Secondary MLR Research Articles

Selective abrogation of alloreactivity via priming in the presence of aphidicolin, a specific inhibitor of DNA polymerase.

Aphidicolin, a specific and direct inhibitor of eukaryotic DNA polymerase alpha, was used to investigate its impact on immunologic reactions in vitro. Dose response curve of the inhibitory effect was studied in murine and human primary allogeneic responses, as well as the proliferative responses to both PHA and Con A mitogens. The presence of aphidicolin during the allosensitization phase in secondary MLR of mice splenocytes resulted in complete abolishment of the subsequent response directed against the priming alloantigens, whereas alloreactivity to unrelated alloantigen-bearing cells was inhibited to a much lesser degree. The allosensitized aphidicolin-treated cells lost the ability to respond to subsequent PHA stimulation, but were capable of exerting a high responsiveness to Con A. The presence of aphidicolin during the allosensitization phase in secondary MLR of human mononuclear cells resulted in markedly decreased alloreactivity directed against the priming cells, but spared the subsequent response to unrelated alloantigens and to both PHA and Con A mitogenic stimuli. It is suggested that aphidicolin may be used for selective inactivation of proliferating cells without interfering with immunologic functions of other quiescent subsets. Aphidicolin may thus be a useful agent for induction of specific unresponsiveness in experimental models of allogeneic transplantation.

Lymphokine production by murine T cells in the mixed leukocyte reaction.

Although the production of B cell stimulatory factors by cell lines and hybridomas is well established, production of specific lymphokines by normal T cells in response to antigen stimulation has not been analyzed. We have used bioassays and neutralizing mAbs to demonstrate that IL-2, IL-4, and B cell growth factors (BCGF) are produced during primary and secondary MLRs. IL-2 is detected in the first 12 h of both types of MLR. IL-4 and BCGF appear at 24-48 h in the conditioned medium of the primary MLR, and peak by 12 h in the secondary MLR. The amount of IL-4 in the primary response reaches a level that is 10% of that detected in the secondary. In contrast, BCGF production steadily increases over time in the primary MLR, and maximal production is equivalent to that made in the secondary response. Allogeneic dendritic cells and anti-Ig-activated B blasts both stimulated lymphokine production in the primary MLR, whereas small B cells were weak. In the secondary MLR, all three cell populations stimulated the production of IL-2, IL-4, and BCGF. Therefore, the release of several defined B cell stimulating factors can be detected in the conditioned media of responding primary T lymphocytes.

Possible role of neuraminidase in activated T cells in the recognition of allogeneic Ia.

In a primary MLR, predominant stimulators in spleen cells are adherent cells and not B cells, although B cells are one of the cell types expressing a large amount of Ia molecules. Our previous experiments showed that T cells treated with neuraminidase (Nase) responded to an allogeneic Ia on B cells. In our experiments, the relationship between the responsiveness to the allogeneic Ia molecules on B cells and Nase activity of T cells was examined. The results showed that T cells increased in Nase activity with the acquisition of the reactivity to Ia on B cells. T cells from normal mice increased in Nase activity after the incubation for 3 days or more in MLR, and these T cells responded to allogeneic Ia on B cells. However, T cells from mice genetically deficient in Nase responded poorly to the Ia on allogeneic B cells even after the incubation in MLR for 3 days. T cells incubated for 3 days in MLR decreased in electrophoretic mobility, indicating the decrease of net negative charge of the cells, and increased in their binding of peanut agglutinin which has been reported to bind to galactosyl residues exposed on T cell surface by removing sialic acids. These results suggest that Nase in T cells was activated by the cultivation in MLR for 3 days, and sialic acids of some molecules on T cell surface were removed by the enzyme and, in turn, T cells acquired the responsiveness to allogeneic B cells in a secondary MLR. Thus, Nase was suggested to play a regulatory role in the recognition of Ia molecules in T cells.

