Immunological functions of human T-lymphoid cell line (MOLT). I. Release of immunosuppressive factors from the mixture of MOLT-4 cells and sheep red blood cells.

A short term incubation of the mixture of established human T-lymphoid cells (MOLT) and sheep red blood cells (SRBC) resulted in the release of factors which nonspecifically suppressed the response of mouse spleen cells against heterologous erythrocytes in vitro. Neither human B-cell line (RPMI 1788), nor the supernate of MOLT cell suspension in the absence of SRBC had such suppressive effects. The supernate of the mixture of MOLT cells with chicken red blood cells (CRBC) did not suppress either anti-CRBC or anti-SRBC responses of mouse spleen cells. Since CRBC did not form rosettes with MOLT cells, it is suspected that the origin of the production of these factors might be MOLT cells forming SRBC rosettes. Some of these factors are dialysable.

Specific transplantation antigens of mouse sarcomas induced by adenovirus type 12.

A specific isograft resistance against three different mouse adeno 12 sarcomas was demonstrated in mice treated with four to eight doses of viable or X-ray-killed adeno 12 mouse tumors. Whole-body X-ray irradiation with 350 R 24 hr previous to the test challenge did not abolish the resistance, indicating that it was due to a true anamnestic immune reaction. This was further proven by the finding that similar treatment with tumors of other origin did not induce any immunity, nor did the treatment with adeno 12 tumor material induce any immunity against two neoplasms of Schmidt-Ruppin-Rous viral origin. The previous report by Trentin et al. (17) that adeno 12 infection leads to a specific transplantation immunity was fully confirmed. When the specificity of this virus-induced immunity was studied it was discovered that besides adeno virus type 12, type 7 and probably type 18 also gave the same type of resistance while adenovirus type 5 did not. A contamination of the adeno 7-infected mice with adeno type 12 was excluded by testing pooled sera from these animals for anti-adeno 12 CF or HI antibodies.

Rearrangement and expression of the alpha, beta, and gamma chain T cell receptor genes in human thymic leukemia cells and functional T cells.

Using cDNA and genomic probes representing the alpha, beta, and gamma chain of the human T cell receptor genes, we have examined the structure and expression of these genes in 14 human leukemic T cell lines, representing different stages of thymic differentiation, and 15 functional human T cell clones. Rearrangement of the gamma and beta chain genes was found in all of the functional T cell clones and all but one (P30/OKUBO) thymic leukemia cell line; all of the lines that had rearrangement of the beta chain expressed beta mRNA. Expression of the alpha chain was found in all of the functional T cell clones examined, while rearrangement of the alpha chain gene, using currently available probes to the J region, could be shown in 10 of 13 functional clones. In contrast, expression of the alpha chain was found in 6 of 10 leukemic T cell lines, while rearrangement was found in six of these nine cell lines. Of the 14 leukemic cell lines studied for rearrangement of the alpha chain, rearrangement was found in six cases. The data obtained with the cell lines are consistent with an ordered rearrangement and expression of the gamma, beta, and alpha chains of the T cell antigen receptor (TcR) genes. The leukemic cell lines used in the present study have previously been characterized with regard to cell surface antigens and intracellular enzymes. Based on those results a scheme of thymic development was proposed. The developmental stages identified by those studies are not in complete agreement with stages of T cell development, as determined in the present study using molecular probes.

Isolation and transmission of human retrovirus (human t-cell leukemia virus).

Nine new isolates of human T-cell leukemia-lymphoma virus (HTLV) were obtained from cells of seven patients with malignancies of mature T cells and from two clinically normal relatives of a T-cell leukemia patient. These people were from the United States, Israel, the West Indies, and Japan. The virus was detected in the fresh T cells and was isolated from the established T-cell lines. Each isolate is closely related to the first HTLV isolate, and all the new HTLV isolates were transmitted into normal human T cells obtained from the umbilical cord blood of newborns.

Deregulation of c-myc by translocation of the alpha-locus of the T-cell receptor in T-cell leukemias.

