Macrophages as targets for inhibition by cyclosporine

In order to understand the mechanism of immunosuppression by cyclosporine, its effects on macrophage-mediated antigen-specific T cell activation (IL-2 production) were studied in vitro. While cyclosporine (CsA) present during the macrophage-T cell coculture inhibited antigen presentation effectively, pretreatment (2 hr) of macrophages with the drug also caused marked inhibition regardless of the antigen concentration and order of drug/antigen addition. Pretreatment of T cells caused only modest inhibition. With macrophage pretreatment, the structural analog cyclosporine-G had the same inhibitory activity as cyclosporine (cyclosporine-A), whereas dihydro-cyclosporine-D and cyclosporine-H were inactive. Cyclosporine demonstrated saturable binding to macrophages suggesting the existence of CsA-binding sites. A 50% inhibition of IL-2 production was achieved with 10(-6) M CsA and 60-70% of the binding sites were occupied at this concentration. CsA-treated macrophages did not release inhibitory material and the drug did not appear to be transferred from the macrophages to the T cells during the coculture. Although antigen-specific T cells could bind to drug-treated macrophages, they did not produce IL-2. Collectively, these results suggest that CsA has a direct effect on macrophages that subsequently interferes with IL-2 production at a stage following T cell antigen recognition.     

Analysis of cloned T cell function. II. Differential blockade of various cloned T cell functions by cyclosporine

Cyclosporine has profound suppressive effects on selected in vitro functions of cloned T lymphocytes. Cyclosporine inhibits the antigen-induced proliferation of the helper T cell clone 12-11. The effective dose required to reduce this response by 50% (ED50) is 28 ng/ml. In contrast, the proliferation of clone 12-11 induced by exogenous growth factors in secondary mixed lymphocyte culture supernatant (2 degrees MLC SN), is relatively insensitive to cyclosporine (ED50 = 4600 ng/ml). Furthermore, cyclosporine abrogates both antigen-induced and mitogen-induced secretion of lymphokines by clone 12-11, indicating that cloned helper T cell function is sensitive to cyclosporine even when interactions between specific alloantigens and their cell surface receptors are bypassed with mitogen. The suppressive effect of cyclosporine is not limited to helper T cell clones. The cytolytic T lymphocyte (CTL) clone 5MD2-2 is also sensitive to cyclosporine. Again, cyclosporine (100 ng/ml) blocks the antigen-driven, but not the exogenous lymphokine-driven, component of clone 5MD2-2 proliferation. This suppression does not result from the occlusion of antigen receptors or from antigen deformation by cyclosporine, because clone 5MD2-2 remains capable of antigen-specific cytolysis in the presence of cyclosporine concentrations that can suppress its proliferation. Finally, the ability of clone 5MD2-2 to remove IL-2 activity from culture media, a function that is significantly enhanced by contact with specific alloantigen, is not influenced by suppressive cyclosporine concentrations.     

Role of macrophages in the immune response to hepatocytes

The purpose of this study was to determine the role of host macrophages in the development of allospecific cytolytic T cells (allo-CTLs) in response to purified allogeneic MHC Class I+, Class II- hepatocytes in vivo in hepatocyte sponge matrix allografts (HC-SMA). Depletion of antigen-presenting cells (APCs) from responder splenocytes in mixed lymphocyte hepatocyte culture (MLHC) inhibits the development of allo-CTLs in response to purified hepatocytes. First the ability of sponge macrophages to function as accessory cells in indirect presentation of hepatocyte Class I antigen was tested in MLHC. We found that addition of irradiated sponge cells (a source of sponge macrophages) restored the development of allo-CTLs in MLHC depleted of responder APCs. Therefore, radioresistant sponge macrophages can function as accessory cells in MLHC. We next employed silica as an immunotherapy targeted against host macrophages and assessed the effect on development of allo-CTLs in HC-SMA. We found that local (intrasponge) silica treatment completely inhibited the development of allo-CTLs in HC-SMA. Combined local and systemic silica treatment resulted in inhibition of allocytotoxicity comparable to local silica treatment alone in the doses tested. We conclude that host macrophages which infiltrate HC-SMA can function as accessory cells in vitro in MLHC and that both infiltrating host macrophages and lymphocytes participate in the development of an alloimmune response to purified hepatocytes in vivo. This interaction may involve indirect antigen presentation of hepatocyte Class I antigen by macrophages to host lymphocytes which accumulate in HC-SMA.     

