One-way Mixed Lymphocyte Reaction Research Articles

Phenotyping of HLA-D-region products by negative and positive mixed lymphocyte reactions

Individuals of known HLA phenotype were used to examine the specificity of secondary proliferative (restimulation) responses in lymphocyte populations which had been primed previously in long-term, one-way mixed lymphocyte reactions (MLR). The objective was to identify the genetic region responsible for this secondary mitogenic effect. Restimulation responses between unrelated and related individuals from a family with a recombinant betweenHLA-B andD produced similar results. In both cases responses were grouped into zero, intermediate, and positive. Restimulation was by three types of cell: those with HLA-D types compatible with the responder; those with HLA-D types specific to the primary stimulator; and those with HLA-D types of a third-party cell. Compatible responses fell into the zero category and the intermediate category, third-party responses fell into the intermediate category, and specific responses fell into the positive category. Our conclusion was that HLA-D products evoked secondary mitogenic responses, while structures coded for by the HLA-A and B regions appeared to have no effect.

"Physiological interaction" does not explain the H-2 requirement for recognition of virus-infected cells by cytotoxic T cells.

B10.A (H-2Kk, H-2Dd) ectromelia-immune T cells from secondary responses in vitro were protent killers of both infected L929 (H-2Kk H-2Dk) and infected P-815 (H-2Kd, H-2Db) target cells. Specific competition with unlabelled targets showed that two separate T cell subsets were responsible for lysis of infected L929 and infected P-815 cells. One hypothesis to account for this (a form of "physiological interaction") is that T cells which kill one target e.g. infected L929) display only one out of two possible self-complementary recognition structures, in this example the H-2Kk alloantigen, not H-2Dd, whereas T cells that lyse infected P-815 targets display only H-2Dd, not H-2Kk. This hypothesis was tested and seems untenable because of the following results: A.TH (H-2Ks, H-2Dd) ectromelia-immune, secondary cytotoxic T cells which killed infected SJL/J (H-2Ks, H-2Ds) targets were themselves inactivated by pre-incubation with SJL/J cytotoxic T cells generated in one-way mixed lymphocyte reaction (MLR) against BALB/c (H-2Kd, H-2Dd). A.TL (H-2Ks, H-2Dd) ectromelia-immune secondary cytotoxic T cells which killed infected BALB/c targets were themselves inactivated by BALB/c cytotoxic T cells generated in MLR against SJL/J. Thus, virus-immune T cells which lyse infected targets by virtue of shared H-2K are also displaying H-2D alloantigen, and vice versa.

Specific loss of stimulator activity following mixed lymphocyte reactions is due to cytotoxic cells.

Experiments were performed to test the hypothesis that stimulating antigens in the mixed lymphocyte reaction (MLR) are expressed in a restricted fashion on lymphocytes. Thus one-way MLR between spleen cells from congenic mouse strains differing at H-2 were treated with 5-bromo-2-deoxyuridine (BrdUrd) and light to inactivate dividing cells. The ability of the residual cells to act as responder or stimulator cells in a subsequent MLR was assessed. As expected, a specific loss of responder cell activity occurred under these conditions. Furthermore, such cells, following irradiation, would not stimulate fresh spleen cells from the strain against which they initially responded, but would stimulate spleen cells, following irradiation, would not stimulate fresh spleen cells from the strain against which they initially responded, but would stimulate spleen cells of unrelated mouse strains. This was shown to be due to the generation of cytotoxic T cells detectable in 51Cr release assays in the primary MLR, despite BrdUrd and light treatment and irradiation. Furthermore, loss of stimulator activity mapped to the H-2K region of the H-2 complex rather than the strong MLR-stimulating I-A or I-B subregions. These experiments provide no evidence of restricted expression of MLR-stimulating antigens on lymphocytes.

TRANSFORMATION OF LYMPHOCYTES FROM PATIENTS AWAITING CADAVER RENAL TRANSPLANTS

The response of lymphocytes from prospective kidney transplant recipients to inactivated donor lymphocytes (one-way mixed-lymphocyte reaction [M.L.R.]) and to phytohæmagglutinin (P.H.A. response) was measured before transplantation of cadaver kidney allografts in 78 and 75 cases respectively. M.L.R. results from patients whose grafts had failed within 3 or 6 months compared with M.L.R. results from patients whose grafts continued to function at the same times showed only slight differences irrespective of whether the data was expressed as a relative response or a mitotic index. Because of the large overlap in values, it was concluded that the M.L.R. test was of limited value in predicting graft prognosis. Pretransplantation P.H.A. responses from patients whose grafts failed within 3 or 6 months were significantly different from those shown by patients whose grafts remained functioning at those times. It is suggested that lymphocyte responsiveness to P.H.A. before transplantation may be of value in predicting the fate of renal allografts.

