Suppression of the human allogenetic response in vitro with primed lymphocytes and suppressive supernates.

Human lymphocytes primed in vitro by allogeneic cells develop lymphocyte populations with different functions. Cells with a memory of the allogeneic contact and cytotoxic effectors have been identified previously. We now report on a third lymphocyte population generated by repeated in vitro sensitization. This is of suppressor lymphocytes that can inhibit the primary proliferation of unsensitized lymphocytes. Our experiments indicate that these human suppressor cells are most probably T lymphocytes, adherent to glass or nylon wool, and radioresistant. They derive from both the large blast cells and the small, nondividing lymphocytes that are observed on day 7 of the allogeneic response. The suppressor cells release suppressor factor(s) upon restimulation. Studies on the specificity of the suppression have shown that suppressor cells are specific to the HLA-DR antigens presented by stimulator lymphocytes and that they probably release the suppressor factor only when confronted with the specific HLA-DR antigen. However, when the suppressor factor is produced, the proliferative response to any stimulating cell is inhibited regardless of its HLA-DR antigens. On the other hand, the suppressor factor can only suppress the proliferation of lymphocytes from some individuals. This restriction suggests that suppression can only occur when the producer of the suppressor factor and the responding lymphocytes that are being tested, have some identities in common. No evidence in favor of an HLA-D restriction has been obtained so far.

Structural organization of the hCTLA-1 gene encoding human granzyme B.

Cytotoxic T lymphocytes (CTLs) and natural killer/lymphokine-activated cells produce granzymes, a family of serine esterase proteins located in cytoplasmic granules. These might be involved in different cytotoxic pathways. We report the structural organization of the human gene encoding granzyme B (hCTLA-1). A 4.75-kb genomic DNA fragment containing all the sequences of granzyme B-encoding cDNA clones has been sequenced. The gene is composed of five exons and four introns. A comparison with the genomic organization of murine CCP1/CTLA-1 showed very similar structure and a 76% nucleotide homology in the coding sequences. This suggests that both genes may have a common ancestor. No typical regulatory element was detected in the 1160 bp upstream from the ATG start codon. The detection of a second locus related to hCTLA-1 is also described.

Perforin and granzyme B expression is associated with severe acute rejection. Evidence for in situ localization in alveolar lymphocytes of lung-transplanted patients.

To evaluate rejection episodes in lung-transplanted patients, we analyzed 31 bronchoalveolar lavage specimens for lymphocyte levels and lymphocyte expression of two intracytoplasmic activation markers, perforin, the pore-forming lytic protein, and granzyme B, a member of the serine esterase family. Using anti-human granzyme B and perforin mAbs, we show that their expression in alveolar lymphocytes is correlated with the severity of rejection as assessed by histological parameters and the patients' clinical status. The presence of these molecules may provide a prognostic parameter that will facilitate the patients' monitoring, particularly in cases with minimal acute lung rejection susceptible to rapid progression to severe rejection.

In vitro allostimulation of peripheral blood mononuclear cells generates autologous reactive T lymphocytes: analysis at the clonal level.

Cells from two siblings within family PER with a maternal crossing-over were used as responder and stimulator cells; they differed in class II MHC gene products. The responder cells were cloned and studied for their specificity. All the clones were T4+, most of them, were allospecific cytotoxic T-cell clones, some were only proliferative. Among the 62 clones that have been screened for their phenotype and function, we could expand, in our cloning system, two autoreactive T-cell clones specific for the self-class II MHC products. The results indicated that this autoreactivity was also observed in serum-free medium, which excludes any serum components susceptible to alter the self-class II MHC products. For the autorecognition, the two clones used the T3-Ti complex, and only one of them, the T4 structure.

What is a T-cell clone? Effect of rIFN on T-cell clone function and T-cell receptor gene rearrangement.

A T4+ proliferative, noncytotoxic cloned line acquires specific lytic function by treatment with recombinant interferon alpha or gamma. Simultaneous with the acquisition of this new cell function, a rearrangement of the T-cell receptor alpha gene occurs. These changes necessitate a revised concept of a T-cell clone regarding its T-cell receptor gene configuration and cell function.

Allospecific proliferative human T-cell clones acquire the cytotoxic effector function after three months in culture, in IL-2 conditioned medium.

