyclophosphamide (4-HC) were compared to the sensitivity of leukemic progenitor cells (L-CFU) to this drug. The results were expressed as the dose of 4-HC needed to kill 90% (TD 90) of the progenitor cells. The mean TD 90 were respectively for N-CFU-GM : 59 (+/- 11 S.E.M.) nM ml-1 and for L-CFU 79 (+/- 6 S.E.M.) nM ml-1. Thus, L-CFU were equally sensitive to 4-HC as N-CFU-GM. Moreover, the mean TD 90 for LR-CFU-GM was 87 (+/- 5 S.E.M.) nM ml-1. Thus, the sensitivity of N-CFU-GM and LR-CFU-GM did not differ significantly from that of L-CFU. These results are not encouraging for the use of 4-HC in vitro to eliminate the residual leukemic cells from autologous bone marrow of AML patients in complete remission. The sensitivity of L-CFU was modified neither by previous cytoreductive therapy (different from cyclophosphamide) nor by the time elapsed since diagnosis of AML.     

Retinoic acid induction of CD38 antigen expression on normal and leukemic human myeloid cells: relationship with cell differentiation

Differentiation in the hematopoietic system involves, among other changes, altered expression of antigens, including the CD34 and CD38 surface antigens. In normal hematopoiesis, the most immature stem cells have the CD34+CD34 -phenotype. In acute myeloid leukemia (AML), although blasts from most patients are CD38+, some are CD38 -. AML blasts are blocked at early stages of differentiation; in some leukemic cells this block can be overcome by a variety of agents, including retinoids, that induce maturation into macrophages and granulocytes both in vitro and in vivo . Retinoids can also induce CD38 expression. In the present study, we investigated the relationship between induction of CD38 expression and induction of myeloid differentiation by retinoic acid (RA) in normal and leukemic human hematopoietic cells. In the promyelocytic (PML) CD34 -cell lines, HL60 and CB-1, as well as in normal CD34+CD34 -hematopietic progenitor cells RA induced both CD38 expression as well as morphological and functional myeloid differentiation that resulted in loss of self-renewal. In contrast, in the myeloblastic CD34+ leukemic cell lines, ML-1 and KG-1a, as well as in primary cultures of cells derived from CD34+-AML (M 0 and M 1 ) patients, RA caused an increase in CD38+ that was not associated with significant differentiation. Yet, long exposure of ML-1, but not KG-1, cells to RA resulted in loss of self-renewal. The results suggest that while in normal hematopoietic cells and in PML CD34 -cells induction of CD38 antigen expression by RA results in terminal differentiation along the myeloid lineage, in early myeloblastic leukemic CD34+ cells, induction of CD38 and differentiation are not functionally related. Since, several lines of evidence suggest that the CD38 -cells are the targets of leukemic transformation, transition of these cells into CD38+ phenotype by RA or other drugs may have therapeutic effect, either alone or in conjunction with cytotoxic drugs, regardless the ability of the cells to undergo differentiation.     

Myeloperoxidase gene expression and regulation by myeloid cell growth factors in normal and leukemic cells

Myeloperoxidase (MPO) is present in azurophilic granules which appear in the promyelocyte stage of differentiation, and is the most common functional protein of myeloid cells. With progress in molecular biology, the expression and regulation of MPO have been clarified in normal myeloid and leukemic cells, not only by enzymatical activity but at the gene level MPO expression is affected by the differentiation of myeloid cells, and has been suggested to be regulated by myeloid cell growth factors, such as granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor and interleukin-3. In the past decade the signal transduction from their receptors has been clarified. This review describes the expression and regulation of the MPO gene in myeloid cells including myeloid disorders, such as myeloid leukemia or myelodysplastic syndromes, The effects on MPO by myeloid growth factors and signal transduction from their receptors are also presented.     

Indirect induction of differentiation of normal and leukemic myeloid cells by recombinant interleukin 1

Different clones of myeloid leukemic cells can be induced to differentiate to mature macrophages or granulocytes by different normal hematopoietic regulatory proteins. The present experiments with recombinant IL-1 alpha and recombinant IL-1 beta show that, (a) that there are clones of myeloid leukemic cells which can be induced to differentiate to mature cells by the myeloid cell differentiation-inducing protein MGI-2 and can also be induced to differentiate to mature macrophages and granulocytes by both types of IL-1; (b) this IL-1-induced differentiation is mediated by endogenous production of differentiation-inducing protein MGI-2; (c) IL-1 and MGI-2 induce production of GM-CSF in these leukemic cells; and (d) IL-1 also induces cell differentiation and production of MGI-2 and GM-CSF in normal myeloid precursor cells. The results indicate that IL-1 induces differentiation indirectly.     

HLA class I antigen cell surface expression is preserved on acute myeloid leukemia blasts at diagnosis and at relapse.