Serum-free culture of hamster lymphoid cells and differential inhibition of lipopolysaccharide stimulation by isologous serum.

The optimal conditions for serum-free cultures of hamster lymphoid cells were determined. The cells responded to both the nonspecific thymus-derived lymphocyte mitogen concanavalin A (Con A) and the nonspecific bone marrow-derived lymphocyte mitogens lipopolysaccharide (LPS) and dextran sulfate (DxS). Normal MHA hamster serum was shown to specifically inhibit the response to LPS and DxS by 95% without inhibiting the response to Con A. The serum also did not inhibit one-way mixed lymphocyte reaction. Incubation of cells in 5% serum for short periods of time, followed by serum-free culture, did not lead to inhibition. The serum inhibited the LPS response by 65% even when added 24 h after initiation of the culture. The activity was associated with the high-molecular-weight material on Sephadex G-200 fractionation of serum. The inhibitory fraction did not possess antibody activity to LPS. The possibility that the material is an alpha2-macroglobulin is discussed.

Regulation of immune responses: I. Effects of cyclic AMP and cyclic GMP on immune induction

Agents that raise intracellular cAMP levels (dibutyryl cyclic AMP, aminophylline, adenosine and butyric acid) increase the magnitude of an in vitro primary humoral immune response when added at 10 −3 M during the first 12 hr of a 108 hr culture. Under the same conditions, cGMP has no direct effect but inhibits cAMP-mediated stimulation. DbcAMP (10 −3 M or 10 −4 M), present from 0 to 12 hr, also increases the number of cytotoxic lymphocytes in CBA/J (H-2 k) spleen cell cultures stimulated in a one-way mixed lymphocyte reaction with DBA/2J (H-2 d) spleen cells. The dbcAMP effect is antigen-dependent in both humoral and cell-mediated immunity and antigen-specific in the case of humoral responses.

The synergistic effect of synthetic polynucleotides and mercaptoethanol in amplifying the murine mixed lymphocyte reaction.

Tritiated thymidine incorporation of one-way murine mixed lymphocyte reactions was compared in the presence of mercaptoethanol and/or poly AU. Each of these agents amplified specific responsiveness in this system without altering reaction kinetics and the combined use of these agents enhanced specific thymidine incorporation beyond that achieved by either alone. These agents may be useful in those situations where increased sensitivity of murine mixed lymphocyte reactions is required.

Effects of Sodium Periodate Modification of Lymphocytes on the Sensitization and Lytic Phases of T Cell-Mediated Lympholysis

Sensitization of mouse splenic lymphocytes in vitro with sodium borohydride, suggesting that the biologic effects of sodium periodate are-treated autologous spleen cells stimulated a one-way mixed lymphocyte reaction and led to the generation of thymus-derived cytotoxic effector cells. These effectors were capable of lysing in 4 hr periodate-treated syngeneic and, to a lesser extent, periodate-treated allogeneic target cells. These results suggest that sensitization by periodate-treated autologous cells could result either from a specific reaction to modified self components or from a nonspecific mitogenic stimulation. Effector cells generated by allogeneic sensitization were detected on periodate-modified targets, irrespective of the H-2 antigens expressed by the targets. The effects of periodate modification on both stimulator and target cells were reversible by sodium periodate are dependent on the formation of a free aldehyde group on cell surface glycoproteins. Pretreatment of stimulator cells with neuroaminidase prevented the effect of periodate treatment, suggesting that the sensitization involves oxidized sialic acid residues. During the 4-hour 51Cr-release assay periodate-treated targets could be used to detect cytotoxic effector cells of any specificity. Fresh spleen cells and lymphocytes cultured for 5 days without antigen or in the presence of lipopolysaccharide did not lyse periodate-treated targets. An increasing level of cytotoxicity was detected on periodate-treated targets when the effector cells were generated, respectively, by stimulation with concanavalin A, by sensitization with periodate-modified autologous cells. Although the lysis of periodate-treated targets is itself nonspecific, effector cell specificity could be determined by selective blocking of the lytic phase with cells syngeneic to the stimulators. These results indicate that a nonspecific interaction can occur between lymphocytes and periodate-treated target cells, but that this interaction leads to lysis only when the lymphocytes were activated to become cytotoxic effectors.

Inhibition of in vitro immune responses by a fraction from seminal plasma.

