Quantitative studies on the mixed lymphocyte interaction in rats. 3. Kinetics of the response.

The proliferative interaction of cultured rat lymphocytes of immunogenetically disparate origin-the mixed lymphocyte interaction-was employed as an experimental model to examine the initial stages of the immune response mechanism. Using mixed cultures of cells derived from parental strain and F(1) hybrid rats, in which only the parental lymphocytes respond, the following observations were made on the magnitude and kinetics of the reaction. After initiation of the cultures, there was a latent period of approximately 40 hours during which time no mitotic activity was detected. This inactive phase was followed by a period of proliferation in which previously nondividing cells entered the mitotic cycle for the first time. Activity in the cultures, as detected by incorporation of radioactive thymidine and measured by radioautography or scintillation spectrometry, increased exponentially with a doubling time (T2) of 9-10 hr. In this exponential proliferative phase, lasting approximately 100 hr, the dividing cells underwent a series of rapid sequential divisions with a generation time (Tc) of 8 hr, and few, if any, dropped out of the mitotic cycle. In addition to the cells which first entered mitosis at the beginning of the proliferative phase and then proceeded through multiple divisions, significant numbers of new, previously nondividing cells continued to enter the mitotic cycle during the entire exponential growth phase. The total number of these newly responsive, first division cells throughout the total culture period amounted to 1-3% of the original parental cell inoculum. This is a surprisingly large proportion of peripheral blood lymphocytes with demonstrable reactivity to a particular antigen system, if it is assumed that these first division cells in vitro are functionally related to the hypothetical antigen-sensitive cells which proliferate and differentiate into immunological effector cells. At present there is no entirely satisfactory explanation for this large number of reactive cells in the mixed lymphocyte interaction.

Quantitative studies on the mixed lymphocyte interaction in rats. IV. Immunologic potentiality of the responding cells.

Studies were designed to provide some explanation for the unexpectedly large proportion (2%) of parental rat strain peripheral blood lymphocytes that are reactive in the mixed lymphocyte interaction (MLI) to a strong homologous transplantation isoantigen(s) present on cells from an F(1) donor. The possibilities considered involve nonspecific activation and multispecific reactivity on the part of the responding cells. The essential findings of this study were: (a) In 3-way mixed cultures, lymphocytes obtained from tolerant animals were not "recruited" to proliferate in the presence of cells from normal, isologous donors which were in the process of responding to F(1) cells bearing the tolerance-inducing antigens. With the use of chromosome markers and normal and tolerant parental strain donors of different sexes, the responsive cells were identified and proved to be derived from the normal and not the tolerant donor. (b) The magnitude of the proliferative response is increased additively when potentially reactive cells are exposed to two antigen systems simultaneously. On the other hand, doubling the "gene-dosage" of the genetic determinants of the H isoantigens employed had no effect on the responding cells. (c) A state of induced immunologic tolerance to one H isoantigen system did not alter the response capacity of cells from such a donor to an alternative antigen system. (d) Mixed cultures of heterologous cells from human and rat donors displayed a proliferative response which was less than that of homologous mixed cultures from human or rat donors. Prior sensitization of rat donors with human cells, however, greatly increased the mitotic activity of rat lymphocytes stimulated with human cells. These results suggest that the large number of responsive cells in the MLI do not include a significant number recruited or activated in some nonspecific manner. Rather, they appear to be fully specific in their response capacities so that a given lymphocyte does not react to a multiplicity of different antigens. The degree of proliferation depends on the number of different antigen systems presented to the responding population and not on the number of genetic determinants or "gene dosage" of a given isoantigen system. Finally, on a cell-for-cell basis, the peripheral blood lymphocyte population contains more cells reactive to histocompatibility isoantigens within the species than to heterologous antigens of a different species.

Quantitative studies on the mixed lymphocyte interaction in rats. V. Tempo and specificity of the proliferative response and the number of reactive cells from immunized donors.

