Antigen-specific T lymphocytes efficiently cluster with dendritic cells in the human primary mixed-leukocyte reaction

Experimental conditions have been developed to detect the efficient interaction of antigen-presenting cells and antigen-specific CD4+ T lymphocytes early in the human primary mixed-leukocyte reaction (MLR). When monocytes are depleted from the stimulator population, it is evident that small numbers of allogeneic dendritic cells form multicellular aggregates with responsive T cells. B cells and monocytes in allogeneic stimulator populations do not appear to form aggregates in the first 2 days of the MLR. Upon return to culture, most of the lymphocytes that have clustered with dendritic cells become IL-2 responsive, proliferating lymphoblasts. The nonclustered cells exhibit little growth, while mixtures of clusters and nonclusters proliferate comparably to clusters alone. Cluster-derived lymphocytes respond rapidly to rechallenge with foreign leukocytes from the original donor but are greater than 90% depleted of reactivity to other "third party" donors. Nonclustered lymphocytes, in contrast, are greater than 90% depleted in specific reactivity but respond normally to third party. Therefore antigen-specific (alloreactive) resting CD4+ lymphocytes efficiently and selectively aggregate with dendritic cells. Dendritic-T-cell aggregates represent a stable microenvironment in which the MLR begins and might be useful in the experimental analysis of early events in the sensitization phase of cell-mediated immunity in man.     

Effect of anti-beta2-microglobulin on antigen and allogeneic lymphocyte-induced proliferation of human lymphocytes.

The effect of anti-beta2-microglobulin (beta2m) on the mixed lymphocyte reaction (MLR), and an antigen-induced proliferative response was studied. Anti-beta2m IgG and Fab' fragments completely inhibited the MLR. Preincubation of stimulator or responder cells with anti-beta2m suggested that the major effect of anti-beta2m may be on the responder cell population. A clear-cut effect on responder cells was demonstrated by showing that anti-beta2m completely inhibited a MLR in which the stimulator population was a beta2m negative lymphoblastoid cell line. Anti-beta2m also inhibited PPD-induced proliferation of sensitized lymphocytes. The kinetics of this inhibition indicated that anti-beta2m added within the first 18 hr of stimulation was effective in inhibiting the proliferative response. These data are discussed in light of the hypothesis that beta2m may be a subunit of an antigen receptor on T cells.     

Functional differences between cells from MLR colonies grown in soft agar cultures

This report describes a method of growing soft agar colonies of human T lymphocytes activated in the MLR. Two types of colonies were demonstrated: lower colonies grew within the agar layer, and upper colonies grew on the surface of the agar layer. Three days of priming the lymphocytes in the MLR and the use of supernatants of day-3 MLR cultures to provide T cell colony growth factor were necessary for optimal colony formation. Lymphocytes obtained from colonies were grown in long-term (2 to 4 weeks) cultures to generate sufficient numbers of cells to be tested in different functional assays. Cells from both types of colonies exhibited PLT activity. Upper colony cells showed considerably higher CML activity than lower colony cells (mean percent cytotoxicity 37 +/- 5 vs 6 +/- 3). Cells from both types of colonies contained radiosensitive suppressor cell activity that inhibited the primary MLR. The suppressor cell effect of lower colony cells was specific for the original stimulator, but upper colony cells displayed nonspecific suppressive effects. For both types of colony cells, it appeared that suppressive effects were unrelated to the CML activity of these cells. These data suggest that the soft agar colony assay offers a promising approach to separate subpopulations of lymphocytes activated in the MLR.     

Induction of allograft tolerance after total lymphoid irradiation (TLI): development of suppressor cells of the mixed leukocyte reaction (MLR).

We searched for the presence of suppressor cells of the MLR in C57BL/Ka leads to BALB/c chimeras. The chimeras were made with total lymphoid irradiation (TLI) and marrow transplantation. Spleen cells from the old chimeras inhibited the MLR of BALB/c responder cells against C57BL/Ka stimulator cells. Inhibition was specific for the stimulator cells, since no effect on the MLR was observed with C3H or BALB.C3H stimulator cells. Maximal inhibition was achieved when the responder cells in the MLR shared the H-2 haplotype of the chimeric recipient. Spleen cells obtained from chimeras young 30 to 40 days after BM transplantation inhibited the MLR nonspecifically, since similar marked inhibition was observed regardless of the H-2 haplotype of the responder or stimulator cells. The finding of antigen-specific and nonspecific suppressor cells is similar to that observed in mice rendered tolerant to bovine serum albumin after treatment with TLI.     