Two human T-cell leukemias carrying a t(8;14)(q24;q11) chromosome translocation were studied for rearrangements and expression of the c-myc oncogene. For one leukemia, rearrangement was detected in a region immediately distal (3') to the c-myc locus; no rearrangements of c-myc were observed in the second case (DeF). However, studies with hybrids between human and mouse leukemic T cells indicated that in the leukemic cells of DeF, the breakpoint in chromosome 14 occurred between genes for the variable (V alpha) and the constant (C alpha) regions for the alpha chain of the T-cell receptor. The C alpha locus had translocated to a region more than 38 kilobases 3' to the involved c-myc oncogene. Since human c-myc transcripts were expressed only in hybrids carrying the 8q+ chromosome but not in hybrids containing the normal chromosome 8, it is concluded that the translocation of the C alpha locus 3' to the c-myc oncogene can result in its transcriptional deregulation.

T- and B-lymphocytes in chronic lymphocytic leukemia: correlation with clinical and immunologic status of the disease.

T- and B-lymphocyte count were correlated with clinical and immunologic status of 59 patients with chronic lymphocytic leukemia. The mean total T-cell count was slightly lower than normal in patients in complete remission, within normal limits in those in partial remission, and significantly higher in those with active disease. The mean total B-cell count, however, was slightly elevated in patients in complete remission and those in partial remission, and markedly elevated in those with active disease. The T-cell count correlated well with the duration of disease in patients in remission: The mean count was within normal range in those patients with disease of less than 3 years, whereas for those patients with disease of 3-12 years, a significant reduction was observed. The T-cell count was well correlated with the status of skin test response of patients with either complete or partial remission; the B-cell count did not correlate with immunoglobulin levels in patients with this disease.

Rabbit antiserum against a non-T, non-B leukemia cell line that carries the Ph1 chromosome (NALM-1): antibody specific to a non-T, non-B acute lymphoblastic leukemia antigen.

Rabbit immune sera against human cells of a non-T, non-B leukemia cell line bearing the Ph1 chromosome (NALM-1) were characterized. After proper and exhaustive absorption, the sera were tested by indirect membrane immunofluorescence on a panel of 19 human hematopoietic cell lines with T-, B-, or non-T, non-B cell surface characteristics. The absorbed sera reacted specifically with NALM-1 cells but not with cells of another Ph1-positive cell line, K-562. Four of the 6 leukemia T-cell lines, 2 of the 4 leukemia-lymphoma B-cell lines, all of 4 acute lymphoblastic leukemia (ALL) lines with non-T, non-B cell characteristics, and uncultured cells of all patients with non-T, non-B cell ALL were reactive with the antisera. A cross-absorption study of the antisera suggested that a single antigenic complex is involved in this antigen specificity. The antigen involved appears to be a common non-T, non-B ALL one that has been described previously.

Electron microscope study on human lymphocyte-sheep erythrocyte rosettes.

Nonimmune rosette formation of human peripheral blood lymphocytes and cultered MOLT 3 and MOLT 4 cells with sheep red blood cells was studied by transmission electron microscopy. At the cell contact area of the rosette, lymphoid and red cell membranes were parallel and 80-100 A apart. The inner leaflet of the lymphoid cell membrane seemed denser, and amorphous substance attached to its cytoplasmic face. The cell contact area was 100-1000 nm wide and frequently on the lymphoiid cell body rather than on microvilli, though some cells extended microvilli near red cells.

Quantitation of human thymus-leukemia-associated antigen in established hematopoietic cell lines by radioimmunoassay.

A competitive radioimmunoassay for a saline-soluble human thymus-leukemia-associated antigen (HThy-L) was applied for quantitation of this antigen in leukemia and normal hematopoietic cell lines. Highly increased quantities of HThy-L were detected in all T-cell leukemia lines tested, regardless of the presence or absence of receptors for sheep erythrocytes. This elevated level of HThy-L in combination with high terminal deoxynucleotidyl transferase and adenosine deaminase activities and the presence of a T-lymphocyte-specific surface antigen appear to represent stable phenotypic characteristics of T-cell lines. Most normal B-cell lines had low quantities of HTy-L. The level of HThy-L was slightly elevated in a considerable number of lymphoma B-cell lines and in all non-T, non-B leukemia cell lines tested. No relationship existed between quantities of HThy-L and an expression of different surface immunoglobulin isotypes in B-cell lines. Low quantities of HThy-L were detected in leukemia myeloid and myeloma cell lines as well as in B-cell leukemia lines originating from patients with B-cells acute lymphoblastic leukemia. Apparently, the increased quantities of HThy-L in T-cell leukemia lines may be related to certain stages of T-cell differentiation at which leukemia cell transformation occurs.