These studies were designed to determine what influence prior immunization with homologous H isoantigens might have on the subsequent proliferative activity of lymphocytes from these animals in the mixed lymphocyte interaction. The results demonstrate the following: (a) Subcutaneous immunization with splenic cells from donors differing at the major H locus accelerates the tempo of the proliferative response to F(1) cells bearing these same antigens in the MLI, whereas antigen given systemically reduces the proliferative response. (b) The altered proliferative behavior is specific for the immunizing antigens. (c) The period after immunization during which the MLI displays an altered tempo is a short one, lasting not longer than 3 wk. (d) Whether they are derived from previously immunized or from normal donors, the proportion of lymphocytes responsive in the MLI is the same, even though the response profiles are different. These results suggest that in comparison to immune responses to other types of antigens, immunologic reactivity to the major H isoantigens already involves a large number of antigen-reactive cells in normal animals and that the proportion of these cells is not increased as a result of immunization. Rather, lymphocytes from immunized animals respond more rapidly to the presence of these antigens.

Studies on the life history of lymphocytes. I. The life-span of cells responsive in the mixed lymphocyte interaction.

The life history, within the rat, of lymphocytes responsive to histocompatibility isoantigens in the mixed lymphocyte interaction was examined by the use of in vivo labeling with tritiated thymidine and radioautography. Lymphocytes in the peripheral blood and H-ARC (mitotic figures in the MLI) were compared with respect to the frequency of labeled cells and the median grain count. The following conclusions were drawn from this study: (a) Although some can be considered long-lived, the majority of H-ARC are the products of recent divisions in the body. (b) Adult thymectomy does not eliminate the production of long-lived lymphocytes, some of which are H-ARC. Hence, in addition to direct origin in the thymus, H-ARC, as well as other lymphocytes of the long-lived lymphocyte population, may derive from already existing thymus-derived cells in the circulation and thymus-dependent areas of the secondary lymphoid tissues. (c) Sublethal X-irradiation (600 R) in combination with adult thymectomy does not eliminate the capacity to produce some long-lived lymphocytes, however, few if any are H-ARC. (d) H-ARC and other long-lived lymphocytes appear to go through a series of rapid multiple divisions before they enter the circulation. Thereafter, long-lived lymphocytes appear to undergo intermittent single divisions which decrease both the frequency and median grain count of labeled cells gradually with time. On the other hand, labeled H-ARC maintain a more stable grain count despite a rapid decrease in frequency with time. This is taken to indicate that H-ARC are less likely to undergo occasional single divisions during their life-span, but may undergo periodic rapid sequential divisions. A speculative model is developed from these data on the life history of H-ARC which may be of predictive value in future studies and which can be tested against known facts.

Quantitative studies on the mixed lymphocyte interaction in rats. II. Relationship of the proliferative response to the immunologic status of the donors.

The influence of the immunologic status of the cell donors on the proliferative behavior of rat lymphocytes in the mixed lymphocyte interaction has been studied. Mixed cultures of cells from various parental and F(1) combinations having morphologically distinguishable sex chromosomes exhibited unidirectional proliferative reactivity. The mitotic figures were predominately of parental origin. Lymphocytes from donors made tolerant at birth to homologous transplantation isoantigens were specifically unreactive against cells bearing antigens of the tolerance inducing strain, but not to indifferent third party homologous lymphocytes. Cells from animals that had been surgically thymectomized at birth exhibited a markedly and sometimes totally diminished reactivity against homologous lymphocytes. Presensitization of the cell donors resulted in a curtailment of proliferative reactivity in cultures with cells bearing the immunizing antigens. This may reflect the destructive properties that lymphocytes from sensitized animals are known to possess. The results of these experiments show that the proliferative activity of lymphocytes in the mixed lymphocyte interaction accurately reflects the immunologic status of the cell donors, and these findings provide further support for the premise that the mixed lymphocyte interaction represents a primary immunologic response by cells in culture against homologous cells bearing histocompatibility antigens.