Mixed lymphocyte reaction in mice genetically selected for high (Hi/PHA) or low (Lo/PHA) responsiveness to phytohemagglutinin.

Lymph node cells from Hi/PHA and Lo/PHA mice were evaluated for proliferative response after stimulation by allogeneic lymphocytes (MLR) originating from four inbred strains of different H-2 haplotype (C57B1/6, DBA/2, CBA, A). Reactivity to MLR and PHA were compared in these two lines and in the four inbred strains. The high and low responder status of Hi/PHA and Lo/PHA, as determined by T mitogens lymphocyte responsiveness, was also observed when one measured T responsiveness after MLR. Values obtained with the four inbred strains are included in the range of those measured in Hi/PHA and Lo/PHA cells when stimulated by PHA as well as by allogeneic cells. In contrast, when used as stimulator cells, Hi/PHA or Lo/PHA lymphocytes induce an equivalent proliferative response versus every responder inbred strain studied. These experiments support the hypothesis of a common genetic control of proliferative response following PHA or MLR stimulation. The genes implicated would be different from those coding for I region associated antigens.     

Immunologic effects of whole-body ultraviolet (uv) irradiation. III Defective splenic adherent cell function in alloantigen-stimulated T-cell proliferation

The daily exposure of a mouse to ultraviolet (uv) radiation causes a selective depletion of Ia-bearing adherent cells in that animal's spleen. This depletion manifests itself in functional deficiencies in the presentation of protein antigens and haptens to T cells. The present studies demonstrate a defect in splenic adherent cells (SAC) from uv-irradiated mice resulting in defective alloantigen presentation. We show that unfractionated splenocytes and SAC from uv-irradiated mice show decreased stimulatory activity in allogeneic MLR. We then utilize this phenomenon induced by uv radiation to characterize the stimulator cell in the M locus (Mls) determinant-driven MLR. We show that the stimulator cell in Mls determinant-driven MLR is an adherent cell and demonstrate that this stimulator cell bears Ia determinants by showing that whole spleen cells and SAC from mice treated with uv radiation are inefficient stimulators of the Mls determinant-driven MLR. The importance of the Ia determinant on the stimulator cells in Mls determinant-driven MLR is corroborated by the demonstration that a monoclonal antibody directed at this determinant fully blocks the Mls determinant-driven MLR. The significance of these studies to the problem of alloreactions in vivo is discussed.     

Lack of generation of killer cells in the mixed lymphocyte reaction between mitogen-stimulated and unstimulated autologous lymphocytes.

The present study has demonstrated that the Con A-activated cell-mediated autologous mixed lymphocyte reaction (MLR) is not associated with the generation of cytotoxic effector cells that kill autologous targets. Thus, the suppression of antibody production of PWM stimulated lymphocytes by autologous Con A-activated suppressor cells cannot be explained by detectable cytotoxicity. We have further demonstrated that the stimulator cell in this system is a nonadherent non-T cell.     

Requirement for the location of both appropriate and irrelevant H-2 antigens on the same stimulator cell for unspecific DNA-synthesis inhibition by the H-2-antigen-primed,specific suppressor T cells

Specific suppressor T cells (SSTC), primed in vivo with H-2 antigens, have been shown previously to inhibit DNA synthesis in the one-way, three-cell mixed lymphocyte reaction (MLR) provided that (a) the stimulator cells bear the priming H-2 antigens, and (b) the responder cells possessIC+S regions homologous to those of the SSTC. Anti-B10.A BlO.A(2R) SSTC (anti-D^d) and anti-A.AL A.TL SSTC (anti-K^k) are shown here to be able to inhibit the DNA synthesis triggered in MLR, not only by the corresponding antigens, D^d and K^k, respectively, but also by irrelevant, third-party H-2 and Mls products provided that the corresponding and third-party antigens are presented on the same stimulator cell. If stimulatorH-2 regions, whose products interact with SSTC and responders, are located on different stimulator cells within the particular MLR, SSTC activity is not elicited. Participation of cytotoxic T lymphocytes in DNA-synthesis suppression is ruled out. Direct contact or location of the inhibited responder cell very close to SSTC is considered to be required for the development of SSTC activity.     