Human leukocyte antigens (HLA) class I molecules restrict the interaction between cytotoxic T cells and target cells. Abnormalities in HLA class I antigen expression and/or function may provide tumor cells with a mechanism for escaping immune surveillance and resisting T cell-based immunotherapies. The potential for applying T cell-based immunotherapy in the treatment of acute myeloid leukemia (AML) has stimulated interest in analyzing HLA class I antigen expression on leukemic blasts in this disease. Little information is available in the literature. We have analyzed HLA class I antigen expression on bone marrow samples from 25 newly diagnosed AML patients by indirect immunofluorescence staining with monoclonal antibodies. Five of these patients were also studied at relapse. Leukemic blasts were resolved from normal lymphocytes by staining with antiCD45 antibody; CD45 expression is dim on leukemia cells, but bright on lymphocytes. HLA class I antigen expression was higher on leukemic blasts than on autologous lymphocytes in all but one case. Moreover, there was no significant change in HLA class I antigen expression at relapse. These results suggest that abnormalities in HLA class I antigens are infrequent in AML and should not represent a major obstacle to the application of T cell-based immunotherapies in this disease.     

RIZ1表达对髓性白血病细胞株凋亡的影响

背景与目的RIZ1是近年来发现的抑癌基因,在多种实体瘤如乳腺癌、肝癌和胃癌中RIZI均表达下降或无表达。本研究探讨了RIZ1转录对髓性白血病细胞凋亡的作用。方法采用RT-PCR法检测5种髓性白血病细胞株即AML193、KG-1、KG-1a、K562、Ery-1中RIZ1mRNA转录状况。将含RIZ1全长cDNA的重组质粒pRIZ1RH转入该基因表达缺乏的细胞株中,以制备强制性基因表达细胞模型。细胞株导入单纯载体(pcDNA3.1)作为对照。于基因导入后24h应用FCM细胞凋亡率。结果该5种细胞株中,AML193与K562呈RIZ1mRNA无转录或低转录,但导入pRIZ1RH24h后则出现转录或转录明显增加,且测得该两细胞株凋亡率分别为(22.7±0.7)%、(28.6±1.2)%,均高于相应对照组[分别为(11.7±1.6)%、(9.0±0.8)%],差异均有显著性(P<0.01)。结论RIZ1的转录能够诱导AML193和K562髓性白血病细胞株凋亡,提示该基因可作为其失活的髓性白血病患者之基因疗法的重要靶分子。     

Reduced expression of HLA class I and II antigens in colon cancer

The expression of HLA class I and II antigens was studied by immunohistochemistry in (a) specimens of colon cancer from 25 patients, (b) normal colonic mucosa obtained 5-10 cm away from each tumor, and (c) colonic mucosa from 13 normal individuals. Thirteen of the tumor specimens had normal epithelium adjacent to the cancer, which thus served as an internal control. The expression of HLA class I antigens in colon cancer was dramatically reduced compared to control (P less than 0.0001): undetectable in 28%, diminished in 68%, normal in 4%. The expression of class II antigens was also reduced in cancer (P less than 0.0001 for all when compared to normal), being undetectable in most (HLA-DP 64%, HLA-DQ 72%, HLA-DR 68%). In 44% of the cancers all three HLA class II antigens were undetectable; in 92% at least one class II antigen was undetectable; and in 20% both class I and class II antigens were undetectable. No cancer specimen had a completely normal HLA phenotype. The expression of other surface antigens was preserved in cancer tissues and, therefore, loss of HLA antigens was not due to a nonspecific decline in surface molecules. When glands of normal mucosa immediately adjacent to cancer were compared to those of normal controls, significantly reduced expression of only HLA class I antigens (P = 0.0149) and HLA-DP (P = 0.034) was found. The expression of the HLA antigens in colonic mucosa remote from the cancer was no different from that of normal controls. Our data show extensive and significant reduction in the expression of HLA antigens in colon cancer; its potential relationship to immunosurveillance in cancer is discussed.     

Cytotoxic activity of an anti-transferrin receptor immunotoxin on normal and leukemic human hematopoietic progenitors