Differences in purine metabolizing enzyme activities in human leukemia T-cell, B-cell and null cell lines.

Clear metabolic differences between T- and B-cells were demonstrated. Both adenosine deaminase (ADA) and nucleoside phosphorylase (NP) activities increased during logarithmic growth and then decreased in T-cells, but remained essentially constant during the growth cycle of B-cells. When these enzyme activities were examined in a number of T-cell, B-cell, and null cell lines, ADA activity was clearly higher in T-cells as compared to all others. With NP, the opposite appeared to be true, although the differences were much smaller and not statistically significant in all instances. No clear differences were found in the isoenzyme distributions of both enzymes in the various cell types.

Chromosome evolution of near-haploid clones in an established human acute lymphoblastic leukemia cell line (NALM-16).

A cell line has been established from blood lymphoblasts of a female patient with acute lymphoblastic leukemia (ALL) shown to have near-haploid (27 chromosomes) cells in the bone marrow. The findings about the cell line were: 1) The frequency of near-haploid cells in culture decreased with time from 98.2% when the culture was started to 5.4% 15 months later. 2) Most of the other cells except the near-haploid ones were hyperdiploid, i.e., duplicates of the cells with near-haploid chromosome constitutions. 3) Chromosome evolution was seen in the near-haploid clones. The possible ancestor clone (clone A) had 27 chromosomes, one of each pair except no.10, 14, 18, and 21, which were disomic. A suggested evolution process is: clone A yields clone B (26 chromosomes: clone A, -no.10) yields clone C (27 chromosomes: clone B, +X) yields clone D (26 chromosomes: clone C, -no.21), clone E (28 chromosomes: clone C, +NO.20). Clone B and D, each with 26 chromosomes, appeared to contain the lowest number of chromosomes, appeared to contain the lowest number of chromosomes ever described for human somatic cell clones in vitro. 4) Changes in the constitutions of the hyperdiploid cell clones were preceded by evolution and changes in the near-haploid clones. 5) In near-haploid cells with 2 X-chromosomes, 1 exhibited late DNA replication; in hyperdiploid cells with 3-5 X-chromosomes, 2 were non-late DNA-replicating. 6) Fresh (uncultured) and cultured leukemia cells were antigenically typical non-T, non-B or common type ALL cells (positive for la-like and null-type ALL antigens and negative for surface membrane immunoglobulin).

Dependency of sister chromatid exchange in T- and B-cells on the incorporation of deoxyribonucleosides into chromosomal DNA.

Human T-cells (CCRF-HSB2) did not incorporate tritiated thymidine ([3H]TDR)--1.0-5.0 muCi/ml--into the nuclei, where.as they readily incorporated tritiated deoxycytidine (E13H]CDR). When contamination with pleuropneumonia-like organisms was ruled out, these findings strongly suggested a deficiency of the enzyme thymidine kinase in the cells. Human B-cells (CCRF-SB) and normal T-lymphocytes (NTL) readily incorporated [3H]CDR, [3H]TDR, and tritiated 5-bromodeoxyuridine, and they clearly exhibited differential staining of the sister chromatids (SCD). When nonisotopic bromodeoxyuridine (BUDR), 10(-6)-10(-4) M, was used with the B-cells and NTL, SCD were clearly evident and sister chromatid exchange (SCE) was relatively infrequent; when the concentration was 10(-7) M, SCD staining was poor but the frequency of SCE was high. SCE frequencies in NTL, measured by autoradiography after incorporation of [3H]CDR, were the same as SCE frequencies measured by staining with BUDR at 10(-4) M. In the case of CCRF-HSB2, 10(-4) M BUDR produced relatively high frequencies of SCE as did 10(-7) M BUDR with the former two cells. However, [3H]CDR with CCRF-HSB2 gave relatively low frequencies of SCE, of the magnitude observed after 10(-4) M BUDR was used with NTL and the B-cells. Thus the high frequency of SCE in CCRF-HSB2 cells may have been due to the staining property of chromosomes that had incorporated low levels of BUDR.

