Alloantigens In Mixed Lymphocyte Culture Research Articles

Cytolytic activity and regulatory functions of inhibitory NK cell receptor–expressing T cells expanded from granulocyte colony-stimulating factor–mobilized peripheral blood mononuclear cells

AbstractInhibitory natural killer cell receptor (NKR)–expressing cells may induce a graft-versus-leukemia/tumor (GVL/T) effect against leukemic cells and tumor cells that have mismatched or decreased expression of HLA class I molecules and may not cause graft-versus-host disease (GVHD) against host cells that have normal expression of HLA class I molecules. In our study, we were able to expand inhibitory NKR (CD94/NKG2A)–expressing CD8+ T cells from granulocyte colonystimulating factor (G-CSF)–mobilized peripheral blood mononuclear cells (G-PBMCs) by more than 500-fold using stimulation by an anti-CD3 monoclonal antibody with interleukin 15 (IL-15). These expanded and purified CD94-expressing cells attacked various malignant cell lines, including solid cancer cell lines, as well as the patients' leukemic cells but not autologous and allogeneic phytohemagglutinin (PHA) blasts in vitro. Also, these CD94-expressing cells prevented the growth of K562 leukemic cells and CW2 colon cancer cells in NOD/SCID mice in vivo. On the other hand, the CD94-expressing cells have low responsiveness to alloantigen in mixed lymphocyte culture (MLC) and have high transforming growth factor (TGF)–β1– but low IL-2– producing capacity. Therefore, CD94-expressing cells with cytolytic activity against the recipient's leukemic and tumor cells without enhancement of alloresponse might be able to be expanded from donor G-PBMCs.

The alloantigen-specific immunosuppressive activity of estradiol-chlorambucil conjugate (KM2210) and its beneficial effect on allogeneic bone marrow transplantation in mice.

KM2210, a conjugate of estradiol and chlorambucil (CBL), which was originally developed as an anti-breast cancer agent, inhibits proliferative response of human mononuclear cells to alloantigens in mixed lymphocyte culture in a dose-dependent manner, but has no effect on their response to phytohemagglutinin. Neither estradiol benzoate nor CBL alone showed these unique actions. The suppressive effect of KM2210 on MLC was abrogated by adding of anti-transforming growth factor-beta (TGF-beta) antibody to the culture, but was not affected by the addition of interleukin-2, suggesting that KM2210, unlike CBL, displays its actions via TGF-beta. In experimental allogeneic bone marrow transplantation using mice, daily oral administration of KM2210 (2 mg/kg/day) for 30 days posttransplant significantly inhibited the alloantigen-specific immune reactions. Furthermore, the survival rate of the KM2210-treated mice was significantly higher than that of the cyclosporine-treated (2 mg/kg/day, p.o.) mice, and no adverse effect of KM2210 on hematopoietic recovery was found. These results strongly suggest possible clinical benefits of KM2210 as a new immunosuppressive agent for the prevention and treatment of graft-versus-host disease and other allospecific immune reactions.

Reconstitution of cytolytic alloreactivity with N-[4-[(4-fluorophenyl) sulfonyl]phenyl]acetamide (CL 259,763) in animals immunocompromised by cyclophosphamide

A novel synthetic immunopotentiator, i.e. N-[4-[(4-fluorophenyl)sulfonyl]phenyl]acetamide (CL 259,763), was investigated for its potential in reconstituting the cell-mediated immune response of animals whose immunologic system had been severely depressed by cytoreductive agents. It was demonstrated that lymphocytes from mice which had received 300 mg/kg of cyclophosphamide (CY) immediately following antigen sensitization had a reduced capability of responding to alloantigens in mixed lymphocyte culture and failed to generate effective cytolytic T-lymphocytes (CTL) capable of destroying appropriate tumor target cells in a cytotoxicity assay. However, treatment of these immunocompromised animals with CL 259,763 produced a significant restoration of alloreactivity, as evidenced by an enhancement of the CTL response. Although effective doses of CL 259,763 ranged from 20 to 300 mg/kg, the optimal effect was observed at 75 mg/kg. Findings from a time course study indicated that the maximum restoration occurred when CL 259,763 was given to mice 2–5 days after, but not before or simultaneously with, CY treatment. Both the immunoimpairment by CY and its reversal by CL 259,763 appeared not to be antigen specific. The lessened immunoreactivity of CY-treated mice was explicable by the presence of suppressor cells in their spleens. These suppressors were able to adhere to plastic and resisted treatment with anti-Thy 1.2 antibody, indicating a macrophage characteristic. Flow cytometric analysis indicated a quantitative depletion of all T-lymphocytes, including Thy-1.2(+), Lyt-1(+), Lyt-2(+) and L3T4(+) subsets in the spleens of CY-treated mice; however, a population of Mac-1(+) cells was markedly expanded. More importantly, administration of CL 259,763 to CY-treated animals significantly, although not completely, corrected the imbalanced cell distribution patterns toward normalcy in most instances examined. These results suggest that CL 259,763 is an immunorestorative agent capable of rescuing the immune system from CY-induced immunodepression and thus may be considered potentially useful in the treatment of patients who are undergoing cytoreductive chemotherapy.