A potassium ionophore (Nigericin) inhibits stimulation of human lymphocytes by mitogens.

Nigericin, an ionophore that exchanges K+ for H+ across most biologic membranes, reversibly inhibited the proliferative response of human lymphocytes to phytohemagglutinin (PHA). Inhibition occurred at nigericin concentrations of 10(-8) M or greater, and only during the early event of mitogenesis. There was no effect if nigericin was added 24 h or later after the initiation of PHA-stimulated cultures. The effect was not the result of toxicity or impaired mitochondrial respiration. At similar concentrations, nigericin also inhibited lymphocyte responses in mixed lymphocyte cultures and to other mitogens including concanavalin A, pokeweed mitogen, and the calcium ionophore A23187. The findings support the view that one or more transmembranous events, mediated by changes in cation flux and/or membrane potential, are critical in the initial stages of lymphocyte mitogenesis.

Proliferation in vivo of T lymphocytes reactive to histocompatibility alloantigens: a correction.

Rat lymphocytes in mixed cultures can reutilize tritiated thymidine from labeled granulocytes. Shortly after thymidine injections in vivo, major effects on the frequency of labeled lymphocyte mitoses in peripheral blood cultures are introduced by 10-20% polymorph contamination, even though transfer of label via supernates is not demonstrable. Cold thymidine in the cultures prevents reutilization, and has permitted reevaluation of several previous conclusions concerning the life history of lymphocytes reactive to major histocompatibility alloantigens (HARC). Rather than being predominantly recently divided cells, HARC do not appear to have an age distribution, in blood or lymph, significantly different from the general recirculating lymphocyte population. However, the ability of immunization across strong allogeneic differences to increase markedly the proportion of young HARC among the specifically responsive population has been confirmed.

The clonal evolution of tumor cell populations.

It is proposed that most neoplasms arise from a single cell of origin, and tumor progression results from acquired genetic variability within the original clone allowing sequential selection of more aggressive sublines. Tumor cell populations are apparently more genetically unstable than normal cells, perhaps from activation of specific gene loci in the neoplasm, continued presence of carcinogen, or even nutritional deficiencies within the tumor. The acquired genetic insta0ility and associated selection process, most readily recognized cytogenetically, results in advanced human malignancies being highly individual karyotypically and biologically. Hence, each patient's cancer may require individual specific therapy, and even this may be thwarted by emergence of a genetically variant subline resistant to the treatment. More research should be directed toward understanding and controlling the evolutionary process in tumors before it reaches the late stage usually seen in clinical cancer.

Regulation of bcl-2 proto-oncogene expression during normal human lymphocyte proliferation.

The bcl-2 and c-myc proto-oncogenes are brought into juxtaposition with the immunoglobulin heavy chain locus in particular B-cell lymphomas, resulting in high levels of constitutive accumulation of their messenger RNAs. Precisely how the products of the bcl-2 and c-myc genes contribute to tumorigenesis is unknown, but observations that c-myc expression is rapidly induced in nonneoplastic lymphocytes upon stimulation of proliferation raise the possibility that this proto-oncogene is involved in the control of normal cellular growth. In addition to c-myc, the bcl-2 proto-oncogene also was expressed in normal human B and T lymphocytes after stimulation with appropriate mitogens. Comparison of the regulation of the expression of these proto-oncogenes demonstrated marked differences and provided evidence that, in contrast to c-myc, levels of bcl-2 messenger RNA are regulated primarily through transcriptional mechanisms.

Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation.

From an acute B-cell leukemia cell line, a DNA probe was obtained that was specific for chromosome 18 and flanked the heavy chain joining region of the immunoglobulin heavy chain locus on chromosome 14. This probe detected rearrangement of the homologous DNA segment in the leukemic cells and in follicular lymphoma cells with the t(14:18) chromosome translocation but not in other neoplastic or normal B or T cells. The probe appears to identify bcl-2, a gene locus on chromosome 18 (band q21) that is unrelated to known oncogenes and may be important in the pathogenesis of B-cell neoplasms with this translocation.