L-asparagine requirements of human T-lymphocytes and B-lymphocytes in culture.

The characterization of two human T-lymphocyte lines revealed that they required exogenous L-asparagine for cell growth, whereas all four B-cell lines studied were L-asparagine independent. T-cells were 800-2,000 times more sensitive to Escherichia coli L-asparaginase than were B-cells. The cytotoxic effects of a high concentration of L-asparaginase on B-cells were not related to the hydrolysis of L-asparagine but were due to heat-labile and heat-resistant substances in the enzyme. The findings were consistent with reports that L-asparaginase is effective in suppressing cellular immunity and inducing remission in patients with acute lymphocytic leukemia, mainly a non-B-cell disease. Thus these cell lines provide in vitro models for the study of a nutritional approach to chemotherapy or immunotherapy.

Differential chemotherapeutic susceptibility of human T-lymphocytes and B-lymphocytes in culture.

After previous work from this laboratory revealed that asparaginase was 800-2,000 times more inhibitory against human T-lymphocytes in culture than against B-lymphocytes, a similar further study of 13 chemotherapeutic and immunosuppressive agents was done. Cytosine arabinoside and 5-fluorouracil also had differential inhibitory activities on human T- and B-cells in culture. On the basis of the dose producing 50% inhibition of viable cell growth on day 5, cytosine arabinoside had 45-80 times more inhibitory activity against T-cells than against B-cells. In contrast to asparaginase and cytosine arabinoside, 5-fluorouracil had 10-20 times more inhibitory activity against B-cells. The rest of the chemotherapeutic and immunosupressive agents tested had minor or no differential activity. These findings indicated that T-cell response to asparaginase and cytosine arabinoside and B-cell response to 5-fluorouracil may be exploitable for the differential immunosuppressive effects presumed to be active in vivo. In addition, such differential responses may predict differential tumor cell behavior against these chemotherapeutic agents by T- and B-cell neoplasms in vivo.

High terminal deoxynucleotidyl transferase activity in a new T-cell line (RPMI 8402) of acute lymphoblastic leukemia origin.

High activity of terminal deoxynucleotidyl transferase (terminal transferase) was found in a new "thymus-dependent" cell line (RPMI 8402) which is of acute lymphoblastic leukemia origin. This enzyme resembled the terminal transferase from other human cells in all its properties including Km (0.7 x 10(-6) m for dGTP). The high activity of this enzyme in RPMI 8402 and fresh acute leukemia lymphoblasts, in contrast to the low activity of this enzyme reported for "thymus-independent' cells, suggested that this cell line may have originated from leukemia cells. Moreover, the high activity of terminal transferase in RPMI 8402 cells should make feasible large-scale purification of this enzyme for detailed studies.

Enzyme markers in acute leukemias: advances during the last decade.

Besides the recent advances in immunology that provided important information for the characterization of leukemia cells, enzyme marker analysis is another area of progress in leukemia research. Assays of enzyme activities, both quantitative enzyme levels and qualitative isozyme changes, are of value in the classification of leukemias. The interest in the study of enzyme markers is due not only to technical improvements and new biochemical possibilities, but also to the involvement of enzyme marker analysis in the so-called "multiple marker analysis," which combines traditional and recently developed techniques in leukemia research. The aims of this review are to describe well-known and potential enzyme markers as well as to stress the relevance of enzyme marker analysis combined with multiple marker analysis for leukemia subclassification and the understanding of normal hematopoietic cell differentiation.

Acute lymphocytic leukemia-associated cell membrane antigen.

