Lymphocyte-activating Determinants Research Articles

Recognition of major histocompatibility complex antigens on cultured human biliary epithelial cells by alloreactive lymphocytes

We have developed an in vitro system to study the interactions between biliary epithelium and lymphocytes using cultured human biliary epithelial cells. No class II antigens were detected by immunoperoxidase staining of the normal biliary epithelial cells, but alloactivated lymphocyte culture supernatants were able to induce class II expression. The activity of the supernatants was blocked with an anti-gamma-interferon monoclonal antibody. In addition, recombinant human gamma-interferon alone induced the expression of class II antigens and increased the intensity of class I staining of cultured biliary epithelial cells. Biliary epithelial cell-induced proliferation of alloreactive T lymphocytes demonstrated that the major histocompatibility complex molecules carry functional lymphocyte-activating determinants. The recognition of major histocompatibility complex determinants was confirmed by monoclonal antibody-blocking studies and by stimulation of an alloreactive T-cell clone. However, the biliary epithelial cells were much less potent stimulators than arterial endothelial cells tested in the same assay system.

Constitutive Functional Heterogeneity without Detectable Somatic Mutation of Antigen Receptor Genes in Helper T Cell Clones: Possible Regulation by Novel HLA Class II “DY” Determinants*

The majority of interleukin 2-dependent HLA class II alloreactive human CD4-positive helper T-cell clones (TCC) presented the phenomenon of constitutive intraclonal heterogeneity, as reflected by limiting dilution analysis of changing frequencies of autonomously proliferative cells within the monoclonal population. Moreover, at 30-35 population doublings, these TCC lost their allospecific proliferative and helper capacity (phase I) and instead acquired strong antigen-non-specific suppressive activity (phase II). The TCR was still expressed at the same level on the cell surface. As shown by stable and identical rearrangement profiles of their T-cell receptor beta- and gamma-chain genes in both phases I and II, this constitutive change of function was probably not due to somatic mutation of the genes coding for the antigen-specific receptor. Moreover, antigen specific reactivity was retained in phase II TCC by the criterion of specifically stimulated secretion of granulocyte/macrophage colony stimulating factor. The preprogrammed intra-clonal functional flexibility is explained here by invoking a novel regulatory mechanism mediated by a fourth group of HLA class II determinants. When TCC were used as stimulators with the appropriate primed responders, it could be demonstrated that phase I autonomously proliferative non-suppressive T-helper lines failed to express certain novel lymphocyte activating determinants (LADs), whereas after their acquisition of suppressive function (phase II) these LADs were present. These stimulatory moieties appeared to represent a novel class II-like structure as established by serology, immunochemistry and functional characterization employing monoclonal antibodies to block stimulation. These operationally designated "DY" determinants are primarily and thus far exclusively involved in the induction of antigen non-specific suppressor cells. It is proposed that the intra-clonal changes of T-cell function described here are mediated by a switch in the utilization of qualitatively distinct class II-like restriction elements. The functional status of the phase I TCC can be altered by a switching over to the engagement of "DY" determinants in phase II. Thus, an autostimulatory and self-maintaining suppressive network may operate in extended inductive phases of human HLA restricted T-cell responses.

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.

Lack of correlation between lymphocyte activating determinants and HLA-DR on acute leukaemias.

The expression of allogenic lymphocyte-activating determinants (LAD) on 25 acute leukaemias has been compared with the expression of cell-surface antigens identified by HLA-DR allo- and xeno-antisera. The close correlation between LAD and DR known to occur on normal lymphocytes was not found in leukaemias. Twenty-two LAD+ leukaemias included 2 DR- cases, whilst 2 LAD- leukaemias were DR+. With the exception of 3 leukaemias all were strongly beta 2 microglobulin+. No correlation was found between the % DR+ cells and the level of lymphocyte stimulation. Separation of leukaemia cells on Ficoll gradients into fractions containing different proportions of DR+ cells did not correlate with LAD expression. Furthermore, antisera to DR antigens only partially blocked leukaemic LAD. The results support the notion that LAD on acute leukaemias are not necessarily associated with or identical to HLA-DR antigens, and that the lymphocyte activating capacity of HLA-DR may be modulated.

Enhancement of the mixed lymphocyte reaction by in vivo treatment of stimulator spleen cells with anti-IgD antibody.

