Activated Suppressor Cells Research Articles

Blockade of CCR5 in melanoma: An alternative immune checkpoint modulator.

Melanoma is a deadly tumor, which in recent years has been successfully treated with immune checkpoint inhibitors as PD-1/PD-L1 and CTLA-4 inhibitors and targeted therapy as BRAF and MEK inhibitors. However, immunotherapy poses deleterious side effects and pursuit of new therapeutic targets is warranted. As knowledge of tumor immunology advances, such targets are being recognized. C-motif chemokine receptor-5 (CCR5) is a receptor found on immune cells whose effects impact the immune response both to induce inflammation and to activate suppressor cells causing an anti-inflammatory effect. CCR5 is well known as a target for HIV therapy where its blockade is efficient and safe, it is also known that its mutation CCR5delta32 is for the most part non-pathological to its carriers. In oncology, activation of the CCR5 receptor has been observed in high-stage disease and CCR5 blockade has been associated with an increased immune response. In this letter, we build up the rationale to utilize CCR5 as a therapeutic target for metastatic melanoma.

Phenotypic analysis of lamina propria lymphocytes. Predominance of helper-inducer and cytolytic T-cell phenotypes and deficiency of suppressor-inducer phenotypes in Crohn's disease and control patients.

To better define the nature of intestinal T cells, the phenotypes of isolated lamina propria lymphocytes (LPL) were determined in both Crohn's disease patients and control patients using combinations of monoclonal antibodies that have been found to correlate with particular immunoregulatory functions. Isolated LPL and autologous peripheral blood lymphocytes (PBL) were stained with multiple combinations of monoclonal antibodies and studied by dual immunofluorescence flow cytometry. In LPL, compared with PBL, there was a significant increase in the proportion of T cells having the Leu-3+, Leu-8- and Leu-3+, 2H4- phenotypes (associated with helper-inducer function) and a corresponding decrease in the proportion of T cells having the Leu-3+, Leu-8+ and Leu-3+, 2H4+ phenotypes (associated with suppressor-inducer function). It was also found that in LPL, compared with PBL, the percentage of cells with the Leu-2+, Leu-15+ phenotype (associated with suppressor-effector function) was significantly lower. However, the percentage of T cells with the Leu-2+, 9.3+ phenotype (associated with cytolytic function) was similar in PBL and LPL in control patients. There were no major differences comparing Crohn's disease patients with control patients, except that the proportion of Leu-2+, 9.3+ lymphocytes was higher in PBL in Crohn's disease patients. These results show that the lymphocyte subpopulations in the lamina propria differ from those in peripheral blood in having predominantly the phenotypes of helper-inducer and cytolytic T cells, whereas the phenotypes of suppressor-inducer cells and activated suppressor cells are less frequently observed.

Tumor hypoxia drives immune suppression and immunotherapy resistance

Tumor hypoxia predicts poor outcomes across all cancers and has long been recognized as a critical source of resistance to both chemotherapy and radiotherapy. Despite the success of T cell immune checkpoint blockade in treating melanoma, aggressive adenocarcinomas of the prostate and pancreas are largely resistant to CTLA-4 and PD-1 antibody therapy in the mouse and in man. We find that hypoxic zones of these tumors resist infiltration by T cells even in the context of robust infiltration of T cells in normoxic areas of the same tumor (e.g. in the context of T cell checkpoint blockade). Beyond this lack of accessibility to tumor-specific T cells, hypoxia drives the establishment of a highly interdependent network of immunosuppressive stromal cells. Among these, we find myeloid-derived suppressor cells and myofibroblasts to be the critical populations which act together to suppress T cell responses and mediate immunotherapy resistance. Evofosfamide is a hypoxia-specific chemotherapeutic pro-drug which is activated only in the hypoxic cores of tumors and thus can be co-administered with immunotherapy. We find that Evofosfamide-driven disruption of hypoxia zones sensitizes prostate cancer to antibody blockade of CTLA-4 and PD-1 in both transplantable and genetically-engineered murine models of prostate cancer. Co-administration of Evofosfamide and α-CTLA-4/α-PD-1 promotes tumor rejection in a significantly larger percentage of mice than either therapy alone. Mechanistic studies reveals that loss of immune resistance is a consequence of re-oxygenation of hypoxia zones which results in 1) loss of active myeloid suppressor cells, 2) reduced suppressive capacity of new myeloid immigrants, 3) loss of suppressive activation of myofibroblasts, and 4) enhanced infiltration of effector T cells. Therefore, this combination of hypoxia disruption and T cell checkpoint blockade has immense potential to render some of the most therapeutically resistant cancers sensitive to immunotherapy.