Allostimulated T lymphocytes were cloned by micromanipulation and expanded in IL-2 conditioned medium. Three T3+,T4+,T8-, clones called BJ1, BJ4, and BJ37, were extensively studied. The BJ1 cells were able to proliferate and kill the specific target. The BJ4 and BJ37 cells were able to proliferate with the specific restimulator but could not kill even in lectin-dependent cell-mediated cytotoxic assay; however, they acquired the specific cytolytic activity in the 6-day culture when fresh irradiated autologous peripheral blood mononuclear cells as feeder cells were added to the specific irradiated Epstein-Barr virus transformed cell line, in the presence of recombinant IL-2. This observation strongly suggested that the culture conditions could be involved in the differentiation of proliferative clones into cytotoxic T lymphocyte (CTL) clones, by the lymphokines, either present in the IL-2 conditioned medium or secreted by the mixed allogeneic irradiated feeder cells. Moreover, it was shown that the acquisition of the cytolytic function could be blocked by the monoclonal antibody LeoA1, previously described and which recognized the TLiSA1 structure involved in the CTL differentiation.

Granzyme B-gene expression: a marker of human lymphocytes "activated" in vitro or in renal allografts.

Human killer cells contain cytoplasmic granules in which serine esterases, or granzymes, and perforin have been identified. We have studied the induction of granzyme B-gene expression in peripheral blood lymphocytes and large granular lymphocytes activated by various stimuli in vitro as well as in cellular infiltrates at the site of renal allografts in patients with and without rejection. Using in situ hybridization, kinetic experiments have shown that granzyme B mRNA is an early marker of in vitro cell activation. Data obtained in vivo also indicate the presence of granzyme B mRNA-bearing cells, which argues in favor of the induction of granzyme B gene in cells activated in vivo.

A five-generation family with sacral agenesis and spina bifida: possible similarities with the mouse T-locus.

In man, a malformation that recalls some of the defects associated with T/t mutants in the mouse is sacral agenesis. We report on a family with a high incidence of sacral malformation, ranging from a complete absence of the sacrum (SA), with or without spina bifida aperta, to a spina bifida occulta (SBO) that could only be detected by x-ray. The condition appeared in a man with four children who were all affect, and thereafter, to varying degrees, in 17 of his 28 descendants. Segregation analysis has been performed in this family, using the Elston and Stewart transmission probability model [1971]. The two traits (SA and SBO) were first studied separated and then together. A fully penetrant major dominant gene is show to cause SA. When the phenotypes SA and SBO are considered together, Mendelian transmission is rejected. This could be explained genetically by two alternative hypotheses: genetic heterogeneity or a dominant major gene transmitted in excess by heterozygotes (tau Aa A = 0.896), suggesting a segregation distortion property of an allele at a T-like locus.

HLA-DR-specific suppressor cells after repeated allogeneic sensitizations of human lymphocytes in vitro.

In conclusion, DR-specific suppressor cells can be induced by repeated in vitro sensitizations. They were able (1) to decrease a secondary proliferation, (2) to suppress consistently, in a primary proliferative assay, when added as third cells (primed twice against a DR antigen [PLT II] and gamma-irradiated), the response of unprimed cells towards stimulating cells, which share a DR specificity with the priming cell of the PLT II. The suppression follows the D part of the recombinant haplotype within an HLA-B/D recombinant family and is specific for the DR antigen used twice as stimulator for production of the PLT II.

Close genetic relationships between determinants of the HLA-D region detected by MLR-I, MLR-II, and B-lymphocyte serology.

The human Ia equivalent (Ly-Li system) or a very closely linked gene plays a preponderant role in secondary MLR, and also has an additive effect on the intensity of the primary MLR. PLTs developed in Li phenoidentity seem to detect still other determinants.

Bone marrow transplantation in 65 patients with severe aplastic anemia.

This articles summarizes the experience of the Hospital Saint-Louis Bone Marrow Transplantation Team of bone marrow transplantation in severe aplastic anemia. Sixty-five consecutive patients have been transplanted with an HLA identical sibling marrow. Various conditioning regimen have been used. Conditioning regimen using high dose Cyclophosphamide alone or associated with Procarbazine and anti-thymocyte globulin gave a high number of bone marrow graft rejection. Therefore, a conditioning regimen using Cyclophosphamide and total body irradiation with lung shielding has been used for the last three years. This regimen suppressed bone marrow graft rejection. The main problems remain graft versus host disease and intercurrent infections. Despite these complications, 50 per cent of the patients become long term survivors and are apparently cured of their initial disease.