Antisera were raised in New Zealand White rabbits against non-B, non-T acute lymphocytic leukemia (ALL) cells coated with antilymphocyte serum. Following minimal absorption with chronic lymphocytic leukemia (CLL) cells, the antiserum reacted mainly with non-B, non-T ALL cells. The following numbers of patients had leukemia cells that reacted with the ALL antisera: 13 of 18 with ALL, 3 of 27 with acute myelocytic leukemia, 1 of 8 with chronic myelocytic leukemia (CML), and 0 of 12 with CLL. The positive CML was a patient in CML blast crisis. Normal peripheral blood B- and T-lymphocytes and normal bone marrow were negative. Reactions of the anti-ALL serum (136K) were compared with the reactions of a rabbit anti-B-cell antiserum (63K) that reacted with approximately 70% of leukemia cells. Cultured lymphoblastoid cell lines from normal donors were negative by both cytotoxicity and immunofluorescence tests. However, by immunofluorescence testing, 8 of 17 known malignant lines from a variety of lymphoproliferative disorders were positive; 4 of these lines were of T-cell origin. By immunoprecipitation and polyacrylamide gel electrophoresis, the ALL antigen appeared to consist of a single polypeptide chain of approximately 98,000 daltons. The anti-ALL antiserum was not cytotoxic for normal myeloid stem cells (colony-forming units).

Establishment of a hairy cell leukemia cell line carrying Tac antigen and phagocytic activity with B-cell characteristics.

A hairy cell leukemia cell line designated "Hair-M" was established in a suspension culture derived from the peripheral blood of an 86-year-old Japanese male with a diagnosis of hairy cell leukemia. The Hair-M cells were identified as having prominent hair-like cytoplasmic projections by examination with phase-contrast and scanning electron microscopy. These cells displayed ruffled membranes and stublike microvilli similar to those observed on the surfaces of cells in the peripheral blood of the patient. Immunologic and cytochemical studies on the Hair-M cells confirmed derivation from the clone of the patient's leukemia cells. Although the cultured Hair-M cells had definite B-cell characteristics, such as IgG kappa-chains on the surface and in cytoplasm, they also demonstrated Tac antigen, which is usually expressed on activated T-cells, and myelomonocyte antigens determined by OKM-1 and MCS-1 monoclonal antibodies. Other cell surface markers, including E(-), IgGFc(-), IgMFc(-), C3R(+), Ia-like antigen(+), OKT9(+), OKT10(+), and terminal deoxynucleotidyl transferase(-), were detected; no Epstein-Barr virus-determined nuclear antigen was detected. The karyotype of the Hair-M cells was determined to be 46XY with -11, -14, and two marker chromosomes. The Hair-M cells also had phagocytic activity to rabbit anti-human IgG serum-coated polyacrylamide gel particles.

A non-T, non-B human leukemia cell line (NALM-1): establishment of the cell line and presence of leukemia-associated antigens.

A permanent hematopoietic cell line, designated NALM-1, was established from the peripheral blood of a patient who was in blastic crisis of Ph1-positive chronic myleocytic leukemia. By means of a panel of specific xenoantisera, the NALM-1 cells were found to express a specific antigen of acute lymphoblastic leukemia and blast leukemia-associated antigen. The cells exhibited no cell-surface receptors for sheep erythrocytes, IgG, or complement; neither cell-surface immunoglobulins nor cytoplasmic immunoglobulin were observed. Furthermore, normal T-cell or B-cell antigens, detectable by the antisera used in this study, were not found in the NALM-1 line.

Cytogenetic study of a new Ph1-positive cell line (NALM-1).

The chromosomes of a cell line (NALM-1) derived from the leukocytes of a patient with chronic myelocytic leukemia (CML) were examined with several banding techniques. The modal chromosome number was 46 and the cells contained a Philadelphia chromosome (Ph1), due to the standard translocation of the missing segment of the long arm of chromosome No. 22 onto the distal end of the long arm of chromosome No. 9, i.e., t(9;22) (q34;q11). The Ph1-positive modal cells of the NALM-1 line also had two common marker chromosomes, an extra X-chromosome, and missing chromosomes in groups No. 7, 9, and 15. Immunologic examination of the NALM-1 cells revealed them to have non-T-non-B (null) surface characteristics. An antigen specific for cells of acute leukemia and a human la-like antigen were detected. These facts suggested that the NALM-1 cell line originated from CML cells and maintained the cytogenetic and Immunologic characteristics of such cells.