Production of antigen-specific human monoclonal antibodies: comparison of mice carrying IgH/kappa or IgH/kappa/lambda transloci.

Here we compare human monoclonal antibody (MAb) production from mouse strains that carry disruptions of their endogenous mouse IgH/IgK loci and harbor human IgM + Igkappa(BABkappa) or human IgM + Igkappa + IgA transloci (BABkappa,lambda). We found that whereas both strains proved effective for the isolation of antigen-specific IgM antibodies, many of the IgM MAbs elicited from BABkappa comprise human mu chains that are associated with mouse lambda chains. In contrast, BABkappa,lambda mice gave rise to fully functional, polymeric human IgM antibodies comprising both human IgH and human IgL chains. Therefore, the inclusion of a human Iglambda translocus (in addition to the human IgH + Igkappa transloci) not only diminishes problems of endogenous mouse Iglambda expression but also provides a strain of mice that yields fully human MAbs to a wide range of antigens, as witnessed by the isolation of MAbs to human blood cells, tumor cell lines, and an immunoglobulin idiotype.

Delimitation of the proliferative stages in the human thymus indicates that cell expansion occurs before the expression of CD3 (T cell receptor).

Precursor of T lymphocytes undergo proliferation and maturation under the influence of the thymic microenvironment. In our study, we have attempted to determine the distribution of human postnatal thymocytes in division according to their stage of differentiation. Our data show that about 11.5% of all thymic cells are in S/G2/M phases, and that a subset of the cortical and precortical subpopulations contains most of the dividing cells. Rate of cell division is maintained at high levels from the prethymocyte precursor along the successive stages of differentiation represented by CD1+CD3-CD4-CD8- and CD1+CD3-CD4+CD8- cells. The percentage of dividing cells is maximal in an intermediate subset of CD1+CD3-CD4+CD8-CD45RO+ cells defined by the distinct expression of class I HLAdim/high molecules, which could contain cells in transit from prethymocytes to double-positive cortical cells. The CD3- fraction of the double-positive cortical cells contains most of the dividing thymocytes, although the rate of division within this subset is much less than that of the precursor CD1+CD3-CD4+CD8- cells. In a linear scheme of differentiation, cell division stops at or near the point of initiation of CD3 expression. These results suggest that in human thymus cell expansion takes place before the initiation of the positive selection process. According to this view the stringency of the selection process would require the previous generation of a large number of precursors to permit the production of sufficient numbers of mature T cells.

Differential effect of tumour necrosis factor on human thymocyte subpopulations.

We have studied the effect of tumour necrosis factor (TNF) on purified human thymocyte subpopulations. For this purpose human thymocytes were purified by negative selection with three rounds of several antibodies plus complement. TNF was able to co-stimulate in a dose-response manner the proliferation of single positive (SP) CD3+ CD4+ or CD3+ CD8+ thymocytes in the presence of optimal doses of interleukin-2 (IL-2), phytohaemagglutinin (PHA), anti-CD3 antibodies or phorbol esters. However, CD1+ CD3low CD4+ CD8+ cortical thymocytes did not proliferate significantly in response to any stimulus alone or in combination. The TNF proliferative effect on SP thymocytes was blocked by an anti-IL-2R alpha antibody. In addition, TNF enhanced the expression of the IL-2R alpha but not IL-2R beta on the cell surface of CD1- CD3+ SP thymocytes over the levels induced by the other primary stimuli, inducing as a consequence, an increase in the number of high affinity IL-2R. Furthermore, TNF was able to increase IL-2R alpha mRNA levels on SP thymocytes. On the other hand, TNF was mitogenic in the absence of any other stimulus for CD1- CD3- CD4- CD8- prethymocytes, as was IL-2, and this proliferation was not blocked by anti-IL-2R alpha antibodies. Furthermore, the proliferation of this subset in response to IL-2 and TNF was additive. TNF was able to increase directly the cell surface expression of both chains, IL-2R beta and IL-2R alpha, and the IL-2R alpha messenger RNA (mRNA) levels of CD1- CD3- CD4- CD8- prethymocytes. In summary, our results suggest that TNF may have an important role as a co-stimulatory signal in some human thymocyte subpopulations by inducing the expression of IL-2R.

Apoptosis in human thymocytes after treatment with glucocorticoids.