Effects of cyclosporin a on in vitro lymphocyte response to autologous or allogeneic stimulation: Influence of HLA phenotypes

In this study we investigated whether the interindividual variability of lymphocyte sensitivity to cyclosporin A (CsA) could be controlled by the HLA region. The models used were the in vitro primary and secondary autologous (AMLR) and allogneic mixed lymphocyte (MLR) cultures of cells from 32 healthy subjects from our HLA reference panel. Our results show that CsA inhibited primary allogeneic MLR to a much greater extent than primary AMLR (−81 ± 2% vs −38 ± 8%, P < 0.001). The same pattern was observed when cells harvested from CsA-treated primary cultures were rechallenged in secondary cultures with the original sensitizing stimulator cells (−40 ± 6% vs −17 ± 9%, P < 0.05). No differences were observed in primary autologous and allogeneic cultures among responders of different HLA phenotypes. In contrast, the secondary responses did vary according to the HLA types: in secondary AMLR, CsA-priming did not lower, or even enhance, the proliferative responses of DR5 + and/or DR2 + lymphocytes (+7 ± 13%), whereas it significantly lowered the responses of DR2 −5 − cells (−46 ± 8%). In secondary MLR, lymphocytes proliferation was lowered by CsA-priming in all but DRW11(5) + subjects (−45 ± 7% vs +2 ± 23%, P < 0.05). It is concluded that the individual HLA phenotype influences the pattern of lymphocyte sensitivity to CsA.

In vitro immunomodulatory effects of placental substances on preparatory MLR and resulting modifications of lymphocyte reactivities

The immunomodulatory effects of murine placental extracts (PE) were studied in vitro using mixed lymphocyte culture (MLC) and resulting cell-mediated lympholysis (CML). The results showed that preparative cultures in the presence of PE syngeneic to the responding cells led to a low secondary MLR response with a concomitant generation of suppressor cells. At the efferent phase, cells from the same preparative culture showed a weaker cytotoxic activity than controls cultured in the absence of extract. Furthermore, the induction of regulatory cells able to inhibit CTL in vitro activity was also observed. The active substances can be found in the 30% ammonium sulphate precipitate as well as in some gel filtration fractions showing several main bands from 115 to 43 kDa in SDS-PAGE.

Resistance to suppression and acquisition of memory by human T cells

Human memory cells acquire resistance to several types of suppressor cells, including MLR generated suppressor cells. These data suggest one possible mechanism of that resistance, namely retention of IL-2 receptors in the resting state. Cells from a 7-day MLR were separated on a single step percoll gradient. All proliferating cells were found in the interface. Pellet cells were nondividing. Interface and pellet cells had equivalent memory function in a secondary MLR. Thus, there appear to be at least two subpopulations of memory cells, including one that is primed without undergoing division. These subpopulations are functionally distinct. Interface memory cells were 30–50% more resistant than pellet memory cells to MLR generated suppressor cells. On culture day 10, neither pellet nor interface cells displayed significant spontaneous proliferation but exogenous Interleukin 2 (IL-2) produced up to five times as much proliferation in interface cells as in pellet cells. Further, FACS analysis with an anti-TAC equivalent antibody also showed that significantly more interface cells have surface receptors for IL-2. Thus, cells that had previously divided continue to have more and/or higher affinity receptors for IL-2 even after return to the resting state. If a mechanism of suppression in the mixed lymphocyte reaction is to reduce the synthesis/release of Il-2, memory cells may acquire their relative resistance to this suppression by virtue of the increased IL-2 sensitivity of this discrete subpopulation.

Human suppressor cell induction in vitro: preferential activation by class I MHC antigen.