We studied the inhibitory effect of a fraction of bovine seminal plasma on in vitro lymphocyte responses to specific and nonspecific ligands. Seminal plasma was fractionated by Sephadex G-100 chromatography, and the inhibitory component was found to have a molecular weight of above 100,000 daltons. This fraction was devoid of lymphocytotoxicity and could inhibit, in a dose-dependent mode, both the primary antibody response of normal mouse spleen cells to the thymus-dependent antigen sheep erythrocytes and the one-way mixed lymphocyte reactions. Concanavalin A-induced proliferative T-cell responses were also inhibited by the same fraction. The inhibition of the T-lymphocyte responses to Con A was complete at suboptimal concentrations of the mitogen and could only partially be overcome at supraoptimal concentrations. Four other fractions in seminal plasma were also studied in this particular system, three being without effect and one being stimulatory. The inhibitory fraction was also found to inhibit the polyclonal B-cell responses induced by fetal calf serum. The importance of the present findings for the immunophysiology of reproduction are discussed.

Cell-Mediated Immune Responses in Vitro

Abstract The role of cellular proliferation in the development of cytotoxic lymphocyte (CL) responses in one-way mixed lymphocyte reactions was investigated by using tritiated thymidine of high specific activity to kill proliferating cells. To develop maximum CL responses, responding lymphoid cells must proliferate for approximately 72 hr; thereafter, precursors of CL appear to differentiate into active CL without further proliferation. Different alloantigen-sensitive precursor cell populations participate in the CL responses to each of two sets of stimulating alloantigens. When cells responding to one set of alloantigens were selectively destroyed after incorporating the hot thymidine, the surviving cells retained the capacity to develop a normal CL response to the second set of alloantigens.

The mixed lymphocyte reaction: Selective activation and inactivation of the stimulating cells

Allogeneic cells pretreated for 48 hr with 2 × 10 −6 M ouabain have lost the capacity to show the mixed lymphocyte reaction (MLR). Analysis of various combinations of cells in the one-way MLR revealed that this effect was on the stimulating cells and not on the responding cells. Pretreatment of cells from both donors with 10 −7 M ouabain caused no change in incorporation of labeled thymidine into DNA during the first 5 days of mixed lymphocyte culture; thereafter, as incorporation by the controls declined, that of the pretreated cells continued to increase. This effect was also on the stimulating cells and not on the responders. The irreversible effects of ouabain are thus either to activate or inactivate the stimulating cells depending on the concentration of the drug; there is little or no effect on the responding cells.

MLC stimulatory capacity and production of blastogenic factor in patients with chronic lymphatic leukemia and Hodgkin's disease

The aim of the present study is to evaluate the stimulatory capacity of chronic lymphatic leukemia (CLL) and Hodgkin's disease (HD) lymphocytes in the one-way mixed lymphocyte reaction (MLR) and to investigate whether, in patients with these diseases, a correlation exists between the degree of stimulatory capacity and production of a blastogenic factor. These studies might help to resolve the controversy about the nature of the stimulating cells and to offer some insight into the mechanism of the stimulation in the MLR. The significant observations from this study are summarized as follows: (1) Stimulatory capacity of CLL lymphocytes was intact or increased, while their responding capacity was markedly depressed. (2) In our group of patients with advanced Hodgkin's disease, both stimulating and responding capacities were imparied. (3) In these patients a correlation existed between the degree of stimulatory capacity and production of a blastogenic factor. This observation would raise the possibility that the production of a blostogenic factor may in some way be involved in the stimulation of responding lymphocytes in mixed cultures. Our results, which show a dichotomy between MLC stimulatory and responding capacities of CLL lymphocyte, may suggest that different factors are involved in stimulation and response.

MLC Stimulatory Capacity and Production of a Blastogenic Factor in Patients With Chronic Lymphatic Leukemia and Hodgkin’s Disease

The aim of the present study is to evaluate the stimulatory capacity of chronic lymphatic leukemia (CLL) and Hodgkin's disease (HD) lymphocytes in the one-way mixed lymphocyte reaction (MLR) and to investigate whether, in patients with these diseases, a correlation exists between the degree of stimulatory capacity and production of a blastogenic factor. These studies might help to resolve the controversy about the nature of the stimulating cells and to offer some insight into the mechanism of the stimulation in the MLR. The significant observations from this study are summarized as follows: (1) Stimulatory capacity of CLL lymphocytes was intact or increased, while their responding capacity was markedly depressed. (2) In our group of patients with advanced Hodgkin's disease, both stimulating and responding capacities were imparied. (3) In these patients a correlation existed between the degree of stimulatory capacity and production of a blastogenic factor. This observation would raise the possibility that the production of a blostogenic factor may in some way be involved in the stimulation of responding lymphocytes in mixed cultures. Our results, which show a dichotomy between MLC stimulatory and responding capacities of CLL lymphocyte, may suggest that different factors are involved in stimulation and response.