The injection of anti-IgD antibody into mice has been shown to increase the expression of Ia antigens on splenic B cells. These antigens are the most potent lymphocyte-activating determinants (LAD) that trigger proliferation in an H-2-defined mixed lymphocyte reaction (MLR) and may play a role in the recognition of minor lymphocyte-stimulating (MIs) determinants. Therefore, we wished to investigate a possible correlation between apparent quantitative alterations in B cell Ia expression after anti-IgD activation with changes in the functional capacity to present allogeneic major histocompatibility complex (MHC) and MIs antigens to responsive T cells. We observed that the capacity of splenocytes removed 24 hr after the in vivo injection of anti-IgD to stimulate T cell proliferation across an H-2 barrier was most frequently enhanced two- to fourfold when a suboptimal concentration of stimulator cells was used or an early time point in the MLR was examined. In contrast, the capacity of splenocytes to stimulate across an MIsa, d difference after exposure to heterologous or hybridoma anti-IgD antibody often was increased 10-fold or more. Optimal MLR stimulatory capacity was induced by injection of 100 to 200 micrograms of heterologous anti-IgD. Augmented MIs stimulatory capacity of spleen cells peaked 24 hr after such treatment and continued to decline from this value at day 3 and at day 7 after injection. In contrast, the H-2 stimulatory capacity increased 1 day after injection of anti-IgD and remained at that elevated level 3 and 7 days after injection. The spleen cells from B cell-defective (CBA/N x DBA/2)F1 male mice were unaffected in their capacity to stimulate across an MIsa barrier after in vivo anti-IgD treatment; however, spleen cells from phenotypically normal (DBA/2 x CBA/N)F1 male mice after exposure to anti-IgD did evidence a considerably enhanced ability to stimulate in an MIsa-defined MLR. Because anti-IgD antibodies presumably have their major initial effect on surface IgD-bearing B cells, these studies suggest that anti-immunoglobulin-activated B cells may have a role (direct or indirect via interaction with accessory cells) in the presentation of allogeneic MHC and MIs antigens to responsive T cells.

Detection of HLA-DR associated PLT determinants by alloreactive lymphocyte clones.

This study deals with alloreactive T-cell clones which recognize cellular determinants associated with HLA-DR antigens. Two clones, CB55 and DS56, exhibited a PLT specificity that was perfectly associated with DR5. On the other hand, clones CB7, DS1 and HS1 showed PLT reactivity with approximately one-half of the DR5 positive cells and none of the DR5 negative cells, whereas clone MD4 largely reacted with the other half of DR5 positive cells. Another MLR culture generated two alloreactive clones DS6 and DS9 with PLT specificity for DR2. However, these clones did not respond to DR2 cells, which were also positive for the DR2-associated HLA-B7 and B18 antigens. Monoclonal antibody (mAb) inhibition studies showed heterogenous patterns, whereby monomorphic non-DR mAbs inhibited the DR2-associated PLT clones while the DR5-associated PLT clones were inhibited by different groups of anti-DR and non-DR mAbs. These observations suggest the existence of several lymphocyte-activating determinants associated with HLA-DR antigens. This diversity may be an important consideration in studies of the role of HLA-DR in immune mechanisms and transplant compatibility.

Activation of human alloreactive cytotoxic precursor T lymphocytes

The requirements for allogeneic T-cell activation have been studied in experiments with T and/or B cells as stimulator. Although target determinants (TDs, defined by CTL effectors in CML) are present on B and T cells used as target cells, this study indicates that TDs are functionally different when expressed on B and T cells used as stimulator cells, as only B cells can activate CTL precursors. Further, the study confirms that inducing TDs and strong lymphocyte-activating determinants (LADs, defined by proliferation in MLC) can be distinct structures found on two different stimulator B cells. The study suggests that binding of cytotoxic precursor T cells to TDs per se does not allow any detectable activation or start of proliferation and differentiation but requires another function of the stimulator cells in the non-T-cell compartment. The nature of this function is unknown, but it is the background for the first signal received by the TD-specific clones of CTL precursors, resulting in the expression of growth receptors for T-cell growth factor or interleukin 2 which is the second signal necessary for clonal expansion and differentiation.

Immunogenetic analysis of the HLA-D region: Serological and cellular detection of the MB system

The HLA-D region contains three serologically defined systems: DR, MB, and MT. The antigens of MB and MT are strongly associated with groups of DR antigens. This study dealt with the detection of MB by lymphocytotoxicity testing using B cell alloantisera obtained from multiparous women. Selective absorptions of anti-MB and anti-DR sera have demonstrated that MB antigens are distinct and that DR antigens show no crossreactivity. Primed lymphocytes (PLT cells) were generated from mixed leukocyte cultures whereby the stimulator had one incompatible DR-MB combination. Frequently such PLT cells showed specificity not only for DR but also for MB antigens. The latter could be blocked by preincubation of the secondary stimulator with anti-MB serum. Also, it was possible to generate alloreactive lymphocyte clones with specificity for MB. These data suggest that MB antigens may function as lymphocyte-activating determinants. The immunogenetic relationship between DR, MB, and MT suggests a multilocus control of HLA-D region products involved with lymphocyte activation.