Human peripheral blood lymphocytes heated at 45 degrees C for 1 hr were found to continue to express all the serologically detected class II MHC antigens (HLA DR, MT, MB) but not to stimulate proliferation in primary or secondary MLR. Such cells did, however, stimulate the formation of potent suppressor cells. Three additional stimulator cell models for the presentation of either class I antigen only (purified platelets and purified T cells) or class I antigen plus nonimmunogenic class II antigen (D/DR-compatible cells) gave identical results. Supernatants from cultures stimulated with any of these cell types had significantly reduced IL 2 activity when compared to control MLR. The suppressor cells generated in such cultures were not restricted to the class I or class II MHC antigen of the original stimulator. These data are interpreted to mean that 1) the class II epitopes detected by alloantisera and the epitopes that serve as lymphocyte-activating determinants are metabolically or conformationally distinct, and 2) that presentation of class I MHC antigen alone or in conjunction with nonimmunogenic class II MHC antigen preferentially stimulates the formation of suppressor cells. It is hypothesized that the latter may be an additional mechanism that contributes to the efficacy of matching for class II determinants in human renal transplantation.

Differential reactivity of human lymphocytes allosensitized in vitro in hormonally defined medium or medium supplemented with plasma

We compared the in vitro allogeneic response of human lymphocytes cultured in hormonally defined medium (HDM) with that of those cultured in plasma-containing medium (PCM). Differences occurred only in secondary MLR or PLT and in cell-mediated cytotoxicity in terms of specificity, which was either greater or modified when HDM was used.

Primary and secondary MLR characterization of three allelic variants of HLA-Dw7 segregating in a single family

Three of four parental haplotypes of a kindred from the Old Order Amish religious isolate carried HLA-DR7 and specificities of the HLA-Dw7 “cluster”. Intrafamily primary and secondary mixed lymphocyte responses clearly distinguished the three Dw7-related allelic specificities. Two of the specificities fall within the Dw11 crossreacting group, designated here as Dw11 “short” (Dw11S) and Dw11 “long” (Dw11L), while the third is more closely related to Dw7. Reaction patterns in this family illustrate the complexity of antigen recognition in primary and secondary mixed lymphoyte responses and the important role played by the responder cell in generating discriminatory primed lymphocyte typing reagents.

Organisation génétique et polymorphismes de la région HLA-D

Summary The HLA-D region contains several genes encoding proteins which, after glycosylation, constitute at least two class II molecules (human Ia: Iah), each composed of two chains (α and β). The region comprises at least 4 closely linked genes: DRα, DRβ, DCα and DCβ, the order of which is unknown. The first molecule described is DR, which carries the DR1 to DRw10 allelic series. It is composed of an α chain—which is not (or is only slightly) polymorphic (MW⋍34,000) — and a highly polymorphic β chain (MW⋍29,000) carrying the allospecificity. The DR molecule is the human homologue of the mouse IaE molecule. The DC molecule carries the DC1, 3 and 4 specificities (also called MB1, 2 and 3). It is made up of an α chain (MW⋍32,000) and a β chain (MW⋍28,000). Both chains are biochemically polymorphic, β more so than α. It is not known which chain carries the allotypic specificities. The DC (MB) molecule is homologous to the IaA murine molecule. A third allelic series with 3 specificities MT1, 2 and 3 (MT2 and MT3 are also called BR3 and BR4) could be carried by a third Iah molecule not yet formally demonstrated. Another possibility is that the 3 allelic series (DR, DC and MT) could be carried by two Iah molecules. The SB locus is centromeric to HLA-DR, from which it is separated by recombination. Its product is an Iah molecule made up of an SBβ chain (demonstrated) associated with an SBα chain (postulated). Five SB allelic forms initially recognized by secondary MLR have been described. Some of these were also serologically detected. SB is similar in structure and function to DR and DC and thus has to be included in an enlarged HLA-D region. The HLA-D region initially defined by a cellular technique (primary MLR), was then analysed by biochemical and serological methods. The relationship between a postulated HLA-Dw product defined by cellular techniques and DR and DC products serologically detected, remains controversal. One interpretation would be that there is no HLA-Dw product per se, and that the HLA-Dw phenotype defined by primary MLR is the result of the allotypic make-up of the DR, DC and perhaps SB molecules carried by the cells present in the MLR. According to this hypothesis, HLA-Dw would be composed of multiple determinants, one of which (DR) could be immunodominant. The biochemical and allotypic analysis of the products of the HLA-D region are important in the understanding of their biological role: presentation of the exogenous antigen to the T cells, and thus regulation of the immune response. A more detailed analysis of the allogeneic response can now be made and applied to the definition of a better histocompatibility for kidney or bone marrow transplantation.

