Effect of monoclonal antibodies (MoAb) to class I and class II HLA antigens on lectin- and MoAb OKT3-induced lymphocyte proliferation

We have examined the effect of several monoclonal antibodies (MoAb) to monomorphic determinants of class II HLA antigens, and MoAb to monomorphic determinants of class I HLA antigens and to beta-2-microglobulin (beta 2-mu) on lectin- and MoAb OKT3-induced proliferation of human peripheral blood mononuclear cells (PBMNC) and cultured T cells (CTC). Some, but not all, anti-class II HLA MoAb inhibited the proliferative response of PBMNC to MoAb OKT3 and pokeweed mitogen (PWM). The degree of inhibitory effect varied considerably. This effect was not limited to anti-class II HLA MoAb since anti-class I HLA MoAb and anti-beta 2-mu MoAb also inhibited MoAb OKT3- or PWM-induced proliferative responses. In contrast, the response of PBMNC to phytohemagglutinin (PHA) and concanavalin A (Con A) was not blocked by any anti-class II HLA MoAb. However, some anti-class II HLA MoAb also inhibited the proliferative response of CTC plus allogeneic peripheral blood adherent accessory cells (AC) to PHA or Con A as well as to MoAb OKT3 or PWM. This may be attributable to the substantially greater class II HLA antigen expression by CTC than by fresh lymphocytes. Pretreatment of either CTC or AC with anti-class II HLA MoAb inhibited OKT3-induced proliferation. In contrast, pretreatment of CTC, but not AC, with anti-class I HLA MoAb inhibited the proliferative response of CTC to OKT3. Pretreatment of CTC with anti-class I HLA MoAb inhibited PHA-, Con A and PWM-induced proliferation, to a greater degree than the anti-class II HLA MoAb. It appears as if lymphocyte activation by different mitogens exhibits variable requirements for the presence of cells expressing major histocompatibility determinants. Binding of Ab to membrane markers may interfere with lymphocyte-AC cooperation, perhaps by inhibiting binding of mitogens to their receptors or by interfering with lymphocyte and AC function. We also have examined the role of class II HLA antigens on CTC by depleting class II HLA-positive cells. As expected, elimination of class II HLA-positive AC with anti-class II HLA MoAb plus complement caused a decrease in proliferation of CTC in response to all the mitogens tested. In contrast, elimination of class II HLA-positive CTC was shown to clearly increase proliferation of CTC, perhaps because this may deplete class II HLA-positive suppressor cells.     

Lymphokine regulation of human lymphocyte proliferation: Formation of resting G0 cells by removal of interteukin 2 in cultures of proliferating T lymphocytes

The question of whether lymphocytes which have once been activated and have completed one or several cell cycle(s) can return to the G₀ phase and stay ready for a new activation (G₀-G₁ transition), rather than simply die, was investigated. To do so interleukin 2 (IL-2) was removed from cultures of continuously proliferating human T lymphocytes and the formation of resting (G₀) cells was measured. Kinetic analyses in freshly prepared peripheral blood lymphocytes (PBL) revealed that the onset of detectable RNA synthesis and the appearance of structures binding the anti-Tac antibody occurred simultaneously. This allowed the expansion of the definition of G₀ T lymphocytes as cells having a low RNA (and DNA) content, and no Tac antigen. When cultured human T cells proliferating continuously by means of IL-2 were characterized in terms of their distribution in the cell cycle, 7 days after the initial PHA stimulation, it could be demonstrated that very few cells were in the G₀ phase, supporting the concept of direct S/G₂/M-G₁ transition. However, when IL-2 was removed from the cultures, the [³H]thymidine incorporation per 10⁴ cells and correspondingly the number of cells in the S/G₂/M and G₁ phases were reduced drastically and during the following 72-hr period, the number of G₀ cells increased markedly. Restimulation of such in vitro formed G₀ cells, under conditions permitting observation of their shift from the G₀ to G₀ phase, demonstrated that most cells could respond normally. Based on these observations, it was concluded that IL-2 not only ensures T-lymphocyte survival and proliferation, but IL-2 starvation induces many continuously proliferating T lymphocytes to stop cycling and to return to the G₀ phase of the cell cycle where they remain functional.     