The process of cellular iron uptake involves a specific receptor for the plasma carrier transferrin and a pathway of receptor-mediated endocytosis. Transferrin receptor expression is closely related to the rate of cell proliferation, and conjugates between anti-transferrin receptor monoclonal antibodies and toxins have been shown to have potent cytotoxic activity. We have constructed an anti-transferrin receptor immunotoxin by conjugating the anti-transferrin receptor monoclonal antibody B3/25 to a ribosome-inactivating protein, the saporin-6 (SO6), which is derived from the seeds of the plant Saponaria officinalis. The immunotoxin B3/25-SO6 was tested for in vitro cytotoxic activity against the human cell lines K-562 and HL-60 and against normal human bone marrow hematopoietic progenitors and acute myeloid leukemia clonogenic cells. The immunotoxin proved to be an effective inhibitor of K-562 and HL-60 clonogenic cell growth, in vitro colony formation being completely inhibited at immunotoxin concentrations ranging from 10(-7) to 10(-10) M. B3/25-SO6 markedly reduced the recloning efficiency of HL-60 clonogenic cells at 10(-12) M. Exposure of HL-60 cells in suspension culture to 10(-9) M B3/25-SO6 for 48-72 h completely abolished their clonogenic potential. The immunotoxin was also found to be cytotoxic against normal human bone marrow progenitor cells (burst-forming unit-erythroid and colony-forming unit-granulocyte, macrophage) in a dose-dependent manner. However, exposure of normal colony-forming unit-granulocyte, macrophage in suspension culture to 10(-9) M B3/25-SO6 for 72 h resulted in only 50% suppression of their clonogenic potential. Finally, B3/25-SO6 was found to be a potent inhibitor of in vitro growth of acute myeloid leukemia clonogenic cells. The cytotoxic effects of B3/25-SO6 were shown to be specific, since both saporin alone and irrelevant immunotoxins did not have any effect in the cellular systems examined. We conclude that the immunotoxin B3/25-SO6 has dose-related cytotoxic effects on both normal and leukemic human hematopoietic progenitors. Since there are substantial differences between normal and leukemic progenitors with respect to the proportion of cycling cells and the expression of transferrin receptors, B3/25-SO6 or similar immunotoxins may have clinical application in bone marrow-purging procedures.     

Comparison of the sensitivity of normal and leukaemic myeloid progenitors to in-vitro incubation with cytotoxic drugs: a study of pharmacological purging

The sensitivity of myeloid leukaemic colony forming cells (AML-CFC), to five cytotoxic drugs has been compared in two culture systems with the sensitivity of normal myeloid progenitor cells (GM-CFC). No increased sensitivity was found for AML-CFC to any of the chemotherapeutic agents studied. AML-CFC were significantly less sensitive than normal GM-CFC to mafosfamide at the doses commonly used to purge bone marrow autografts. It is suggested that AML cells probably display similar sensitivity to cytotoxic agents as normal myelopoietic cells at a similar stage of differentiation. Hence complete elimination of the leukemic clone by pharmacological purging may be incompatible with bone marrow re-engraftment. We conclude that purging AML autografts with any of the agents examined has little scientific basis.     

HLA class I and class II expression of pulmonary adenocarcinoma cells and the influence of interferon gamma

Background: The clinico-biological significance of HLA (both class I antigen and class II one) expressed on tumor cells still remains controversial. Methods: Tumor cells were freshly separated from 33 surgical specimens of pulmonary adenocarcinoma. The tumor cells were incubated for 24 h in the presence or absence of IFN-γ (130 International Units/ml). After incubation, the cells were cytocentrifuged onto glass slides and immunostained with either an anti-HLA class I (A, B, C) monoclonal antibody or anti-HLA class II (DR) one. Results: In 22 of 33 cases (66.7%), the HLA class I were individually expressed by more than 60% of tumor cells while so were the HLA class II in 15 (45.4%). No significant correlation was observed between the HLA class I expression and the HLA class II one. The proportion of HLA class I-positive tumor cells correlated with neither the grade of histological differentiation nor the stage of disease. In contrast, the proportion of HLA class II-positive tumor cells correlated with both the grade of histological differentiation and the stage. In most cases, IFN-γ was found to increase the proportion of class II-positive tumor cells as well as that of class I-positive cells. Conclusions: The above findings thus suggested that the HLA class II expression might therefore represent a manifestation of cellular differentiation and that IFN-γ may, as a result, have the potential to differentiate cancer cells.     

Differential sensitivity of leukemic and normal hematopoietic progenitors to the killing effect of hyperthermia and quercetin used in combination: Role of heat-shock protein-70

Autologous bone-marrow transplantation (ABMT) is widely used in the treatment of acute leukemias where a matched sibling donor is not available for allogeneic transplantation. However, a major problem in ABMT is relapse, and ex vivo purging may be very important in preventing it. We show here that quercetin enhances the growth-inhibitory effect of hyperthermia (HT) in AML (19 cases) and ALL (6 cases) leukemic blasts. Furthermore, the inhibitory effect of this combined treatment resulted in leukemic-cell apoptosis. On the contrary, normal hematopoietic progenitors were neither growth-inhibited nor induced to apoptosis by HT-plus-quercetin treatment. To explain this difference in sensitivity of leukemic and normal hematopoietic progenitors, we analyzed the effect of quercetin on heat-induced expression of heat-shock protein-70 (HSP-70), which has been shown to be important in regulating thermosensitivity. We found that quercetin inhibits heat-induced HSP-70 expression both at protein and at mRNA levels in AML and ALL blasts. In normal CD34⁺ progenitors, the combined treatment with HT and quercetin did not reduce HSP-70 expression and did not induce cell apoptosis. Considering the difference in heat sensitivity of normal CD34⁺ and leukemic progenitors in the presence of quercetin, the combined use of HT and quercetin could constitute a purging protocol for ABMT. Int. J. Cancer 73:75–83, 1997. © 1997 Wiley-Liss, Inc.