Specific cytotoxic activity of T lymphocyte clones derived from a patient with gliosarcoma

Eleven lymphocyte clones were established from the peripheral blood lymphocytes of a patient with gliosarcoma by means of autologous tumor stimulation and the limiting-dilution technique with recombinant interleukin-2. Ten of the 11 clones were cytotoxic against the autologous tumor cell line GI-1. Seven of the 10 clones were also cytotoxic against allogeneic brain-tumor lines and HeLa cells, one clone was cytotoxic against several target cells, and two clones were specifically cytotoxic against GI-1 and allogeneic brain-tumor cells. One of the 11 clones was not cytotoxic against any target cells tested. Lymphokine-activated killer cells induced by recombinant interleukin-2 alone exhibited cytotoxic activity against all target tumor cells tested. Surface phenotypic analysis revealed that all lymphocyte clones expressed CD3 antigen, some expressed CD4 antigen, and others expressed CD8 antigen. These clones seemed to be antigen-specific cytotoxic T lymphocyte clones. Analysis with these antigen-specific cytotoxic T lymphocyte clones may be useful in the elucidation of tumor-specific or tumor-associated antigens on autologous tumor cells.     

A comparison of the inhibitory effects of immunosuppressive agents cyclosporine, tetranactin, and didemnin B on human T cell responses in vitro.

The agents cyclosporine, tetranactin (TN), and didemnin B (DB) were compared for their ability to inhibit proliferative human T cell responses in vitro, using anti-CD3, PHA, alloantigen, or tetanus toxoid as stimuli and using monocytes or Langerhans cells as antigen-presenting cells/accessory cells (APC/AC). We found that all three agents suppressed T cell activation in a dose-dependent fashion, irrespective of the stimulus of APC/AC type used. Both T cells and APC/AC were affected by the drugs. DB appeared to be the most potent suppressive drug (IC50 = 1-4 ng/ml), whereas CsA and TN exerted approximately similar potency (IC50 = 50-60 ng/ml). Remarkably however, DB was toxic at a concentration of 10 ng/ml, which is quite close to the inhibition-inducing dose. No toxicity was observed with CsA and TN at doses up to 5000 ng/ml. The agents TN and DB could interrupt ongoing T cell responses and could block responsiveness to exogenous recombinant IL-2. Expression of IL-2 receptors was slightly inhibited by all three drugs. Expression of MHC class II molecule HLA-D and of adhesion molecules LFA-1, LFA-3, and ICAM-1 was clearly reduced by DB, giving an explanation for the observed inhibition of cluster formation between T cells and APC/AC. Except for a slight reduction of LFA-3 by TN, CsA and TN did not affect the expression of any of these cell surface markers or the formation of clusters. Differences in the effects of CsA, TN, and DB on immune responses in vitro and on the phenotype of T cells and APC/AC suggest that these immunosuppressive drugs have different inhibitory mechanisms.     

Differential requirements for class II MHC antigen in human T cell activation

We have employed three different models of major histocompatibility complex (MHC) antigen to investigate the role of these antigens in some aspects of human T cell activation in in vitro; specifically the induction of lymphokine synthesis and receptors, and in antigen presentation. Those models were (1) allogeneic platelets (class I alone); (2) D/DR compatible lymphocytes (class I plus nonimmunogenic class II); and (3) allogeneic heat-treated lymphocytes. After heating at 45 C for 1 hr, the latter are completely viable and express both class I (HLA A and B) and class II (HLA, DR, MT/MB) MHC antigens but do not stimulate in the mixed lymphocyte reaction (MLR). These models were compared with conventional irradiated MLR stimulating or antigen-presenting cells. Only the conventional cells were able to stimulate the synthesis of interleukin-1 and interleukin-2. Also, irradiated but not heat-treated non-T cells could present soluble antigen to autologous T cells. This implies that intact and unmodified Ia molecules are required for those responses. On the other hand, heat treated allogeneic lymphocytes and D/DR-compatible lymphocytes but not allogeneic platelets could induce responsiveness to interleukin-2. This function appears to require a heat stable but nonallogeneic component of the class II molecules. Our previous observations employing these models have also shown that there is yet a third category of T cell responses, including memory cell priming and suppressor cell induction, that can occur in the presence of predominantly class I antigen. Taken together, these data illustrate the functional heterogeneity of T cell responses to Ia-like antigens. The possible relevance of these findings to clinical transplantation is discussed.     