In vitro hematoporphyrin (HPD) inhibitory effects on some immunological assays

Hematoporphyrin derivative (Hpd) is a fluorescent dye that is preferentially incorporated by tissues with a high mitotic index, such as tumor cells and blast cells. A cytotoxic effect is produced following light activation. Previous studies have shown a long lasting reversible inhibition of DNA synthesis in Hpd-treated cells that failed to stimulate allogeneic lymphocytes in either primary or in secondary MLR. In this study we report Hpd inhibitory effects on some immunological assays in vitro. Treatment with Hpd of cytotoxic effector cells resulted in inhibition of their lytic activity likely dependent on the loss of binding to target cells. In the same way Hpd treatment inactivated the lytic activity of NK cells. In contrast Hpd-treatment of target cells did not modify the above immunological reactions. Moreover Con A agglutinability, antibody dependent capping as well as E-rosettes were inhibited following an Hpd treatment of relevant cells. Since normal susceptibility to humoral and cell mediated lysis was exhibited by Hpd-treated cells it is unlikely that cell surface molecules were damaged. An inhibitory effect exerted by an Hpd treatment on cell surface movements might explain these findings.

Activity of concanavalin A-induced suppressor cells in human MLR: differential effects on primary MLR, secondary MLR, and memory cell precursors.

The relative sensitivity of the MLR responses of freshly isolated human lymphocytes to Con A-induced suppressor cells (SC) was compared to that of lymphocytes that had been primed previously in vitro. Fresh and primed cells were suppressed 72 and 17%, respectively, when cultured under similar conditions (p less than 0.0005). Titration of SC indicated that equivalent suppression in the two populations would require greater than a 50-fold excess of SC in the primed cells. Neither preincubation of the primed cells for 24 or 72 hr with the SC before restimulation, addition of fresh autologous cells to the primed cells, nor preincubation of the SC with fresh cells before addition to the primed cells increased suppression of primed cells. SC added at the beginning of a primary MLC were, however, very suppressive for both the primary response (67% suppression) and the subsequent secondary response (81% suppression). These data indicate that although the human primary MLR and the precursor of the memory cell are both sensitive to the suppressors induced by Con A, the memory cell itself possess an intrinsic resistance to such suppressors that is not related to simple kinetic phenomena nor to the loss in vitro of an intermediate regulatory cell. Cell depletion experiments suggest that resistance to nonspecific suppression may occur at the level of the helper cell.

Ia antigens are the stimulating determinants in the syngeneic mixed lymphocyte reaction.

The murine syngeneic mixed lymphocyte reaction (sMLR) is a T cell proliferative response to self-antigens on non-T stimulator cells. The nature and specificity of the stimulating antigens was investigated using secondary MLR. Lymph node cells were stimulated in vitro by syngeneic spleen cells. After optimal proliferation occurred, the responding cell population was recovered and restimulated with spleen cells from mice of various haplotypes. The secondary response was found to be specific for the haplotype of the primary stimulator (a self-restricted response). The use of recombinant mice in the secondary stimulation localized the determinant to the I-A and/or I-E subregions of the major histocompatibility complex (MHC). This result was confirmed by the ability of monoclonal antibodies directed to the I-A or I-E subregion products to significantly inhibit the response while alloantiserum directed to products of the K or D regions had no effect.