Treatment of unfractionated human thymocytes in culture with the synthetic glucocorticoid dexamethasone induced cell death, as measured by trypan blue exclusion, after several hours of incubation. In purified subsets of human cortical and medullary thymocytes dexamethasone caused cell lysis with similar kinetics in both populations; 50% of thymocytes were killed after 20-24 h of incubation with the steroid. The mechanism of dexamethasone-induced cell death seems to correspond to apoptosis since degradation of DNA into oligonucleosome-sized fragments could be observed in the cultures treated with the steroid. A certain degree of DNA fragmentation and cell death could also be observed in control cultures of thymocytes. In contrast, peripheral T lymphocytes were resistant to the cytolytic effect of glucocorticoid hormone. The killing of human thymocytes by dexamethasone was inhibited by cycloheximide, suggesting that this cell death program requires a fully operating protein synthesis machinery and perhaps the induction of new proteins.

IL-2 protects against anti-CD3-induced cell death in human medullary thymocytes.

In recent years, several studies have confirmed the clonal elimination of thymocytes with receptors that recognize Ag and MHC molecules present on the membrane of thymic stromal cells, a process that may be relevant to the establishment of self-tolerance. In our work, we show that anti-CD3 treatment of single positive CD4+ or CD8+ human medullary thymocytes (obtained by anti-CD1a plus C) induces their apoptotic death. Some events commonly associated with the early steps of normal activation (IL-2R expression, increase in cytoplasmic Ca2+) are also induced after anti-CD3 treatment. Nevertheless, IL-2 is not secreted by these activated cells. The addition of exogenous IL-2 inhibits the apoptosis induced by anti-CD3. We suggest that the lack of secretion of IL-2 by medullary thymocytes may be a physiologic mechanism implicated in the process of negative selection that leads to tolerance.

Correlated expression of surface antigens in human thymocytes. Evidence of class I HLA modulation in thymic maturation.

The correlated expression of several surface antigens (CD 1, class I HLA, CD3) was examined in human unfractionated thymocytes or selected subsets by using single or double-color flow cytometry. Prethymocyte subpopulations expressed high levels of HLA. A high proportion of cortical cells expressed low levels of either HLA or CD 3 antigens. Most of these HLA+ cortical cells corresponded to the more immature cells and did not express HLA-B loci products. CD 1 a and CD 3 antigens were expressed in a high percentage of cells and the levels of expression of each antigen in individual cells were inversely correlated. These data and the contour of double-color histograms are suggestive of the existence of a single pathway of thymocyte differentiation in which class I HLA expression is switched off around the time of the initiation of CD 3 (and Ti?) expression. We suggest that the anti-major histocompatibility complex (MHC) specificity of the unselected Ti receptor may be incompatible with the expression of MHC products on the cell membrane. At this stage, CD 1 antigens, whose expression is inversely correlated with that of HLA, may fulfill the role of MHC antigens. The latter can be re-expressed later on, once the anti-MHC specificities of the Ti receptors have been selected against. Studies on in vitro modulation of HLA molecules by interferon-alpha did not reveal any correlation to the expression of CD 1 or CD 3 antigens.

Cell surface molecular changes on the activation of human thymocytes.

The changes in the expression of antigen molecules on the cell surface membranes of uncultured (nonactivated) and activated human thymocytes have been studied by flow cytometry and immunoprecipitation techniques. Nonactivated thymocytes do not have the phenotypic profile of a resting population because they express cell proliferation molecules such as the transferrin receptor and the 4F2 antigens (mainly the 100,000 dalton subunit). After activation with IL 2-containing supernatants, mature T3+, T6- thymocytes proliferate and are able to nonspecifically kill different target cells. The activated thymocytes are T3+, T11+, T6-, OKM1- and bear T4 or T8 antigens in mutually exclusive cell subpopulations. They also "de novo" express the IL 2 receptor, and the 210.000/130.000 molecular complex defined by the TS2/7 MAb. Activated human thymocytes express higher amounts of class I and class II MHC antigens, equal T3 and LFA-1, and lower quantities of T11 and T4 molecules than nonactivated thymocytes. Furthermore, activated thymocytes only express the T8 34,000 dalton polypeptide subunit, whereas the nonactivated thymocyte population expressed the T8 34,000 dalton associated with a 46,000 glycoprotein. We have demonstrated that this structural change in the T8 molecule from a complex of two polypeptide subunits of 46,000 and 32,000 does not indeed occur in the activation process but rather in the maturation from T6+ to T6- thymocytes.