Quantitative fluorescence analysis of cyclosporine binding to human leukocytes

The purpose of this investigation was to estimate the binding of cyclosporine at the single-cell level on human peripheral lymphocytes, and to test possible identity of the cyclosporine-binding site with a common receptor of T cell activation. A dansyl-coupled derivative (Dans cyclosporine) was used as a fluorescent probe. The histograms of unseparated, labeled peripheral leukocytes obtained by a fluorescence-activated cell sorter (FACS) showed that Dans cyclosporine stained all leukocytes--but two distinct populations could be separated based on the intensity of fluorescence. The more brightly labeled cells consisted mainly of granulocytes and monocytes, whereas the less-bright cells represented the lymphocyte compartment. Fluorescence microscopy revealed binding on the membrane for both cell populations; the label was, however, rapidly internalized in phagocytes. For both populations binding was saturable, time and temperature dependent, and reversible. Half-saturation occurred at approximately 5 X 10(-7) M (Kd). With respect to lymphocyte subpopulations, no difference of cellular fluorescence was found between unseparated lymphocytes and T cell subsets. In addition, mitogens such as concanavalin A, phytohemagglutinin, phorbol 12-myristate 13-acetate, or OKT3 antibody did not inhibit Dans cyclosporine binding. These results clearly indicate that cyclosporine binds to all peripheral blood lymphocytes, and no preferential binding on T cell subsets can be detected.     

An immunosuppressive effect by synthetic sulfonolipids deduced from sulfonoquinovosyl diacylglycerols of sea urchin

BACKGROUND: It is important to develop new immunosuppressive agents without clinical drawbacks. In this article, we reveal the possibility of a chemically synthetic sulfonolipid that acts as a novel immunosuppressive drug. METHODS: We evaluated the immunosuppressive effect of 3-O-(6-deoxy-6-sulfono-beta-D-glucopyranosyl)-1,2-di-O-acylglycerol (beta-SQDG) that contains a saturated C18 fatty acid, which is designated as beta-SQDG(18:0) by mixed lymphocyte reaction (MLR) and rat allogeneic skin graft. Then, we investigated the mechanism of immunosuppressive effect of beta-SQDG(18:0). RESULTS: beta-SQDG(18:0) inhibited human MLR in a dose-dependent manner without overt cytotoxic effect and prolonged rat skin allograft rejection in vivo. beta-SQDG(18:0) did not inhibit the direct activation of responder T. This reagent could not affect the expression of either major histocompatibility antigen complex (MHC) class I or class II molecules on the cell surface of the stimulator cells, antigen-presenting cells. In contrast, beta-SQDG(18:0) was demonstrated to inhibit the binding among allogeneic lymphocytes. However, the expression of known cell surface accessory and adhesion molecules, such as CD4, CD28, leukocyte function-associated antigen 1, intercellular adhesion molecule 1, and CTLA-4, was not affected by beta-SQDG(18:0) treatment. CONCLUSIONS: beta-SQDG(18:0) might be a new class of the immunosuppressive reagent, and the inhibition of responder T-lymphocyte activation in MLR by beta-SQDG(18:0) may be responsible for certain three-dimensional structures of this reagent or its quinovose binding to sulfonic acid.     