Locus of the alpha-chain of the T-cell receptor is split by chromosome translocation in T-cell leukemias.

Mouse lymphoma cells were hybridized with two human acute T-cell leukemias with a t(11;14) (p13;q11) translocation and the segregated hybrids were examined for the presence of the DNA segments coding for the constant (C) and the variable (V) regions of the alpha chain (C alpha and V alpha) of the T-cell receptor. The C alpha segment was translocated to the involved chromosome 11 (11p+) while the V alpha segment remained on the involved chromosome 14 (14q-). The data indicate that the locus for the alpha chain of the T-cell receptor is split by the chromosomal breakpoint between the V alpha and the C alpha gene segments, and that the V alpha segments are proximal to the C alpha segment within chromosome band 14q11.2.

Location of gene for beta subunit of human T-cell receptor at band 7q35, a region prone to rearrangements in T cells.

The T-cell receptor is formed by two chains, alpha and beta, for which specific clones were recently obtained. In this report the gene for the beta chain of the human T-cell receptor was located on the long arm of chromosome 7, band q35, by means of in situ hybridization. This chromosome region in T cells is unusually prone to develop breaks in vivo, perhaps reflecting instability generated by somatic rearrangement of T-cell receptor genes during normal differentiation in this cell lineage.

Molecular cloning of the chromosomal breakpoint of B-cell lymphomas and leukemias with the t(11;14) chromosome translocation.

The chromosomal breakpoint of chronic lymphocytic leukemia (CLL) cells of the B-cell type carrying the translocated long arms of chromosomes 11 and 14 [t(11;14) (q13;q32)] was cloned. The breakpoint was found to be within the joining segment of the human heavy chain locus on the translocated long arm of chromosome 14. A probe that is specific for chromosome 11 and that maps immediately 5' to the breakpoint on the 14q+ chromosome was isolated. The probe detected a rearrangement of the homologous genomic DNA segment in the parental CLL cells and also in DNA from a diffuse large cell lymphoma with the t(11;14) translocation. This rearranged DNA segment was not present in Burkitt lymphoma cells with the t(8;14) translocation or in nonneoplastic human lymphoblastoid cells. The probe can thus be used to identify and characterize a gene located on band q13 of chromosome 11 that appears to be involved in the malignant transformation of human B cells carrying the t(11;14) translocation. This gene, named bcl -1, appears to be unrelated to any of the known retrovirus oncogenes described to date.

Mapping human autosomes: evidence supporting assignment of rhesus to the short arm of chromosome No. 1.

Rh-negative erythrocytes were found in the blood of an Rh-positive man suffering from myelofibrosis. Nucleated hemopoietic precursors were also circulating in his blood, and these cells had an abnormal chromosome complement from which identifiable chromosome segments had been deleted. Correlation of the serological and cytogenetic findings, combined with previous data, indicates that the Rhesus blood group locus is on the distal portion of the short arm of chromosome No. 1.

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.

Expression of c-myc proto-oncogene in normal human lymphocytes. Regulation by transcriptional and posttranscriptional mechanisms.