Choline is required in the diet of lactating dams to maintain maternal immune function.

Choline demands during lactation are high; however, detailed knowledge is lacking regarding the optimal dietary intake during this critical period. The present study was designed to determine the effects of varying intakes of choline on maternal immune function during lactation. Primiparous Sprague-Dawley rats (n 42) were randomised 24-48 h before birth and fed the following diets for 21 d: choline-devoid (0 g choline/kg diet; D, n 10); 1·0 g choline/kg diet (C1, n 11); 2·5 g choline/kg diet (C2·5, n 10); 6·2 g choline/kg diet (C6, n 11). Splenocytes were isolated and stimulated ex vivo with concanavalin A, lipopolysaccharide (LPS) or CD3/CD28. D and C6 dams had lower final body weight, spleen weight and average pup weight than C1 dams (P< 0·05). There was a linear relationship between free choline concentration in pup stomach contents with maternal dietary choline content (P< 0·001, r² 0·415). Compared with C1 and C2·5, D spleens had a lower proportion of mature T cells and activated suppressor cells, and this resulted in reduced cytokine production after stimulation (P< 0·05). Feeding 6·2 g choline/kg diet resulted in a higher cytokine production after stimulation with CD3/CD28 (P< 0·05). Except for a higher IL-6 production after LPS stimulation with cells from the C2·5 dams (P< 0·05), there were no differences between the C1 and C2·5 dams. For the first time, we show that feeding lactating mothers a diet free of choline has substantial effects on their immune function and on offspring growth. Additionally, excess dietary choline had adverse effects on maternal and offspring body weight but only minimal effects on maternal immune function.

Separation of human CD4 +CD39 + T cells by magnetic beads reveals two phenotypically and functionally different subsets

Objective The ectonucleotidase CD39 is an enzyme involved in adenosine production. Its surface expression on human regulatory T cells (Treg) allows for their flow-cytometry-based isolation from peripheral blood. To further develop and improve this method on a scale supporting translational studies, we introduced capture of CD39 + Treg on magnetic immunobeads. Methods Peripheral blood mononuclear cells (PBMC) obtained from healthy donors were used for negative selection of CD4 + T cells on AutoMACS using antibodies (Abs) specific for all lineage + cells. CD4 +CD39 + Treg were captured by biotin-conjugated anti-CD39 Abs and anti-biotin Ab-coated magnetic beads. Isolated CD4 +CD39 + T cells were phenotyped by flow cytometry for Treg-associated markers: CD39, CD73, FOXP3, CD25, CTLA-4, CCR4, CD45RO and CD121a or for the absence of CD127 and CD49d. CFSE-based proliferation assays and ATP hydrolysis were used to measure Treg functions. Results The purity, recovery and viability of the separated CD4 +CD39 + T cells were satisfactory. The isolated CD4 +CD39 + T cell population consisted of FOXP3 +CD25 + T cells which hydrolyzed exogenous ATP and suppressed autologous CD4 + T cell proliferation and of FOXP3 negCD25 neg T cells without suppressor function. The same two subsets were detectable by flow cytometry in normal PBMC, gating on CD4 +CD39 +, CD4 +CD127 neg, CD4 +CD49d neg or CD4 +CD25 high Treg. Conclusion CD4 +CD39 + Treg capture on immunobeads led to a discovery of two CD39 + subsets. Similar to CD39 + Treg in the peripheral blood, half of these cells are CD25 +FOXP3 + active suppressor cells, while the other half are CD25 negFOXP3 neg and do not mediate suppression.