Typing for Human Alloantigens with the Primed Lymphocyte Typing Technique

Publisher Summary In man, the Major Histocompatibility Complex (MHC) is called “the human leukocyte A (HLA) system, whereas, for example, it is H-2 in mice, Antigen B (AgB) in rats, and B in chickens. The MHCs control strong transplantation antigens, play a fundamental role in the cooperation of cells in thymus-dependent immune responses, contain immune response (Zr) genes and some complement coding genes, and control susceptibility and resistance to a diversity of diseases. The HLA-D antigens are identified by the so-called mixed lymphocyte culture (MLC) reaction (MLR) in which lymphocytes from two different individuals are mixed and cultured in vitro . This reaction is based on the facts that lymphocytes carry both HLA-D antigens and receptors for foreign HLA-D antigens not possessed by the lymphocyte donor, and lymphocytes with such receptors under appropriate conditions will respond in vitro by proliferation, which can be measured by the uptake of certain substances. This chapter mainly summarizes the works dealing with the identification of human lymphocyte activating determinants (LADs) recognized by secondary MLR with special reference to the genetics of the PLT in man.

Association of PLT specificity of alloreactive lymphocyte clones with HLA-DR, MB and MT determinants.

The HLA-D region encodes for several serologically defined systems, including DR, MB, and MT. The antigens of MB and MT are strongly associated with two or more DR specificities. The purpose of this study was to determine the role of MB and MT antigens in lymphocyte alloactivation. A soft agar colony assay was used to generate alloreactive lymphocyte clones primed in mixed leukocyte culture against a stimulator who typed as HLA-DR4,-;MB3,-;MT3,-. In secondary primed lymphocyte typing (PLT) assays, several clones were identified with PLT specificities strongly associated with DR4, MB3, or MT3. The data suggest that HLA-D controls different lymphocyte-activating determinants associated with the serologically defined DR, MB, or MT antigens.

Inhibition of secondary mouse mixed lymphocyte reaction by anti-Ia sera.

Secondary mixed lymphocyte reaction (MLR-II) was studied in A.TH anti A.TL and A.TL anti-A.TH combinations in which stimulation was mainly due to H-21-region differences. In both cases of MLR-II was specifically inhibited by the responder anti-stimulator Ia serum. The level of inhibition was dependent on the ratio of the amount of immune serum to the number of stimulating cells. The inhibitory activity and Ia antibodies were specifically absorbed and eluted together. The results confirm that the lymphocyte-activating determinants of the MLR-II (1) are carried by the Ia molecules and (2) are identical to the serologically defined Ia determinants. - Anti-Ia sera directed against private and public specificities of the stimulating cell induced a higher level of inhibition than anti-Ia sera directed only against public specificities, indicating that both private and public Ia specificities are involved in re-stimulation during MLR-II. - These results, in connection with others, suggest that the receptor of the proliferating T cell recognizes the same Ia determinant as the combining site of the Ia-recognizing antibody.

Lymphocyte subpopulations in man: induction of cytotoxic T lymphocytes in vitro by allogeneic B and T cells.

Testing B and T cells as allogeneic stimulators of cytotoxic T lymphocytes in primary as well as secondary in vitro cultures, reveals that fresh, nonactivated B cells isolated from peripheral blood have an enhanced cytotoxic T cell stimulating capacity compared to T cells, although target determinants are present both on B and T cell blasts. Similarly, the capacity of T and B cells to stimulate proliferation in MLC is also quantitatively different. These results are in accordance with the hypothesis concerning the in vitro generation of alloreactive cytotoxic T lymphocytes, which postulates concurrent stimulation by strong lymphocyte activating determinants and target determinants for the generation of cytotoxic effector T lymphocytes, as both determinants are simultaneously found on B lymphocytes. Three cell experiments performed by coculturing allogeneic stimulating B and T cells with responding T cells show that strong lymphocyte activating determinants found on B cells enhance the cytotoxicity against target determinants on cocultured B cells but not on cocultured T cells, indicating qualitative differences between target determinants on B and T cells with respect to specific CTL stimulating capacity. Furthermore, primed resting CTLs in secondary cultures could unspecifically be restimulated by third party B cells or pokeweed mitogen. These results are the basis for a hypothesis concerning activation of CTLs, postulating nonspecific triggering of cytotoxic precursor cells by lymphocyte activating properties intrinsic to target determinants (TD) on B cells, preferentially activating clones of cytotoxic cells. The clonal proliferation is further unspecifically amplified by products of the T cell recognition of strong lymphocyte activating determinants (LAD).