Disparity of Mixed Lymphocyte Reactivity to Cultured Cells of Human T and B Lymphoid Lines<xref ref-type="fn" rid="fn2">2</xref>

Repeated attempts with difference assays and experimental conditions failed to detect significant peripheral blood lymphocyte reactivity in one-way mixed lymphocyte reactions to irradiated or mitomycin C-treated cells of the "leukemic" T lymphoid line RPMI 8402. In contrast, consistently high levels of peripheral blood lymphocyte reactivity were obtained with cells of six B lymphoid lines established from the same blood sample used to initiate this T lymphoid line. Although attempts to define the reason why these cultured T cells did not initiate a mixed lymphocyte reaction were not successful, evidence indicates that this inability may be an intrinsic characteristic common to three other T lymphoid lines (MOLT-4, CCRF-CEM, and CCRF-HSB-2), also established from patients with relapsed acute lymphoblastic leukemia.

The Response of Rabbit Lymphocytes to Mitogens and Alloantigens: Evidence for T Cell Heterogeneity

Abstract Soluble concanavalin A (Con A), phytohemagglutinin-P (PHA), and pokeweed mitogen (PWM) stimulate DNA synthesis in cells from a variety of normal rabbit lymphoid organs. In lethally irradiated rabbits injected with thymocytes, these responses are virtually normal or, in some instances, increased. In thymus-deprived animals (thymectomy, lethal irradiation, bone marrow shielding) they are virtually abolished. We conclude that the substances tested are specific T cell mitogens in the rabbit, as in other species. The response to Con A is both of higher magnitude and wider distribution among various lymphoid organs than the response to either of the other two mitogens. Responses to PHA relative to Con A vary in different organs, being lowest in thymus and highest in popliteal lymph nodes and manually separated cells of the thymus-dependent area (TDA) of the appendix. PHA-responsive cells were found to have the same organ distribution as cells responding in the one-way mixed lymphocyte reaction (MLR). It is proposed that responsiveness to PHA develops only in differentiated, peripheral T cells and that the PHA/Con A response ratio provides a convenient index in the rabbit for estimating function in these cell populations. The appendix TDA was enriched in cells responsive to T cell mitogens and in the MLR relative to whole appendix as well as to the dome and follicle components of this organ. This finding confirms other evidence that this morphologic element of the gut-associated lymphoid tissue (GALT) is thymus-dependent. A small proportion of rabbit B lymphocytes, limited largely to peripheral lymph nodes, were shown to respond to endotoxic lipopolysaccharide. As is the case in other animals, PWM appears to be mitogenic for both T and B cells in the rabbit.

Studies on the Human T-Lymphocyte Population

Abstract The IgG fraction of a goat antibody prepared against human infant thymus cells and extensively absorbed with pooled human red blood cells, insolubilized human Ig and cells from a patient with chronic lymphocytic leukemia (ATG), was used to study human peripheral lymphocyte populations. ATG inhibited the response of human peripheral lymphocytes to concanavalin A (Con A) but not to pokeweed mitogen (PWM). The F(ab′)2 and Fab′ fragments of ATG were as effective as ATG in inhibiting Con A induced 3H-thymidine incorporation. ATG did not interact with Con A or the receptor for Con A on the lymphocyte but was inhibitory in the absence of complement as a result of its apparent selective cytotoxicity. The antibody also inhibited 3H-thymidine incorporation in the one-way mixed lymphocyte reaction, again without affecting the response of these cells to PWM. The specificity of ATG for the T-lymphocyte population was also suggested by fluorescent labeling experiments which indicated that ATG reacted with only 50 to 80% of human peripheral lymphocytes. These results suggest that ATG is directed toward antigens unique to the human T-lymphocyte population. Moreover, it appears that ATG can be used to study human T-lymphocyte responses and to follow the isolation of T-specific antigens.