Early accumulation of suppressor cell precursors in the spleen of Mycobacterium lepraemurium-infected mice and analysis of their in vitro-induced maturation

Spleen cells harvested from mice infected intraperitoneally with M. lepraemurium 11-17 weeks prior to harvest acquired the capacity to inhibit concanavalin A (Con A) induced proliferation of normal spleen cells when precultured for up to 24 h in mitogen-free medium. The in vivo induced suppressor activity correlated with the length of the preculture period, the time post-infection and the infecting dose. These findings were interpreted as an indication that suppressor cell precursors accumulated in the spleen of infected mice during the early phase of the disease. The interaction of infection-dependent adherent suppressor cell precursors and infection-independent, non-adherent regulatory cells is necessary for the suppressor activity to develop. Both the cells which transmit the inductive signal and the precursor cells which mature into active suppressor cells are radiosensitive, whereas suppressor activity itself is a function of radioresistant adherent cells. Preculture of cells for a short period, before they were cocultured with Con A-stimulated normal spleen cells, allowed the detection of suppressor cells before they were deleterious to the infected host and also turned out to be a relevant in vitro model for characterization of suppressor cell development during M. lepraemurium infection.

Predominance of Type-2 Immune Response in Idiopathic Membranous Nephropathy

Background: The Th1/Th2 paradigm is proving increasingly useful in the understanding of infectious diseases and many autoimmune diseases. Th1 cells predominantly produce interleukin-2 (IL-2) and interferon-γ (IFN-γ) and are instrumental in initiating delayed-type hypersensitivity and activating macrophages. Th2 cells secrete other cytokines, such as IL-4, IL-5, IL-10 and IL-13 that trigger B-cell activation and immunoglobulin synthesis. It has been shown that in patients with membranous nephropathy, there may be a predominance of Th2, because of the presence of IgG, particularly IgG4, which belongs to a subclass of the type-2 immune response, and complement deposits in glomeruli. In this study, we investigated the immunoresponse of helper T cells, i.e. Th predominance in patients with idiopathic membranous nephropathy. Methods: We used flow cytometry to assess the levels of circulating Th cells in patients with idiopathic membranous nephropathy (n = 8) and in normal individuals (n = 23) based on the expression of intracellular type-1 and type-2 cytokines. Because the production of each of these cytokines has a specific time course, we observed the cytokine synthesis at 3, 6, 9 and 12 h after stimulation. Results: The percentages of IL-2+/CD4+ cells from patients with idiopathic membranous nephropathy were significantly lower than those from normal individuals at 6, 9 and 12 h, with the difference becoming more significant over time. IFN-γ+/CD4+ cells and IL-4+/CD4+ cells were not significantly different between the two groups. In patients with idiopathic membranous nephropathy, the percentages of IL-10+/CD4+ cells were significantly higher than those in normal individuals at each point in time. Conclusion: Increased IL-10-producing Th cells may lead to suppression of delayed-type hypersensitivity and activate suppressor cells and IgG4 synthesis, resulting in idiopathic membranous nephropathy.

Modulation of Histamine Type II Receptors on CD8+ T Cells by lnterleukin-2 and Cimetidine