Active enhancement in the primate by bone marrow I. Ia-like determinants on nucleated bone marrow cells.

This study investigates the possibility that the active enhancing properties of bone marrow (BM) cells may be related to Ia-like determinants on their surface membranes. Less chromium was released on a cell per cell basis from nucleated baboon BM cells used as targets in complement-dependent cytotoxicity than from labelled lymph node lymphocytes. On the other hand, BM cells stimulated allogeneic lymphocytes in culture more vigorously than lymph node lymphocytes. BM cells with lymphocyte-activating properties could be enriched by fractionation on a discontinous bovine serum albumin gradietnt. BM cells responded poorly to allogeneic cell stimulation in mixed culture. Platelet absorption studies of an alloantiserum supported the conclusion that nucleated BM cells, and particularly BM fractions collected from the less dense interfaces of a bovine serum albumin gradient, express relatively more lymphocyte-activating or Ia-like determinants than "serologically defined" determinants.

Cellular and genetic restrictions in the immunoregulatory activity of alpha-fetoprotein. II. Alpha-fetoprotein-induced suppression of cytotoxic T lymphocyte development.

Alpha-fetoprotein (AFP), a major component of fetal and newborn sera, was shown to exert significant immunosuppressive activity on the in vitro generation of cytotoxic T lymphocytes (CTLs). This suppression proved independent of the suppressibility of the mixed leukocyte culture activation phase, since strain combinations whose proliferative responses were refractive to AFP-induced suppression also failed to develop demonstrable CTLs in the presence of AFP. Several strain combinations were also found in which normal generation of CTLs occurred in cultures containing AFP. This refractive nature correlated with the presence of nonsuppressible lymphocyte-stimulating alloantigenic systems on the stimulating cell population. These data provide the basis for proposing several possible mechanisms for AFP-induced suppression of T-cell-mediated cytotoxicity, as well as suggesting that the primary target of this suppression is the proliferating helper T cell precommited to respond towards the major histocompatibility complex-associated lymphocyte-activating determinants.

Murine B Lymphocyte Heterogeneity: Distribution of Complement Receptor-Bearing and Minor Lymphocyte-Stimulating B Lymphocytes among Cells with Different Densities of Total Surface Ig and IgM

The distribution of complement receptor-bearing (CR+) and minor lymphocyte-stimulating (Mls)-defined lymphocyte-activating determinants (LAD) among B lymphocytes with different densities of total surface immunoglobulin (sIg) and sIgM was determined. B lymphocytes that bore intermediate densities of sIg and low densities of sIgM had the highest frequency of CR+ cells and were the most active in expressing Mls-defined LAD. The distribution of these and other surface markers on B-lymphocyte subpopulations is discussed.

Continuously proliferating allospecific T cells, lifespan and antigen receptors.

AbstractAllospecific mouse T cells derived from one‐way mixed lymphocyte cultures after 19 months in tissue culture still require allogeneic stimulation for their proliferation. If injected into normal or immunodeficient mice these cells do not form tumors. After proliferation over long time periods or after extensive expansion these T cells tend to lose their cytotoxic activity which cannot be reconstituted by addition of cells from active cultures of a different specificity. Accordingly, the specificity of the cytotoxic and proliferative response was investigated. The cytotoxic activity appears to be specific for the H‐2 SD antigens, while the proliferative response and also the activation of cytotoxicity in resting cultures is only triggered by lymphocyte activating determinants (LAD) coded either inside or outside the H‐2 region.The receptors for LAD on these allospecific T cells appear to be clonally distributed. This finding comes from the observation that old and therefore highly selected lines only react to some LAD while younger ones react to many others as well. A search for immunoglobulin on these functional and antigen‐specific thymus‐derived lymphocytes (T cells) was performed using surface radioiodination, immunoprecipitation and polyacrylamide gel electrophoresis combined with autoradiography. Using various antisera no or very little immunoglobulin molecules were found on these T cells. The small amount of immunoglobulin was attributed to a contamination of allogeneic spleen cells used to stimulate the T killer cells.

Studies on non-H-2 linked lymphocyte activating determinants. I. Description of the cell type bearing the Mls product.