Suppression of allograft immunity by 3,4,5,3',4',5'-hexachlorobiphenyl. II. Effects of exposure on mixed lymphocyte reactivity and induction of suppressor cell activity in vitro.

Previous studies in our laboratory have established the sensitivity of the in vivo allogeneic cytotoxic T lymphocyte (CTL) response to suppression by 3,4,5,3',4',5'-hexachlorobiphenyl[(345)2-HxCB], a toxic, Ah receptor-binding polychlorinated biphenyl isomer. The present studies have examined possible cellular mechanisms for this suppression. A modest dose-dependent suppression of the proliferative response to alloantigen in mixed lymphocyte culture (MLC) was observed with lymphocytes from B6 mice exposed to 10 or 100 mg/kg (345)2-HxCB while the CTL response generated in MLC was significantly suppressed only following exposure to 100 mg/kg (345)2-HxCB. The amount of time between treatment with (345)2-HxCB and sacrifice, which ranged from 2 to 23 days, did not appear to influence the degree of immunosuppression produced by (345)2-HxCB exposure. Mitomycin C-treated lymphocytes from B6 mice treated with (345)2-HxCB were not suppressive when added as third party cells to an independent MLC. However, if the mice were alloimmune, lymphocyte-mediated suppression of the MLC response was observed and directly correlated with the magnitude of the CTL response present in the same population. Thus, (345)2-HxCB-treated mice which had less CTL activity as compared to vehicle-treated mice also had less suppressor activity. Further analysis indicated that stimulator cell lysis by the CTL was likely to be responsible for the inhibitory activity of the alloimmune lymphocytes rather than suppressor cells per se. Avoidance of stimulator cell lysis by using H-2-incompatible MLC stimulator cells revealed the existence of antigen-nonspecific suppressor activity that was greater with lymphocytes from vehicle-treated than from (345)2-HxCB-treated mice, suggesting that both CTL and suppressor cell activities were suppressed by (345)2-HxCB exposure. Direct addition of (345)2-HxCB to lymphocyte cultures in vitro indicated a lack of direct toxicity of (345)2-HxCB on lymphoproliferative responses to mitogen or alloantigen at concentrations equal to or less than 1 x 10(-6) M. Thus, the in-vitro functional integrity of lymphocytes obtained from (345)2-HxCB-treated mice coupled with the lack of a direct lymphotoxic effect of (345)2-HxCB in vitro suggest an indirect mechanism of action for (345)2-HxCB-mediated suppression of CTL activity in vivo. Previous reports implicating suppressor cell induction and/or activation by Ah-receptor-binding halogenated aromatic hydrocarbons that mediate the inhibition of CTL generation were not confirmed in these studies.