Immunosuppressive activity of murine newborn spleen cells. I. Selective inhibition of in vitro lymphocyte activation.

Cell-mediated immune responses to newborn lymphocyte alloantigens were investiated using mitogen activation, mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML). Spleen cells from 1- to 5-day-old (C57BL/6 × Balb/c) F₁ mice co-cultured with maternal strain (BALB/c) splenocytes did not affect DNA synthesis of maternal strain cells in the presence of concanavalin A or phytohemagglutinin. Newborn cells did inhibit the lipopolysaccharide response of maternal strain lymphocytes and these cells also depressed DNA synthesis when added to MLR cultures of BALB/c and C57BL/6 spleen cells. Newborn cells expressed poor stimulatory capacity in semiallogeneic MLR and also caused marked inhibition of DNA synthesis when added to semiallogeneic MLR containing BALB/c (responder) and CB6F₁ adult splenocytes (stimulator). The suppression of MLR by neonatal cells persisted for the first 2 weeks of life and was associated with a soluble factor released during culture. The suppressive activity was almost completely abrogated after depleting the T-cells from newborn splenocytes. However, these same cells did not interfere with the in vitro generation of cytotoxic lymphocytes in the CML assay. The selective immunosuppressive properties of newborn spleen cells may be important during pregancy by protecting the immunologically alien fetus from rejection by the mother.     

Mouse T lymphocytes proliferative responses specific for human MHC products in mouse anti-human xenogeneic MLR

It has been reported that human T cells recognize the polymorphism of murine Ia antigens in the human anti-mouse xenogeneic mixed lymphocyte reactions (MLR). In this study, murine T cell recognition of human Class II antigens of the major histocompatibility complex (MHC) was analyzed in mouse anti-human xenogeneic MLR responses. The xenoreactive murine T cell proliferative response was blocked by adding anti-HLA-DR monoclonal antibody to the xenogeneic MLR culture. The specificity of xenoreactive murine T cells was examined with regard to the secondary and tertiary xenogeneic MLR system. The xenoreactive murine T cells were restimulated by distinct human stimulator cells that had no shared HLA antigens with the stimulator used in the primary MLR. The data presented here show that the murine xenoreactive T cells recognize the shared determinant(s) of HLA-DR antigen on non-T, non-B stimulator cells. The xenoreactive murine T cell proliferative responses were mediated by Thy-1+, Lyt-1+, and Lyt-2- cells. Furthermore, the xenoreactive T cell responses required Ia+ cells, and Ia antigen on accessory cells plays a crucial role in eliciting the xenoreactive responses against human stimulator cells, while Ia+ accessory cells in the responding cell population are not essential for the elicitation of allogeneic MLR responses, as reported previously.     

Suppression of lymphocyte proliferation by a nonspecific factor produced by the Burkitt lymphoma-derived lymphoblast cell line.

The DAUDI lymphoblast cell line derived from a patient with Burkitt lymphoma was obtained from two different sources. One of these (DAUDI-I) produced a factor that inhibited lymphocyte proliferation in both human and mouse regardless of the stimulator, i.e. allogeneic lymphocytes or mitogens. Glutaraldehyde treatment eliminated production of the factor and demonstrated that DAUDI-I was capable of stimulating normal lymphocytes in MLR. A second DAUDI cell line (DAUDI-S) did not produce the inhibitory factor and was capable of MLR stimulation.     

Characterization of a serum inhibitor of MLC reactions. III. Specificity: HL-A-related antibodies.