Apoptosis of allospecifically activated human helper T cells is blocked by calcineurin inhibition

BACKGROUND: Classical transplantation immunosuppression relies heavily upon the interruption of interleukin-2 (IL-2) signaling by calcineurin inhibition. However, recent evidence in murine models suggests that IL-2 is necessary for activation-induced cell death (AICD) of allograft-specific lymphocytes. METHODS: We examined the apoptotic effects of the calcineurin inhibitor cyclosporine A and mTOR inhibitor rapamycin on the apoptotic alterations that occur in allospecifically activated human lymphocytes in a one-way mixed lymphocyte culture (MLC). RESULTS: Cyclosporine increased caspase-3 activation in MLC, which corresponded with a decrease in lymphocyte apoptosis in MLC. Cyclosporine also reduced apoptosis in the CD4+ helper T cell subset, while CD8+ cells had similar or increased apoptosis when compared to controls. In contrast, rapamycin-treated cultures had normal levels of CD4+ T cell apoptosis when compared to control MLC, with decreases seen in CD8+ T lymphocytes. CONCLUSIONS: In humans, blockade of IL-2 receptor signal with rapamycin allows apoptosis of allospecifically activated CD4+ lymphocytes to occur, while blockade of IL-2 production with cyclosporine results in decreased apoptosis in this T cell subset. As helper T cells are integral to the immune response, these results may explain the tolerogenic effects of rapamycin.     

Costimulatory signals necessary for induction of T cell proliferation

Two separate signals are required for induction of T cell proliferation. In an attempt to identify them we used polyclonal T cell activation with Con A, which requires costimulation with autologous accessory cells. The costimulatory activity is not constitutively expressed on accessory cells since such cells fixed immediately after separation from whole blood are unable to provide the necessary signal(s), although such activity is readily expressed after activation by incubation and such cells subsequently fixed will support Con A-induced T cell proliferation. Addition of recombinant IL-1 plus IL-6 to T cell cultures in the absence of accessory cells does not result in T cell proliferation but addition of these factors to cultures containing fixed activated accessory cells results in further increase in proliferation. The expression of the costimulatory activity during incubation is inhibited in the presence of cycloheximide or tunicamycin. The costimulatory activity of fixed activated cells is partially inhibited by antibody against ICAM-1. This inhibition is not reversed by the addition of recombinant IL-1 and IL-6. When accessory cells are preactivated in the presence of chloroquine, they are unable to provide costimulation to T cells but addition of recombinant IL-1 and IL-6 restores their ability to support T cell proliferation. Accessory cells preactivated in the presence of colchicine show an increased ability to provide costimulation to T cells in culture.     

Direct activation of human responder T cells by porcine stimulator cells leads to T cell proliferation and cytotoxic T cell development

Abstract: Functional activities of highly purified T human responder lymphocytes reactive against porcine stimulator cells were studied to evaluate whether porcine stimulator cells can directly activate a xenospecific cellular immune response. Mixed lymphocyte culture (MLC) tests revealed that CD4+ human responder cells proliferate when stimulated in vitro with porcine cells, a reaction similar to that of alloresponses regarding magnitude and tempo. A direct pathway of activation was verified by the requirement for adherent porcine stimulator cells and the partial blocking by monoclonal antibodies against porcine major histocompatibility complex (MHC) class II antigens. An analysis of proliferative CD4+, CD3+, CD16-, and 56- human T cell clones revealed that some clones were seemingly recognizing allele-specific determinants, whereas others could be restimulated by a wide range of porcine stimulator cells. Cytotoxic T cells (CTLs) were generated following direct recognition of pig cell ligands by human T cells in the absence of autologous antigen-presenting cells (APCs). Although the identification of target antigen(s) on the pig cell recognized by the CTL warrants some discussion, the pattern of killing exhibited by the CTLs indicates the recognition of porcine polymorphic determinant(s). The implications of these findings for cellular reactivity against porcine transplants are discussed.     

A reappraisal of the effects of monoclonal antibodies directed at T cell differentiation antigens