Cytokines in the Control of Beta-2 Microglobulin Release. I. In vitro Studies on Various Haemopoietic Cells

The role of cytokines in the control of beta-2 microglobulin release from various haemopoietic cells was studied in vitro. Cell types investigated were resting cells or blasts of the T Cell, B cell or monocyte-macrophage lineage. Mediators used in these experiments were r-IFN-α 2c, r-IFN-γ, r-TNF-α, r-TNF-ß, r-IL 1, r-IL 2 and r-GM-CSF. Nanogram amounts of some of these mediators strongly affected beta-2 microglobulin release in vitro. r-IFN-γ, r-IFN-α 2c and r-IL 2 strongly enhanced and r-TNF-α and r-TNF-ß occasionally increased shedding of beta-2 microglobulin. r-IL 1 and high concentrations of r-IFN-α 2c were inhibitory, whereas r-GM-CSF was ineffective. The impact of antigenic stimulation on beta-2 microglobulin release in mixed lymphocyte culture (MLC) was also studied. Stimulation with alloantigens in MLC greatly enhanced beta-2 microglobulin shedding, and this enhancement could be inhibited by a monoclonal antibody neutralizing human IFN-γ. Among the various cell types studied, macrophages derived from peripheral blood monocytes were most susceptible to cytokine-induced alterations of beta-2 microglobulin shedding. From these data, we conclude that cytokines not only control the static expression of beta-2 microglobulin on the surface of haemopoietic cells but also largely affect their shedding.

Human milk-derived lactoferrin inhibits mitogen and alloantigen induced human lymphocyte proliferation

Purified lactoferrin (LF), isolated from human milk, was tested for its effect on human T-lymphocyte proliferative responses to phytohemagglutinin (PHA) and to alloantigens in mixed lymphocyte culture. LF inhibited proliferation in both assays in a dose-dependent manner. The suppressive effect was not due to LF mediated cytotoxicity since washing cells that had been pre-incubated with LF restored their proliferative activity. LF was most effective in suppressing the PHA response when added within 24 h of culture initiation. Iron saturated LF failed to inhibit PHA-induced proliferation, suggesting that the mechanisms of suppression involve the chelating property of LF. The suppressive effect of LF on T-lymphocyte proliferative response in vitro supports the notion that LF has significant immunoregulatory potential in vivo.

Generation in vitro of alloreactive lymphocytes is suppressed by the addition of spleen cells from mice infected with lymphocytic choriomeningitis virus.

The generation of cytotoxic T cells to alloantigens in mixed lymphocyte culture was suppressed by the addition of spleen cells from adult mice acutely infected with lymphocytic choriomeningitis virus. T cells of surface phenotype Lyt 1+2+ were required for the suppression. Suppressive cells appeared also in lymph nodes, but not in the thymus. In the spleen their number was maximal 6 to 8 days after infection. Infectious virus could not be detected in the suppressive spleen cells. Therefore the virus itself does not seem to cause the effect.

MLC-inhibiting antibodies in mice after blood transfusions.

Balb/c (H-2d) mice were transfused weekly with 0.1 ml of whole blood from C3H/HeJ (C3H) (H-2k) mice. One and two weeks after each transfusion, mice were bled and the sera were collected and pooled. Serum samples from transfused mice were absorbed with erythrocytes and spleen cells from C3H mice, and then heat-inactivated. The presence of antiidiotypic antibodies in these sera was investigated using the mixed lymphocyte culture (MLC) inhibition test, in which spleen cells from normal Balb/c mice were tested for proliferative responses to x-irradiated C3H stimulator cells in the presence of sera from transfused mice. Sera obtained from transfused mice caused significant inhibition of responses in MLC. This inhibition in MLC was specific for stimulator cells from the blood donor (C3H), and little or no inhibition was observed with stimulator cells from third-party SJL mice. In addition, the inhibitory effect in MLC was specific for responder cells from the recipient Balb/c mice and no inhibition was observed with responder cells from blood donor C3H mice. These results suggest that blood transfusions induce antiidiotypic antibodies that can block the T cell antigen-specific receptors and cause inhibition of responses of the recipient mice to blood donor alloantigens in MLC. Thus in this strain combination, disparate for the entire H-2 region, antiidiotypic antibodies developed after 1 blood transfusion. These findings are in contrast to our earlier published results in which antiidiotypic antibodies developed after 3 transfusions in recipient-blood donor combination incompatible for the K and I regions. These data suggest that the development of antiidiotypic antibodies may be related to the level of histoincompatibility at the H-2 complex between recipient and the blood donor.