A mixed leukocyte culture (MLC)-inhibitory serum from a healthy multipara, JH, has been characterized with regard to the specificity of its inhibitory antibody. When added directly to MLC, JH serum will inhibit most combinations. However, when lymphocytes intended as responder or stimulator cells in MLC were preincubated with this serum, the specificity narrowed considerably. Four groups of lymphocyte donors were recognized, depending upon whether their lymphocytes were inhibited as responders, as stimulators, as both, or as neither. Absorptions of inhibitory activity, followed by assay of the absorbed sera in pretested MLC combinations, yielded reliable data for determining which donors' cells shared pertinent antigens. An association of MLC inhibition by JH serum with the HL-A types of the involved lymphocytes was observed and these relationships are summarized in Table 4. The three HL-A specificities identified, W19, W29, and 12, correspond with the HL-A typing of the husband of the serum donor. Various cell types absorbed relevant inhibitory activity (against responder and stimulator functions) in the following order of efficiency: LCL cells, B lymphocytes, T lymphocytes, fibroblasts, and erythrocytes. When the above three HL-A specificities were removed by absorption, the serum was no longer inhibitory in any combinations. Whether the inhibitory activity of JH serum is directly related to anti-HL-A antibodies or to antibodies against closely related MLR determinants will depend to a large extent upon the degree of linkage disequilibria found between W19, W29, and 12 antigens and the MLR locus.     

Induction of allospecific suppressor T cells in vitro.

Incubation of mouse thymic lymphocytes with irradiated allogeneic spleen cells gave rise to suppressor cells. The suppressor activity was assayed by adding the incubated cell mixture to a mixed lymphocyte culture (MLC) in which the responder cells were syngeneic with the sensitized thymocytes and the stimulator cells were syngeneic with the sensitizing spleen cells. Such addition suppressed significantly thymidine incorporation in the mixed lymphocyte reaction (MLR). The suppressor cells were found to carry the θ antigen and to function allospecifically, as shown by cross-testing in three allogeneic combinations. Our data suggest that these cells may originate from immature cortisone-sensitive thymic lymphocytes and also provide some preliminary information concerning their mode of action.     

Analysis of responder cell-cell interactions in the syngeneic mixed leukocyte response

In a previous study we identified the target antigens on stimulator cells in the murine syngeneic mixed leukocyte response (MLR) as self Ia molecules. The experiments reported here utilized analysis of log-log cell number-response titration slopes to study the responder T-cell population in the syngeneic MLR. The data indicated that in the peripheral lymphoid population at least two cell populations interact to produce a murine syngeneic MLR. One of these cell types appeared to be a member of an Lyt 2- subset, another a member of an Lyt 2+ subset. A potential third cell type, which was detected in the presence of (PMA)-induced EL4 supernatants, was found in the thymus, an organ that did not appear to contain either of the other two subsets. Experiments with adult thymectomized and neonatal mice suggested that the cell populations involved in the syngeneic MLR were mature T cells and were not an early differentiation state of T lymphocytes whose ability to be stimulated by Ia antigens alone was a transient event.     

Trophoblast regulation of maternal-paternal lymphocyte interactions

The modification of immunological responses by murine embryonic trophoblast cells was investigated using the mixed-lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) test. In MLR containing Balb/c (responder) and C57BL/6 (stimulator) splenocytes DNA synthesis was markedly reduced in the presence of ectoplacental cone or placental trophoblast cells. These same trophoblast cell populations inhibited the in vitro generation of cytotoxic lymphocytes while cultures containing nontrophoblast regulator cells expressed normal cytotoxicity. DNA synthesis in MLR and cytotoxic activity in CML were not suppressed in cultures containing $\text{3}\text{1}\text{2}$ day blastocyst outgrowths. The potent immunosuppressive properties of the trophoblast may be important during pregnancy by protecting the genetically dissimilar fetus from potentially harmful maternal immune effector mechanisms.     