We examined the effects of monoclonal antibodies (Abs) directed at T3 antigen (expressed on most post-thymic T cells), T4 antigen (helper/inducer subset) and T8 antigen (suppressor/cytotoxic subset). Anti-T3 induced interleukin-2 production and proliferation of peripheral blood mononuclear cells (PBM). Anti-T4 or T8 did not exhibit such properties. Addition of methylprednisolone (MP) or cyclosporine (CsA) to PBM activated with anti-T3 resulted in 79% and 88% suppression of proliferation, respectively. Neither anti-T4 nor anti-T8 mediated significant inhibition of anti-T3-induced proliferation. Primary mixed lymphocyte cultures (MLC) were variably affected by Abs. Anti-T3 augmented proliferation found in primary MLCs at 48 hr and had an inconsistent effect on the proliferative response found at 120-136 hr of culture. Primary cytotoxic T lymphocyte (CTL) generation was consistently suppressed by anti-T3, while natural killer (NK)-cell-like activity was augmented at 72 hr and suppressed after 136 hr of culture. Anti-T4 mediated a dose-dependent suppression of proliferation and CTL generation in the primary MLC and had minimal effect on the induction of NK-cell-like activity. At high concentrations (5000-1000 ng/ml), anti-T8 mediated modest inhibition of proliferation and of the induction of cytolytic activity. Alloimmune memory cells, generated in long-term primary MLCs, were activated by anti-T3 to exhibit specific secondary cytolytic activity and NK-cell-like activity in the absence of the original priming stimulus. Neither anti-T4 nor anti-T8, under identical experimental conditions, activated memory cells. When interrelated, our experimental findings indicated that: (1) the ultimate immunity elicited by anti-T3-T3 antigen interaction is critically dependent upon the immune potential of the cell assessed; (2) MP or CsA can inhibit PBM activation by anti-T3; and (3) anti-T4 might have a role as an immunosuppressant in renal graft recipients.     

Soluble suppressive molecule released by human T lymphocytes induced by heat-inactivated allostimulator cells in the presence of cyclosporine

E-rosette-positive peripheral blood lymphocytes (E+PBL) stimulated initially with heat-inactivated allogeneic lymphoblastoid cells in the presence of cyclosporine (CsA/HI) produce a soluble molecule that suppresses fresh lymphocytes in a primary mixed lymphocyte reaction. Cell lines were derived from the E+PBL cells after one and two weeks of culture. These lines were CD4+ by both FACS and mRNA analysis. The cells produce a potent soluble molecule (supernates often containing greater than 1000 units of suppressive activity per milliliter). The factor has an apparent molecular weight of 90 k and is sensitive to both pH and boiling. The molecule is not the suppressive cytokine TGF beta, based on neutralization with anti-TGF beta antibody and mRNA expression. None of the available cytokines expressed by these cells was suppressive when titrated into an MLR, alone or in combination. These results support the conclusion that CsA/HI-activated T cell lines produce a novel cytokine that is not antigen-specific or MHC-restricted.     

Human memory T cell activation. Effects of monoclonal antibodies directed at interleukin-2, receptors for interleukin-2, or interferon-gamma

Several polyclonal T cell activators can induce the differentiation of quiescent antigen-specific memory T cells (memory cells) into specific secondary cytotoxic T cells, in the absence of the original priming alloantigens. Such activation represents a potential pathway by which immunologically nonspecific signals can elicit specific antiallograft immunity. We therefore investigated the molecular basis for antigen-independent activation of memory cells using as probes recombinant interleukin-2 and monoclonal antibodies directed at IL-2, IL-2 receptors (IL-2R), or interferon-gamma (IFN-gamma). Antigen-specific memory cells, generated in human long-term-primary mixed lymphocyte cultures, were induced to proliferate, exhibit antigen specific secondary cytolytic activity, or produce IFN-gamma by recombinant DNA human IL-2 produced in E Coli. Monoclonal antibodies directed at the IL-2R or at IL-2 inhibited IL-2-mediated proliferation, cytolytic activity, or production of IFN-gamma. Monoclonal antibodies directed at IFN-gamma did not inhibit alloantigen or IL-2 mediated activation of memory cells. Our findings suggest that successful interaction between IL-2 and its receptor expressed on memory cells represents a plausible pathway by which an immunologically nonspecific signal might elicit specific antiallograft immunity. Moreover, therapeutic strategies that include antibodies directed at the IL-2 and/or IL-2R and not antibodies directed at IFN-gamma will inhibit IL-2-dependent alloimmunity.     