Failure of Cell-Mediated Effector Mechanisms in Lung Cancer<xref ref-type="fn" rid="FN2">2</xref>

The status of cell-mediated effector mechanisms was studied in 28 patients with lung cancer (25/28 in stage III). Patients' precultured peripheral blood mononuclear leukocytes, isolated by Ficoll-Hypaque, were tested for lymphocyte proliferation responses to alloantigens in mixed lymphocyte culture (MLC). The influence of autologous patients' sera was studied further on MLC responses from patients and controls. Cell-mediated lympholysis (CML), with the use of allogenic blast cells as targets, and antibody-dependent cell-mediated cytotoxicity (ADCC) against human red blood cells also were tested. Major differences between the cancer patients and controls were not demonstrated by MLC. Inhibition or enhancement of MLC responses by the autologous serum was shown; bimodal influence was significant; 72% of the sera caused inhibition and 28% caused enhancement. CML was depressed in 54% of the patients, and ADCC was depressed in 50%. The decrease in both cytotoxic responses was significant (P less than .005). Thirteen patients died after initiation of the investigative protocol; in 11 of 13, CML or ADCC was diminished. The altered cytotoxic capabilities were more prevalent among the epidermoid type, including the deceased patients. This study provides evidence that a severe impairment of cell-mediated effector mechanisms is frequent in advanced lung cancer and may be associated with poor clinical course and with the histologic type.

Induction of antibodies by blood transfusions capable of inhibiting responses in MLC.

B6AF1 (H-2KbkDbd) mice were transfused weekly with 0.1 ml of whole blood from DBA/2 (H-2d) mice. One week after each transfusion, the mice were bled and the sera were collected and pooled. The presence of antiidiotypic antibodies in these sera was investigated using the mixed-lymphocyte culture (MLC) inhibition test, in which spleen cells from normal B6AF1 mice were treated with sera from transfused B6AF1 mice, washed, and then tested for their responses to DBA/2 stimulator cells. The sera collected following 3 and 4 transfusions caused a significant inhibition of responses in MLC, but sera obtained after 1 and 2 transfusions caused little or no inhibition. This inhibition was specific for stimulator cells from the blood donor (DBA/2) and was not observed against third-party SJL (H-2s) stimulator cells. In addition, the suppressive effect in MLC was specific for responder cells from the recipient B6AF1 mice, and no suppression was observed with responder cells from C3H (H-2k) and SJL mice. Treatment of DBA/2 stimulator cells with the serum caused no inhibition in MLC. The MLC inhibitory activity of the serum decreased gradually from the first to the third week following 4 transfusions, although a significant inhibition was still demonstrable after 3 weeks. These findings suggest that multiple blood transfusions induce antiidiotypic antibodies that can block the T cell antigen-specific receptors and cause suppression of the recipient's responses against the donor's alloantigens in MLC.

Blood-transfusion-induced suppression of cytotoxic T lymphocyte responses in mice.

B6AF1 (H-2KbkDbd) mice were transfused weekly with 0.1 ml of whole blood from DBA/2 (H-2d) mice. One week after each transfusion, spleen and serum samples were collected. Blood transfusions did not induce blood donor alloantigen-specific cytotoxic T lymphocytes (CTL) in spleens of B6AF1 mice. When spleen cells from transfused mice were sensitized to alloantigens in mixed lymphocyte culture in vitro, it was observed that 1-3 transfusions induced suppression of blood donor-specific CTL activity. No suppression of CTL activity was found after 4 transfusions. The cell-mixing experiments demonstrated that the suppression of CTL activity following initial 2 blood transfusions was due to the presence of suppressor cells. The presence of antibodies in sera of transfused B6AF1 mice capable of inhibiting CTL was investigated using the CTL-inhibition test. In these experiments, cytotoxic T lymphoblasts generated in MLC in vitro by culturing normal B6AF1 spleen cells with x-irradiated DBA/2 cells were treated with serum before testing them for cytotoxicity. The antibodies capable of inhibiting CTL responses were demonstrable in sera from transfused mice. Three and four BT sera caused significant inhibition of CTL responses. The CTL-inhibitory antibodies were specific for effector cells of the B6AF1 mice and for target cells of the blood donor DBA/2 mice. These results suggest that the inhibition of CTL responses is caused by antibodies directed against the recognition sites on effector T lymphocytes. The data from this study, therefore, demonstrate that BT cause suppression of the recipient's CTL responses against alloantigens present on the blood donor, and that this suppression is mediated by suppressor cells after the initial 1 to 2 transfusions and by antibodies directed against the CTL antigen-specific receptors after subsequent transfusions.