The non-H-2 linked, lymphocyte activating determinants (LADs), which are coded by the minor (histocompatibility) lymphocyte stimulating locus (Mls), were detected on B lymphocytes, but not on immunoglobuin (Ig) negative or on theta positive T lymphocytes. These determinants, like the complement receptor and surface IgD, develop late in neonatal life. Thus, the Mls coded LADs mark a population of B lymphocytes which are not present in the spleens of newborn to 2-week-old mice. Studies of B lymphocytes derived from the spleens of adult mice indicated that the cells bear the Mls coded LADs have a high density of surface Ig and possess the complement receptor.

Studies on non-H-2-linked lymphocyte-activating determinants: IV. Ontogeny of the MlS product on murine B Cells

The minor lymphocyte stimulating (Mls) locus codes for lymphocyte activating determinants (LADs) on murine B lymphocytes, but not T lymphocytes. This observation was strengthened by a series of techniques which allow deletion and addition of T and B cells. These included the use of cytotoxic antisera such as anti-Thy 1.2, anti-MTLA, anti-MBLA, and complement, and the use of a goat anti-μ antisera, and finally the use of a fluorescence activated cell sorter (FACS). The studies in this report document the organ distribution and the ontogenetic appearance of the surface LADs on the surface of B lymphocytes from DBA/2N (H-2 d, Mls a) and CBA/J (H-2 k, Mls d) mice. Adult-like ability to stimulate H-2 identical BALB/c (H-2 d, Mls b) and C3H/He (H-2 k, Mls c) responder cells appeared at about 4–5 weeks of age. Inability of neonatal cells to induce an Mls-defined MLC was found not to be due to a low frequency of B lymphocytes or to the presence of suppressor cells, but due to the absence of the Mls-coded LADs on their surface. These data support the concept that the Mls-coded LADs are present on adult B lymphocytes and are specific markers of B-cell differentiation, which is preceded by membrane IgM and the δ homologue of human IgD, Ia, and the receptor for the third component of complement.

Generation of suppressor cells in mice immunized with M locus-incompatible lymphocytes.

Alloimmunization of mice with M locus-incompatible lymphocytes resulted in the generation of suppressor cells in the immunized host. Lymph node cells from such alloimmunized mice suppressed the in vitro cytotoxic response of normal cells to H-2 alloantigens. The suppression generated was greater than could be accounted for by dilution of the prekiller cell population with cells possibly devoid of cytotoxic potential from M locus preimmunized mice. Using M locus pseudocongenic mice, the suppressive effect was shown to be largely attributable to M locus determinants; restimulation of suppressor cells in culture with the specific M locus was required for suppression of effector cell generation. The in vivo effect of suppressor cells was tested in a graft-versus-host reaction; injection of M locus preimmunized cells into footpads of F1 hybrid mice suppressed the popliteal lymph node enlargement compared with lymph node size after injection of control preimmunized cells. Although the suppressive effect is mainly attributable to M locus determinants, incompatibility for the DBA/2 antigen may add to the suppression. The study of inhibitory effects on T cell cytotoxicity because of serologically undetectable lymphocyte-activating determinants (Mls) could lead to the better understanding of suppressive mechanisms which may allow the growth of syngeneic tumours.

Studies on Non-H-2-Linked Lymphocyte-Activating Determinants

Abstract Minor lymphocyte-stimulating (Mls) coded non-H-2-linked lymphocyte-activating determinants (LADs) are present on a late developing subpopulation of murine B lymphocytes. Spleen cells derived from CBA/N mice, a strain with an X-linked defect in B cell function, failed to stimulate a response in Mls-defined mixed lymphocyte reactions (MLRs) with H-2 identical CBA/J or C3H/N responder spleen cells. However, both CBA/J and C3H/N spleen cells induced significant responses in CBA/N responder spleen cells. The inability of CBA/N cells to induce an Mls-defined response was not due to the presence of the nonstimulatory Mlsb genotype, since spleen cells of immunologically normal F1 mice derived from crosses of CBA/N and C3H/N mice were stimulatory in MLRs for C3H/N responder cells. Immunologically abnormal CBA/N ♀ × C3H/N ♂) F1 male mice, which are hemizygous for the CBA/N X chromosome, were also unable to induce a response in Mls-defined MLRs. Although the Mls-coded LADs are not X linked in immunologically normal mouse strains, these data indicate that the failure of CBA/N mice to functionally express the Mls-coded LADs is X linked. This characteristic was not secondary to the presence of suppressor cells and is most likely related to the absence of a late developing (mature) subpopulation of B lymphocytes in these mice.