Suppressor cell generation during normal wound healing

We have previously shown that 10 day healing wounds in rats contain wound mononuclear cells (WMNC) which inhibit normal lymphocyte mitogenic and allogeneic responses. In the present study we sought to further characterize the WMNC and define their mechanism of action. Polyvinyl alcohol sponges implanted in wounds were harvested and processed 10 days postwounding. The resultant WMNC suspension contained less than 15% macrophages. By FACS analysis, 69.5 +/- 11.4% (mean +/- SD of eight separate experiments) of the cells expressed the all T cell marker (W3/13), while 47.7 +/- 11.9% stained with the T helper/effector marker (W3/25) and 49.5 +/- 18.8% expressed the T suppressor/cytotoxic phenotype (OX8) (Th/Ts ratio = 0.96 +/- 0.13). When various numbers of WMNC were cocultured with 5 X 10(5) PHA-stimulated rat thymic lymphocytes, as few as 500 WMNC inhibit normal blastogenesis. Long-term (72 or 144 hr) culture of WMNC revealed that they maintain their suppressive activity. Furthermore, the conditioned media of long-term cultures also significantly suppressed thymic lymphocyte PHA blastogenesis, suggesting that the WMNC secrete suppressive cytokines. Large doses of human recombinant IL-2 or indomethacin did not abrogate the inhibitory effect of WMNC. We conclude that the healing wound is normally infiltrated by suppressor lymphocytes which generate immune inhibitory cytokines.     

霉酚酸酯对未成熟树突状细胞抗原递呈功能的影响

目的研究霉酚酸酯(MMF)在体外对小鼠未成熟树突状细胞抗原递呈功能的影响。方法在骨髓来源未成熟树突状细胞的培养过程中加入MMF，表型鉴定之后，做抗原递呈功能分析，观察抗原特异性增殖反应、混合淋巴细胞反应以及T淋巴细胞抗原特异性低反应性。结果以MMF处理的未成熟树突状细胞向T淋巴细胞递呈可溶性抗原的能力减弱，在同基因型的树突状细胞和T淋巴细胞接触条件下不能刺激致敏T淋巴细胞的有效增殖；MMF处理的树突状细胞在体外的同种混合淋巴细胞培养反应中能够维持树突状细胞于未成熟状态，不能充分活化CD4^ T淋巴细胞和诱发TH1反应，诱导了T淋巴细胞对同种抗原的低反应性；MMF处理的树突状细胞诱导的T淋巴细胞低反应性具有同种抗原特异性。结论在体外培养过程中给予MMF干预能够降低未成熟树突状细胞的抗原递呈能力，增强同种未成熟树突状细胞的免疫耐受诱导作用。     

Inhibition of the accessory function of murine macrophages in vitro by cyclosporine

The accessory function of macrophages, which is strictly related to the induction of T cell activation, has been studied to determine whether it is affected by cyclosporine (CsA). Irradiated spleen cells, used as a source of macrophages, were pulsed overnight with beta-galactosidase (GZ) in the presence of CsA. After washing of the pulsed macrophages, cells from a GZ-specific T cell line were added to cultures and 3H-thymidine incorporation was measured 72 hr later. We found that 500 ng/ml CsA present during macrophage pulsing with GZ reduced T cell proliferation to 5%. On the other hand, 100 ng/ml CsA almost completely abrogated the proliferative response when present for the duration of the culture. Similar results were also obtained using antigen-pulsed peritoneal-adherent macrophages to stimulate the T cell line to proliferate, or a T hybridoma clone to produce interleukin-2 (IL-2). The possibility that CsA actually affects interleukin-1 (IL-1) production by macrophages by inhibiting uninvolved T cells could be ruled out. We conclude that CsA-induced inhibition of T cell functions (proliferation and IL-2 production) is partially due to the effect of the drug on the accessory function of macrophages. This immunosuppressive mechanism of action of CsA on macrophages has not been previously described.     

T cell costimulatory blockade: new therapies for transplant rejection.

Optimal T cell responses occur when T cells receive both antigen-specific signals through the T cell receptor and non-antigen-specific costimulatory signals through accessory cell surface molecules. The best understood costimulatory receptor is CD28. Signals through the T cell receptor and CD28 cooperatively induce cytokine gene expression and promote T cell proliferation and survival. Negative signals delivered through a related cell surface receptor, cytotoxic T lymphocyte antigen (CTLA-4), act to terminate immune responses and are required for normal immune homeostasis. This article reviews T cell costimulation, including the CD28/CTLA-4 system and other potential costimulatory pathways (such as CD40/CD154), the role of these pathways in normal immune responses, and the potential for the inhibition of these pathways to induce transplantation tolerance.     