Evidence for the presence of suppressor T lymphocytes in animals treated with cyclosporin A.

Mice sensitized with alloantigens and treated with cyclosporin A (CsA) were incapable of generating antigen-specific cytolytic lymphocytes (CL). Lymphocytes from these CsA-treated animals could not be reactivated upon exposure to the same alloantigens in mixed lymphocyte culture (MLC), whereas their response to a third-party antigen remained intact, suggesting a long-lasting and specific effect of CsA. After being irradiated, these lymphocytes from CsA-treated animals were added to normal MLC and were shown to prevent normal lymphocytes from becoming cytolytic in a dose-dependent and antigen-nonspecific fashion. These suppressor cells were not detected in mice receiving CsA only, indicating that CsA did not induce but rather permitted the expression of suppressor cells possibly generated by allosensitization. The suppressor cells appeared to be T lymphocytes, because treatment with anti-Thy-1.2 antibody and C abrogated their suppressive activity. The present results suggest that activation and/or sparing of suppressor cells by CsA may account for the long-lasting unresponsiveness seen in CsA-treated animals.

Biological specificity of low molecular weight inhibitors of lymphocyte transformation.

Extracts of calf thymus have been shown to contain a number of inhibitors of lymphocyte transformation. A low molecular weight (600 daltons) anionic inhibitor of lymphocyte transformation has been identified and separated from contaminating polyamines and nucleotides. The active fraction inhibited the DNA synthetic response of murine or human T cells to alloantigens in mixed lymphocyte culture and to T-cell-specific mitogens. It was inactive against stimulation of B lymphocytes and several cultured tumor cell lines.

Recruitment of functional subpopulations of T lymphocytes. Initiator T lymphocytes recruit helper T cells for cytotoxic response.

Initiator T (Ti) lymphocytes are defined by their ability to recruit other T cell populations in vivo. In this study the function of T cells recruited into draining lymph nodes following injection of Ti cells primed to alloantigens in mixed lymphocyte culture was examined. The results demonstrate that alloantigen-specific helper T cells that interact with cytotoxic T (Tc) lymphocyte precursors are recruited, as shown by the significantly higher frequencies of helper cells in draining lymph nodes compared with controls. However, neither Tc cells nor their precursors are recruited. Recruitment by Ti lymphocytes is therefore selective for certain T cell subsets. Proposals to explain the mechanism, specificity, and selectivity of recruitment are discussed. We suggest that Ti cells have a central role in both the initiation of T cell-dependent immune responses and in the maintenance of immunologic memory. Their function is the rapid mobilization of T cell subclasses to a regional lymphoid organ where the immune response subsequently develops.

Role of suppressor cells in immunological maturation.

Neonatal mice exhibit high suppressor cell activity that can be measured by the ability of their spleen cells to suppress the in vitro response of adult mouse spleen cells to alloantigens in mixed lymphocyte culture. In this paper we have compared the changes in splenic suppressor cell activity after birth with the changes in immunological maturity, the latter being measured by mixed lymphocyte reactivity of the neonatal spleen cells. The data indicate that there are significant strain differences in the postnatal changes of suppressor cell activity. The results further indicate that four of the five mouse strains tested (C57BL/6, B10.A(5R), B10.A, and DBA/2) demonstrate a good correlation between the decrease in suppressor cell activity and increase in mixed lymphocyte reactivity of mouse spleen cells after birth. In one strain (B10.A(3R)), however, the suppressor cell activity drops to adult levels before full mixed lymphocyte reactivity is achieved. The results suggest that, although suppressor cells may have a role in immunological maturation, other factors may contribute to the development of immunocompetence.

A human soluble suppressor factor affecting lymphocyte responses in vitro.