CD8+ T cells are known to play a major role in regulating immune functions under normal and disease conditions. In this study a radioligand binding assay was used to quantitate histamine type II (H2) receptors on activated T cells. The objective was to examine the expression of H2 receptors on T cells during activation with interleukin-2 (IL-2) and treatment with cimetidine. Activated suppressor T cells induced by concanavalin A+IL-2 showed a significant (p less than 0.01) increase in H2 receptors compared to the control nonactivated T cells. The T cells expressing the H2 receptors were identified as CD8+ cells; those among them that had an enhanced level of H2 receptors were identified as CD25+. Treatment of activated suppressor cells with the H2 receptor antagonist cimetidine at a concentration of 10(-5) M significantly reduced the number of H2 receptors. Suppressor cells induced by Con A+IL-2 were able to suppress both IgG and IgM production that was reversible with cimetidine. Incubation of lymphocytes with 50 U/ml IL-2 alone in 3-day culture significantly (p less than 0.005) enhanced H2 receptor expression. These studies demonstrate that activated suppressor T cells that are CD8+CD25+ have enhanced levels of H2 receptors and can be modulated by cimetidine.

Acceptance of class II major histocompatibility complex disparate skin grafts associated with suppressor cells and elevated langerhans cell numbers

Class II major histocompatibility complex (MHC) molecules are only present on Langerhans cells (LC) in normal murine epidermis. Depletion of this antigen with the chemical carcinogen dimethylbenzanthracene (DMBA) causes I-E disparate B10.A(2R) congenic tail skin to be accepted permanently when grafted onto B10.A(4R) recipients. Adoptive transfer of spleen cells from these recipients into naive syngeneic hosts inhibited the ability of the host mice to reject untreated B10.A(2R) tail skin grafts. Hence DMBA-treated LC depleted I-E disparate skin grafts activate suppressor cells which did not inhibit BALB/c mice from rejecting a B10.A(2R) tail skin graft. In contrast, the tobacco derived carcinogen benzo(a)pyrene (BP) increased the number of epidermal LC but had no effect on either class I or class II MHC disparate skin graft survival time. This confirms that the number of class II MHC-positive LC is critical for the initiation of skin graft rejection; when the threshold level is attained graft rejection proceeds at a maximal rate that cannot be enhanced by raising the number of LC. The tolerant skin grafts had increased numbers of LC; this was not observed in syngeneic grafts and therefore may be related to the active suppression of immunity.

Loss of interleukin-2—Producing intestinal CD4+ T cells in inflammatory bowel disease

Interleukin-2 activity of intestinal lamina propria mononuclear cells is decreased in Crohn's disease and ulcerative colitis patients compared with control patients with noninflammatory bowel disease. Factors that might be responsible for this phenomenon were investigated. Most interleukin-2 activity was produced by helper (CD4+) T cells. These were present in comparable numbers in both inflammatory bowel disease and control cultures, but the frequency of interleukin-2—producing cells was significantly (3–4 times) lower among Crohn's disease and ulcerative colitis than control cells. In agreement with this finding, levels of interleukin-2 messenger RNA were substantially decreased in both forms of inflammatory bowel disease compared with controls. Mucosal CD8+ T cells and plastic-adherent cells were unable to suppress interleukin-2 activity by autologous or allogeneic CD4+ T cells. The rate of interleukin-2 absorption was similar for inflammatory bowel disease and control cells. Induction of interleukin-2 by different stimuli (phorbol ester, phytohemagglutinin, or anti-CD3 monoclonal antibody) before or after incubation under basal conditions (“resting”) failed to normalize the capacity to generate interleukin-2 by Crohn's disease and ulcerative colitis cells. Prostanoids (prostaglandin E2 and 6-keto-prostaglandin F1α) were produced in large amounts in cultures of inflammatory bowel disease cells, but inhibition by indomethacin failed to restore interleukin-2 activity to control levels. Finally, supernatants from Crohn's disease and ulcerative colitis cell cultures failed to suppress interleukin-2 production by control CD4+ T cells. Our results show that the low interleukin-2 activity detected in inflammatory bowel disease mucosa is not caused by activated suppressor cells, excessive lymphokine utilization or immune stimulation, a defective response to activation signals, or production of inhibitory substances. Rather, the low interleukin-2 activity appears to be related to a loss of interleukin-2—producing mucosal CD4+ T cells. It is concluded that abnormalities of intestinal CD4+ T-cell function are associated with the immunopathogensis of Crohn's disease and ulcerative colitis.