Impairment of allograft tumor immunity by isotype-like suppression of antigen-specific T cell factors

PCl-F is an antigen-binding factor that is derived from T cells of picryl chloride (PCl) contact-sensitized mice. Intravenous transfer of PCl-F into naive recipients, followed immediately by PCl challenge, results in the ability to elicit an immediate hypersensitivity-like cutaneous response. This PCl-F-dependent early response is an obligatory step in the mediation of a PCl-specific delayed-type hypersensitivity reaction by late-acting, antigen/MHC-restricted effector T cells. These latter cells recruit a local infiltrate of inflammatory cells by producing chemoattractant lymphokines. Injection of PCl-F also induces a T cell-dependent feedback circuit that ultimately suppresses production of PCl-F, and thus suppresses DTH to PCl. This form of regulation is not antigen-specific, since PCl-F induces suppression of DTH to PCl and to other antigens via suppression of the production of antigen-binding T cell factors necessary for initiation of DTH. This form of regulation does not affect classic, late-acting, lymphokine-producing effector T cells of DTH, or helper T cells for antibody responses. We now report that injection of PCl-F is also capable of inducing suppression of cutaneous hypersensitivity responses to allogeneic and syngeneic tumor cells, and of immune resistance to an allogeneic tumor graft. Our results suggest, therefore, that antigen (tumor)-specific T cell factors play a role in initiation of hypersensitivity responses to tumor cells. Injection of PCl-F suppressed, besides tumor-specific cutaneous hypersensitivity, production of the tumor-specific T cell factor that renders macrophages cytotoxic to tumor cells (i.e., specific macrophage-arming factor or SMAF). Thus, PCl-F injection may impair immune resistance to tumor cells by suppressing initiation of hypersensitivity responses that recruit macrophages, and also by inhibiting production of SMAF that renders macrophages cytotoxic. It is therefore tempting to conclude that the antigen-specific T cell factors that initiate DTH, such as PCl-F and SMAF, belong to the same isotype or group of antigen-specific T cell products that can be regulated by a form of feedback suppression that is isotype-like and inhibits production of these related, antigen-specific T cell factors.     

A CD4+ T cell clone selected from a CML patient after donor lymphocyte infusion recognizes BCR-ABL breakpoint peptides but not tumor cells

BACKGROUND: In patients with chronic myelocytic leukemia (CML), the breakpoint cluster region and fusion between the BCR and the c-ABL genes (BCR-ABL) oncogen product is a potential tumor-specific antigen. Previous studies have shown that T cells specific for the junctional region peptides of the BCR-ABL oncoprotein can be detected in healthy individuals as well as in patients with CML in chronic phase. We assessed whether BCR-ABL- specific T cells could be found in a patient achieving a complete cytogenetic remission after CD4+ donor lymphocyte infusion. METHODS: Using dendritic cells pulsed with BCR-ABL breakpoint peptides as antigen-presenting cells, we stimulated patient peripheral blood lymphocytes to isolate peptide-specific T cell clones present at the time of the cytogenetic response. T cell clones were isolated and the cellular specificity of these cells was examined. RESULTS: A CD3+ CD4+ T cell clone (1F7) that recognizes overlapping p210 junctional peptides presented by HLA-DR molecules was identified and expanded in vitro. Clone 1F7 failed to recognize autologous tumor cells as well as dendritic cells derived from patient CML cells. Clone 1F7 did not inhibit the growth and differentiation of CML precursor cells in a standard colony formation assay. Finally, using a clone-specific probe, 1F7 cells could not be detected in patient peripheral blood at the time of the donor lymphocyte infusion response. CONCLUSIONS: These results suggest that clone 1F7 was selected in vitro using highly potent peptide pulsed dendritic cells but was not representative of the anti-leukemia immune response in vivo. Based on these findings, CD4+ T cells with BCR-ABL specificity do not appear to be mediators of the anti-leukemia response in vivo after donor lymphocyte infusion.