A soluble suppressor factor (SSF) has been demonstrated in the supernatant of normal human peripheral blood lymphocyte cultures that exhibits suppressive activity toward the proliferative response of normal lymphocytes to concanavalin A or alloantigens in mixed lymphocyte culture (MLC) or toward pokeweed mitogen-stimulated immunoglobulin synthesis and secretion in vitro. Suppression of the proliferative response in MLC reached maximal levels when added SSF-containing supernatant approximated 20% by volume of the culture medium. Suppression in the MLC was found to act at the proliferative stage. SSF acts independently of cytotoxicity and is stable at 56 degrees C for 30 min but is inactivated at higher temperatures. Addition of SSF to the MLC as late as day 4 after initiation of the culture results in suppression of transformation. This factor(s) may regulate the magnitude of several immune responses in humans.

Immunocompetence of the residual lymphocytes in frozen blood

Lymphocytes remaining in washed, previously frozen blood were isolated and their immunocompetence was studied on the basis of T- and B-cell markers, mitogenic responses, and mixed lymphocyte culture (MLC). The results showed that freeze-preservation of red cells considerably deteriorated immunocompetence of the residual lymphocytes in addition to preferential removal of polymorphonuclear cells. While approximately 60% of the residual lymphocytes excluded trypan blue dye, their E and EAC rosette formation decreased markedly to 6.4 and 5.2%, respectively. These lymphocytes showed weak proliferation to PHA and Con A stimulation, but failed to respond to alloantigens in MLC. In contrast, they retained stimulating activity to allogeneic lymphocytes in MLC. These observations suggest that some portion of the residual lymphocytes may remain viable enough to possess immunocompetence and recognizable immunogenicity.

Effects of Murine Cytomegalovirus Infection on the Immune Response to a Tumor Allograft

The purpose of this study was to compare the effects of murine cytomegalovirus infection introduced after an EL4 ascites tumor allograft with cytomegalovirus infection accompanying or preceding the allograft. Parameters that were measured included documentation to the host's immune system. Depressed immune response of splenocytes from mice infected at any time before assay was documented by decreased responsiveness to phytohemagglutinin, to lipopolysaccharide, and to an alloantigen in mixed lymphocyte culture. In contrast, animals infected after grafting had enhanced lymphocyte-mediated cytolysis (LMC), enhanced serum-mediated cytolysis (SMC), and larger spleens than did animals that were only grafted and animals that were infected before grafting. Neither a depressive nor an enhancing effect of virus administered after grafting was reflected in vivo in reduced or increased graft clearance. Nonspecific effects of virus increased LMC and SMC in vitro, but the primary effect of viral infection after grafting is immune depression.

Viability of T lymphocytes survived in frozen-thawed human bone marrow.

To know immunocompetence of cryopreserved human bone marrow, an optimal condition for cryopreservation of hemopoietic stem cells was determined on the basis of in vitro colony formation. Viability or immune function of frozen-thawed bone marrow lymphocytes was evaluated by spontaneous rosette formation and sheep red blood cells and mixed lymphocyte culture activity. It was demonstrated that the thawed marrow cells could form E rosettes and respond to alloantigens in mixed lymphocyte culture equally or more than those before freezing. Stimulating activity of cryopreserved marrow cells in mixed lymphocyte culture was not altered through a freeze-thawing process. These observations suggest that bone marrow lymphocytes may survive without losing their immunocompetence after freezing as well as hemopoietic stem cells.

Thymus-dependent lymphocytes in human bone marrow.

Human bone marrow is known to contain significant numbers of bursa-dependent lymphocytes. The presence of thymus-dependent (T) cells is controversial. Bone marrow cells obtained from healthy volunteers was fractionated by density centrifugation. A lymphocte-enriched subpopulation was shown to be reactive to alloantigens in mixed lymphocyte culture and to contain substantial numbers of T lymphocytes. The T lymphocytes were detected by cell surface markers (rosette formation with sheep RBC) and by response to the mitogens phytohemagglutinin and concanavalin A. Bone marrow T cells exhibited functional characteristics quantitatively different from peripheral blood T cells, suggesting that they may represent a particular subpopulation of T cells. The lymphocyte-enriched fraction additionally contained committed granulopoietic stem cells capable of colony formation in semisolid gel. The presence of T cells in human bone marrow is consistent with findings in other mammals and may explain the high incidence of graft versus host disease in bone marrow transplant recipients.