Aberrant expression of the c-myc gene results from nonrandom chromosomal translocations involving the transcriptionally active antigen receptor gene loci, in particular lymphocytic leukemias and lymphomas, and is believed to contribute to the etiology of these neoplasms. In addition to its expression in abnormal lymphocytes, increased accumulation of c-myc mRNA occurs rapidly in normal B- and T-lymphocytes after stimulation with appropriate mitogens. The mechanisms that mediate these mitogen-induced elevations in c-myc mRNA levels, however, have not been determined for normal B and T cells. By using enriched populations of B- and T-lymphocytes obtained from freshly isolated human tonsils and stimulated with Staphylococcus-A or with phytohemagglutinin, respectively, we observed marked elevations (20-40-fold) in the steady state levels of accumulated c-myc messenger RNA (mRNA) within 1 h of exposure of cells to mitogens; modest increases (three- to fivefold) in the relative rate of transcription of the c-myc gene through protein synthesis-independent (cycloheximide-insensitive) mechanisms; and rapid rates of degradation of mature c-myc mRNAs through protein synthesis-dependent (cycloheximide-sensitive) mechanisms. These findings corroborate previous studies in other cell types and provide evidence for both transcriptional and posttranscriptional control of c-myc proto-oncogene expression in normal human lymphocytes.

Dexamethasone inhibition of interleukin 1 beta production by human monocytes. Posttranscriptional mechanisms.

Dexamethasone is known to have an inhibitory effect on IL-1 production. To determine the mechanism(s) of this inhibition, adherent human blood monocytes were stimulated with Escherichia coli lipopolysaccharide (LPS) (10 micrograms/ml) in the presence of dexamethasone. Nuclear transcription run-off assays showed that LPS induced IL-1 beta gene transcription two- to fourfold and that this induction was unaffected by dexamethasone exposure (10(-5) M). The lack of dexamethasone's transcriptional effects was further supported by the absence of any significant change in IL-1 beta mRNA accumulation between LPS-stimulated monocytes exposed or unexposed to dexamethasone, as determined by Northern blot analysis. Posttranscriptionally, dexamethasone was found to have multiple effects: slight prolongation of IL-1 beta mRNA half-life, moderate inhibition of translation of the IL-1 beta precursor, and profound inhibition of the release of IL-1 beta into the extracellular fluid. The data indicate that IL-1 beta is first translated as the 33,000-D pro-IL-1 beta protein, the predominant intracellular form, and the processed to a 17,500-D IL-1 beta protein before or during extracellular transport. The major inhibitory effects of dexamethasone appear to be directed at the translational and posttranslational steps involved in these events.

Karyotypic differences between primary cultures and cell lines from tumors with the human T-cell leukemia virus.

Chromosome abnormalities were studied in primary cultures and in established T-cell lines from patients with human T-cell leukemia virus (HTLV)-positive leukemia or lymphoma. The present findings, and data from other laboratories, indicated that primary cultures of the HTLV-positive neoplastic cells nearly always showed a chromosomally abnormal clone, whereas most established cell lines had an apparently normal karyotype. These differences included circumstances in which the same blood specimen was used for both types of culture or in which separate specimens were obtained within a short time span. These observations indicated that many cell lines from HTLV-positive leukemia or lymphoma may be derived from nonneoplastic T-cells that were transformed in vitro by the leukemia virus; human T-cells newly infected with HTLV were suggested to have an in vitro growth advantage over the HTLV-infected tumor cells.

Changes in clonal expression during the course of acute leukemia: possible subsets in childhood leukemia.

We studied cell surface markers and chromosomes in the leukemia cells of a boy with the initial diagnosis of acute lymphocytic leukemia during 18 months from diagnosis to demise. During this time he received induction therapy, underwent bone marrow transplantation, and relapsed. The leukemia cells expressed three membrane phenotypes during different stages of disease: T-cell at diagnosis; T-cell, B-cell, and monocyte during the induction period; T-cell in the first relapse after bone marrow transplantation; and T-cell and B-cell during the terminal stage. Some cells expressed markers of two cell types, indicating a common origin of these cells. Cytogenetic studies during post-transplantation relapse showed abnormal marker chromosomes that indicated two major sublines. However, there was enough sharing of other aberrant chromosomes to suggest that these two populations presented sublines within the same neoplastic clone. We suggest that these leukemia cells were derived from a pluripotential cell prior to differentiation into cells of the lymphoid and monocytic series. This particular case may represent a subset of acute leukemia and may account for the resistance to conventional therapy.