Nucleotide sequence of the split tRNAUAALeu gene from Vicia faba chloroplasts: evidence for structural homologies of the chloroplast tRNALeu intron with the intron from the autosplicable Tetrahymena ribosomal RNA precursor

Summary The gene encoding the tRNA _UAA ^Leu from broad bean chloroplasts has been located on a 5.1 kbp long BamHI fragment by analysis of the DNA sequence of an XbaI subfragment. This gene is 536 bp long and is split in the anticodon region. The 451 bp long intron shows high sequence homology over about 100 bp from each end with the corresponding regions of the maize chloroplast tRNA _UAA ^Leu intron. These conserved sequences are probably involved in the splicing reaction, for they can be folded into a secondary structure which is very similar to the postulated structure of the intron from the autosplicable ribosomal RNA precursor of Tetrahymena. Very little sequence conservation is found in the 5-and 3-flanking regions of the broad bean and maize chloroplast tRNA _UAA ^Leu genes.     

Selection of peptides inhibiting a beta-lactamase-like activity.

A library of random peptide sequences was used to select peptides that inhibit an anti-idiotypic catalytic Ig, immunoglobulin (IgG) 9G4H9, with a beta-lactamase-like activity. This library displays cyclic heptapeptides on the surface of bacteriophages and represents a collection of up to 4.5 x 109 peptides. The first selection step aimed at enriching the library in species that bind to the whole Ig molecule. The second step was to discriminate peptides that bind to part of the molecule other than the active site. Selected peptides were then screened by surface plasmon resonance analysis. Those displaying measurable Kd values were assayed for their ability to inhibit the catalytic Ig.     

Cultures of purified human natural killer cells: Growth in the presence of interleukin 2

We have isolated highly enriched populations of LGL, which are virtually devoid of mature typical lymphocytes (as enumerated by morphological and surface antigen analysis using monoclonal antibodies e.g., OKT3) in comparison to T cells which contain greater than 95% sheep erythrocyte-forming cells and are devoid of LGL and $\text{NK}\text{K}$ activities. Both types of cells grew in the presence of crude or partially purified IL-2. Cultures of LGL could be initiated consistently even in the absence of lectins and the cultured LGL retained their characteristic morphology and cytotoxic activity. However, within 7–10 days after initiation, the cultured LGL changed in surface phenotype to become antigenically indistinguishable from cultured T cells.     

Phenotypic analysis of a large number of normal human bone marrow sample by flow cytometry

Bone marrow aspirates from 48 healthy donors (34 adults, 14 children) were analyzed by flow cytometry (FACS Analyzer) after purification of low-density bone marrow cells (Ld BMC) on a density gradient (d = 1,077) and labelling with 23 anti-hematopoietic cell monoclonal antibodies. Based on physical properties, these Ld BMC could be divided into four different populations called E, My, Mo and L, which comprised 14% +/- 9%, 31% +/- 16%, 10% +/- 5% and 45% +/- 14% of these cells, respectively. The phenotypic analysis of these different populations enabled the identification in E, of erythrocytes (Glycophorin A+, Rhesus D+, but negative for early erythroid differentiation markers such as the transferrin receptor (Tf. R) and the FA6-152 antigen); in My of cells of the myeloid lineage (VIM2+, HLA DR-); in Mo of cells of the monocytic lineage (VIM2+, CD14+) plus some myeloblasts (VIM2+, CD14-, HLADR+) and finally in L of a heterogeneous population including: 1. T lymphocytes labelled to the same extent by CD2, CD3, CD5 and CD6 (28% +/- 10%), B lymphocytes assessed by CD19 and CD20 (12% +/- 8%), Pre-B cells (CD10+ = 8% +/- 7%), less than 5% of "natural killer" cells (CD16+ or Leu7+) and finally, less than 6% of myelomonocytes (CD14+ and/or VIM2+). 2. The erythroid lineage (rhesus D+ = 42% +/- 20%, Tf.R+ and FA6-152+ = 32% +/- 12%). 3. Undifferentiated cells or progenitor cells (CD34+ = 7% +/- 5%). 4. Cells unlabelled by any antibodies (approximately 6%). We observed no difference between bone marrow samples from adults or children, with respect to physical properties, and with all but four immunological markers. A significantly higher proportion of B cells (CD19+ and CD10+) (P less than 0.001) and undifferentiated cells (CD34+ and HLADR+) (P less than 0.02) was observed in children. These data, obtained from a large number of bone marrow samples, could be used to quantify the imbalance of some bone marrow disorders.