Recognition and response to alloantigens in vivo. I. Negative and positive selection of MLR reactivity in murine peripheral blood lymphocytes to major histocompatibility complex and Mls antigens

Specific mixed lymphocyte reaction (MLR) responsiveness to allogeneic major histocompatibility complex (MHC), or minor lymphocyte-stimulating (Mls) determinants, was depleted in the peripheral blood lymphocytes (PBL) obtained from mice 24 to 48 hr after i.v. injection of 5 to 7.5 X 10(7) MHC or Mlsa-incompatible spleen cells, respectively. Results of cell mixture experiments suggest that the generation of suppressor cells was not the explanation for this specific reduction in MLR proliferation occurring with these PBL responder cells. To gain additional insight into parameters involved in the recognition of allodeterminants in vivo, experimental manipulations of the host environment and donor cell inoculum utilized in the negative selection procedure were employed. For example, removal of the spleen in the recipient animal, an anatomic site in which injected allogeneic cells and corresponding host antigen-reactive cells (ARC) are trapped, still permitted the specific depletion in murine PBL of host ARC for donor foreign MHC antigens. This finding may implicate other sites such as the liver where unprimed host alloreactive clones are trapped. In addition, irradiation of allogeneic donor cells significantly reduced their capacity to trap alloreactive T cell clones in vivo, whereas heat treatment of the donor cells completely eliminated this ability, even though the Ia determinants were still expressed, measured by flow cytometry. After the negative selection period, kinetic analysis of proliferation showed that 3, 4, or 5 days after injection of MHC-incompatible allogeneic spleen cells, the PBL of the recipient showed specific hyperresponsiveness to the MHC-haplotype of the donor cells. Interestingly, these primed PBL responder cells had the volume distribution of small resting cells; thoracic duct lymphocytes (TDL), positively selected by adoptive transfer of T cells to irradiated semiallogeneic recipients, are reported to be mainly blast cells. In contrast to the MLR hyperresponsiveness that results from priming with MHC-incompatible splenocytes, PBL, obtained at these later time points from mice primed with Mlsa-incompatible, H-2-compatible splenocytes, showed complete unresponsiveness in MLR to these Mlsa-bearing stimulator cells, as well as some nonspecific reduction in proliferation to MHC-incompatible stimulator cells regardless of their Mls genotype.(ABSTRACT TRUNCATED AT 400 WORDS)     

Suppression of lymphocyte proliferation by a nonspecific factor produced by the burkitt lymphoma-derived lymphoblast cell line

Summary The DAUDI lymphoblast cell line derived from a patient with Burkitt lymphoma was obtained from two different sources. One of these (DAUDI-I) produced a factor that inhibited lymphocyte proliferation in both human and mouse regardless of the stimulator, i.e. allogeneic lymphocytes or mitogens. Glutaraldehyde treatment eliminated production of the factor and demonstrated that DAUDI-I was capable of stimulating normal lymphocytes in MLR. A second DAUDI cell line (DAUDI-S) did not produce the inhibitory factor and was capable of MLR stimulation. Supported by the Children's Leukemia Foundation of Michigan, NIH Grants AI 11013 and AI 11335, and the Kidney Foundation of Michigan.     

Generation of natural killer-like activity in mixed lymphocyte-tumor cell cultures. II. Correlation between expression of HLA-DR antigens on the activated T cells and their cytotoxic capability

In this study, we investigated the role of DR antigens in human mixed lymphocyte reactions (MLR) at the responder cell level. Upon stimulation by allogeneic lymphocytes or leukemic cell lines, a large proportion of T cells underwent blastogenesis and began expressing DR antigens. Analysis by a fluorescence-activated cell sorter revealed that both subpopulations of large activated T cell blasts and of small T lymphocytes became DR+ by synthesis and/or uptake. Depletion of DR+ responder cells from 6-day-old MLRs by treatment with anti-DR monoclonal antibodies (mAbs) plus complement (C) reduced but did not completely abrogate the natural killer (NK)-like activity of the responder lymphocytes, suggesting that the MLR-induced cytotoxic cells include both DR+ and Dr- populations. The expression of NK-like activity by the responder cells was also greatly reduced upon addition of anti-DR mAbs (without C) at the start of the mixed cultures. This effect was observed regardless of the presence of DR antigens on the stimulator cells, indicating that the anti-DR mAbs can interact with the antigens present on both the stimulator and responder populations. These data show that during an MLR, the continued presence of DR antigens on the responding population is essential for the expression and maintenance of the proliferative and cytotoxic capabilities of these cells.     