Parameters for effective use of synthetic double stranded polynucleotides in the amplification of immunologically induced lymphocyte tritiated thymidine incorporation

The present study characterizes parameters of use of synthetic double stranded polynucleotides in the amplification of immunologically induced lymphocyte tritiated thymidine incorporation. Parameters for optimum use both in the one-way mixed lymphocyte reaction as well as in response to soluble antigen are presented. Each lymphocyte population has a range of poly AU which is effective for maximal amplification. Once determined, use of this concentration enables amplification of lymphocyte responsiveness over a broad range of antigen concentration. This optimum range of poly AU concentration appears more specific for the particular lymphocyte population than for the antigen inducing stimulation. When used above optimum concentration, poly AU has an inhibitory effect inversely proportional to the antigen concentration utilized. This study is of practical importance for those investigators studying situations in which minimal responsiveness is being evaluated. It is in this situation, that the relative stimulatory effect of poly AU appears to be greatest.

Effects of immunosuppressive treatment on the in vitro activity of mouse lymphoid cells after stimulation by PHA and allogeneic cells.

The level of stimulation by phytohaemagglutinin (PHA) of mouse lymphocytes of the Balb/c and N strains was dependent on the number of cells and on the lymphoid organ from which the cells were derived. Cells from bone marrow, Peyer’s patches, and the peritoneal exudate showed no increased <sup>3</sup>H-thymidine incorporation when incubated with PHA. No correlation between the number of T cells in an organ and the activity of the cells after PHA stimulation was found. Treatment of animals <i>in vivo</i> with cortisone, cyclophosphamide and X-rays caused a reduction in the cell numbers of spleen, thymus and lymph nodes. The activity of the thymus cells of these animals is strongly enhanced in the PHA stimulation test. The activity of spleen and lymph node cells of animals treated with anti-thymocyte serum or X-rays and of lymph node cells of cyclophosphamide-treated animals is reduced. The spleen and lymph node cells of animals treated with anti-thymocyte serum show a high thymidine incorporation without PHA stimulation. In the mixed lymphocyte reaction a total cell number of at least 10<sup>7</sup> spleen cells was necessary for good stimulation. An optimum was reached after about 4 days. Normal thymus cells were almost inactive in the mixed lymphocyte reaction (MLR). Thymus cells from cortisone-treated animals were very active in the one-way MLR. This result was not obtained with thymus cells of cyclophosphamide-treated animals. Spleen cells from cortisone-treated N mice, but not Balb/c mice showed also an enhanced activity in the MLR.

The immunosuppressive effects of maternal plasma

Although the anatomical barrier which exists between the pregnant female and her fetus appears to be important, a modification of the immune response to histocompatibility differences may also play a role in the symbiosis of pregnancy. The one-way mixed lymphocyte reaction (MLC) expresses the reactivity of one population of cells against the histocompatibility antigens of another population. Therefore, the MLC was used to test the reactivity of newborns' and maternal lymphocytes to each other. It was found that, although maternal lymphocytes reacted to stimulation by cord blood cells, maternal plasma suppressed this response. It was also found that cord blood lymphocytes were hyporesponsive to stimulation by maternal cells as compared to adolescent-mother controls. This hyporeactivity was intensified in the presence of maternal plasma. Further in vitro studies revealed that vaccinia to which the subjects had been previously immunized and suboptimal doses of phytohemagglutinin (PHA) stimulated pregnant females' lymphocytes significantly less well than adult males' cells, and that similarly stimulated cultures prepared with plasma from these females inhibited transformation of their own and male donors' lymphocytes. It appears, therefore, that maternal plasma contains factor(s) which inhibit(s) the MLC reaction, antigenic and PHA in vitro lymphocyte stimulation. This inhibitory effect of maternal plasma may be important in modifying the reactivity of maternal cells to fetal tissue. The inhibition of fetal lymphoid cells in the maternal circulation may also afford protection to the mother in a fetal graft-maternal host reaction.

Ontogeny of cellular immunity: Development in rat thymocytes of mixed lymphocyte reactivity to allogeneic and xenogeneic cells

The one-way mixed lymphocyte reaction of rat thymocytes to allogeneic cells was studied, using ACT and Fischer rat strains. Mitomycin-treated thymocytes or thymocytes from F 1-hybrid rats were used as the stimulating cells. The reactivity of neonatal rat thymus cells was somewhat lower than that of adult rat thymocytes, but was nevertheless quite significant. No reactivity could be determined in neonatal rat thymocytes to mitomycintreated xenogeneic (mouse) cells, although thymocytes from 7-day-old rats reacted equally well to allogeneic and xenogeneic cells. Graft-versus-host reactivity of both rat and mouse thymus cells was also determined. It was found that neonatal and adult rat thymocytes, as well as adult mouse thymocytes, could induce splenomegaly in neonatal mice, but not in neonatal rats. The implications of these findings are discussed in the light of recent theories linking reactivity to histocompatibility antigens to antibody formation.