Alloantigen-Induced Suppressor- and Memory Cells in Chronic Lymphocytic Leukemia

T cell function was evaluated in patients with B cell type of chronic lymphocytic leukemia (CLL). Unseparated peripheral blood lymphocytes (PBL) and T cells from CLL patients stimulated in a primary allogeneic MLR were able to inhibit significantly a second MLR between the original responder (CLL) and stimulator (normal PBL) cell donors. Furthermore, it is shown that T lymphocytes from patients with CLL develop immunologic memory during the course of a primary MLR as evidenced by an enhanced response in secondary MLR. These results are discussed with respect to recently described imbalances of T cell subpopulations in CLL.

Effect of cyclosporin A on human lymphocyte responses in vitro. III. CsA inhibits the production of T lymphocyte growth factors in secondary mixed lymphocyte responses but does not inhibit the response of primed lymphocytes to TCGF.

The mechanism whereby Cyclosporin A (CsA) inhibits secondary mixed lymphocyte responses was assessed. CsA added to secondary MLR cultures inhibited proliferation and induction of cytolytic lymphocyte activity. This inhibition was found to be associated with the inhibition of T lymphocyte stimulating growth factor(s) (TCGF) production in the supernatants of secondary MLR cultures. As little as 1.0 micrograms/ml of CsA added to secondary MLR cultures resulted in no measurable TCGF activity. In contrast, moderate doses of CsA (1.0, 2.5 micrograms/ml), which completely inhibited the secondary MLR response to alloantigen, did not inhibit the proliferative and CML response of alloantigen-primed lymphocytes to these stimulating growth factors. Even at high doses of CsA (20 micrograms/ml), substantial levels of proliferation (50% of control response) and CML induction (60% of control response) were observed when the primed cells were exposed to secondary MLR supernatants containing TCGF activity. It was concluded that inhibition of secondary mixed lymphocyte responses by CsA may be due in part to the inhibition of TCGF production rather than the inhibition of the effect of TCGF on mature cytotoxic T lymphocytes.

Perturbation of early development of the immune system by normal adult lymphoid cells.

Seven-day-old C57BL/10 mice injected with 3 X 10(6) normal adult lymph node cells (NAL) from syngeneic donors were compared with uninjected littermates when 5 to 7 wk old. The direct PFC response to sheep erythrocytes was suppressed by 60%. Responses to PHA and Con A but not LPS were suppressed by 60 to 90%. Primary and secondary (after skin graft rejection) MLR responses were suppressed by 30 to 40%. Active suppressor cells were demonstrable in these mice. Skin grafts incompatible for H-2 were rejected in normal time. NAL derived from adult mice conferred the effect, but not cells from 7-day-old donors. Mice inoculated with Rous sarcoma virus within the 1st day of life and 3 X 10(6) NAL at 7 days of age displayed up to 5-fold increased incidence of primary sarcomas.

Ia-positive stimulator cells are required in primary, but not in secondary, mixed leukocyte reactions against H-2K and H-2D differences.

The expression of I region gene products on stimulator cells in primary and secondary MLR directed against differences determined by the K or D regions of the H-2 gene complex was examined in B10-congenic mice. Treatment of the stimulator cells with anti-Ia antisera and complement (C) eliminates stimulation in primary MLR against K or D region gene products, indicating that the stimulator cells must express Ia antigens on their membranes. Further, stimulation in primary MLR against D region antigens is abolished by treatment of the stimulator cells with C and antisera against either I-A or I-E subregion products, but not by treatment of stimulator cells with C and antisera against I-J subregion products. Therefore, these stimulator cells express on their membranes both I-A and I-E subregion gene products, but not I-J subregion gene products. In contrast, in secondary MLR against K or D region gene products, treatment of stimulator cells with anti-Ia antisera and C does not abolish stimulation, indicating that Ia-negative cells can stimulate primed responder cells. These results indicate that the mechanisms of cell-cell recognition necessary in secondary MLR against K or D region gene products are different from those in primary MLR to the same products.