In vitro alloreactivity of mouse bone marrow lymphocytes

Before characterizing alloreactive cells of the bone marrow, it was necessary to reevaluate the alloantigen response in this tissue. The results of previous studies using the parental-F₁ system in the mixed-lymphocyte reaction (MLR) are open to question because of the recently documented proliferation of F₁ stimulator cells (W. H. Adler, T.Takiguchi, B. Marsh, and R. T. Smith,J. Immunol. 105, 984, 1970; P. F. Piguet, H. K. Dewey, and P. Vassalli, J. Exp. Med. 146, 735, 1977). The culture system was optimized for measuring the MLR of bone marrow lymphocytes enriched on sucrose density gradients. The proliferative response of the enriched fraction (BML) to 2000-R irradiated allogeneic spleen cells was three times as high as the response of unfractionated bone marrow. For maximal responses, antigen concentration had to be twice as high for the BML as for the lymph node, and in a time course study the highest [³H]TdR uptake occurred on Day 3 in BML cultures and on Day 5 in the LN. In lymph node semiallogeneic cultures stimulator cell proliferation can be disregarded, while semiallogeneic BML MLR err significantly on the high side. When BML were matched with allogeneic stimulator cells at the H-2 locus, they gave good MLR responses, provided there was a minor Mls histocompatibility locus difference, while in the lymph node the response was greatly diminished in similar mixtures. The differences in the BML and lymph node alloantigen responses with respect to antigen concentration, kinetics and susceptibility to F₁ and Mls stimulation, suggest that the bone marrow alloantigen response is mediated by a cell population that is different than alloresponsive cells in the lymph node.     

The Mlsd-defined primary mixed lymphocyte reaction: a composite response to Mlsa and Mlsc determinants

Considerable disagreement exists among immunologists regarding the polymorphic nature of the murine Mls system. An estimate of the capacity of a given putative Mls allelic gene product expressed on a stimulator population to elicit proliferation of H-2-compatible Mls-disparate unprimed T cells may vary widely among different groups of investigators. This laboratory has shown previously that preactivation of B lymphocytes in a splenocyte stimulator population by exposure to goat anti-mouse IgD (GaMD) before irradiation dramatically enhanced the in vitro presentation not only of the strongly stimulatory (and highly cross-reactive) Mlsa and Mlsd, but also the more poorly stimulatory Mlsc specificity. Therefore, by the use of GaMD-treated splenocytes that optimally present the various Mls non-H-2 stimulatory epitopes, we attempted in this study to obtain a clearer understanding of Mls polymorphism by re-examining the conflicting claims associated with the mixed lymphocyte reaction (MLR) stimulatory capacity of different Mls specificities. Among H-2k responder cells of the Mls null, Mlsa, Mlsb, or Mlsd genotypes, only T cells from Mlsd-bearing CBA/J mice did not respond to Mlsc determinants present on GaMD-treated C3H/HeJ stimulator cells. Crossing CBA/J with an Mlsc-responsive mouse strain yielded an F1 animal in which nonresponsiveness to Mlsc was dominant. Although Mlsa (AKR/J) and Mlsc (C3H/HeJ) parental T cells both proliferated vigorously to Mlsd (CBA/J) stimulator cells, the Mlsa/c (AKR X C3H)F1 T cells responded poorly to GaMD-treated Mlsd stimulator cells. In addition, Mlsd (CBA/J) T cells were nonresponsive to Mlsa (AKR/J), Mlsc (C3H/HeJ), and Mlsa/c (AKR X C3H)F1 GaMD-treated stimulator cells. Because Mlsa (AKR/J) and Mlsc (C3H/HeJ) specificities are mutually stimulatory, at least limited polymorphism must exist in the Mls system. However, because Mlsa/c (AKR X C3H) and Mlsd (CBA/J) specificities are mutually nonstimulatory, T cell proliferation in an Mlsd-defined primary MLR is most likely due to a composite response to Mlsa and Mlsc epitopes present on CBA/J stimulator cells.