Regulation of Human Interleukin-2 and Interferon-Gamma Gene Expression by Suppressor T Lymphocytes

Concomitant with induction of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) gene expression in human tonsil cells, mitogenic stimulation induces a transient activation of cells able to effectively suppress expression of these genes. Induction of IL-2 and IFN-gamma genes largely precedes appearance of suppressor cell activity, allowing expression of both genes to occur before strong down-regulation is exerted by activated suppressor cells. Suppressive activity induced in one cell population can inhibit IL-2 and IFN-gamma gene expression in another population from the same donor. The distinct nature of suppressor cells is supported by the absence of down-regulation of IL-2 gene expression in a helper cell line, MLA-144; yet, in these cells, negative control can be expressed when active suppressor cells are introduced. Our findings support the concept that actual levels of IL-2 and IFN-gamma gene activity are regulated to a large extent by the differential kinetics of activation of suppressor cells on one hand and of cells expressing the IL-2 and IFN-gamma genes on the other.

Activated suppressor cells inhibit synthesis of interferonγ in patients with multiple sclerosis and normal subjects

Activated suppressor cells inhibit synthesis of interferonγ in patients with multiple sclerosis and normal subjects

Activated suppressor cells inhibit synthesis of interferonγ in patients with multiple sclerosis and normal subjects

Activated suppressor cells inhibit synthesis of interferonγ in patients with multiple sclerosis and normal subjects

Alloantigen-specific T suppressor-inducer and T suppressor-effector cells can be activated despite blocking the IL-2 receptor.

To determine IL-2 requirement for activation of suppressor cells, PBMC were primed in one-way MLR in the presence of 10 micrograms/ml anti-IL-2R beta-chain antibody 2A3 (CD25) or control antibody, then irradiated and added as regulators in a fresh MLR. Cells primed in the presence of antibody 2A3 suppressed the proliferative response to fresh autologous lymphocytes to specific alloantigen but had no effect on the response to cells from third party donors. Priming in the presence of an antibody of irrelevant specificity induced only limited suppressor activity. Activated suppressor cells did not show cytolytic activity specific for the stimulators when tested at the time of the suppressor cell assay. To identify the subset(s) responsible for suppression, cells primed in the presence of antibody 2A3 were separated into CD4+/CD45RA+, CD4+/CD45RA-, and CD8+ subsets, which were irradiated and then tested. The suppressive activity was found predominantly in the CD4+/CD45RA+ subset, whereas CD8+ cells had some activity and CD4+/CD45RA- cells had none. No subset suppressed the response of autologous cells to third-party cells. When primed CD4+/CD45RA+ cells were cocultured with fresh autologous lymphocytes depleted of CD8+ cells, no suppression was observed, indicating that, although the CD4+/CD45RA+ cells can function as inducers of suppressors, they cannot function as suppressor-effectors. Conversely, CD8+ cells activated in MLR in the presence of 2A3 caused suppression, regardless of whether the fresh autologous responder population contained CD8+ cells. CD4+/CD45RA+ and CD8+ subsets isolated after priming in the presence of 2A3 also demonstrated Ag-specific suppression in the generation of cytotoxic T lymphocytes whereas CD4+/CD45RA- cells had no activity. Our data are consistent with the model that suppression of alloreactivity requires the cooperation of two types of cells, a CD4+/CD45RA+ suppressor-inducer and a CD8+ suppressor-effector population. Activated Tsi and fresh Tse or activated Tse alone can suppress lymphocyte proliferation and generation of CTL in response to specific Ag. Activation of Ag-specific T suppressor-inducer and T suppressor-effector cells appears to be relatively IL-2 independent and presumably require one or more other growth factors.

Treatment of spinal cord-induced experimental allergic encephalomyelitis in the Lewis rat with liposomes presenting central nervous system antigens

Chronic-relapsing experimental allergic encephalomyelitis (CR-EAE) in the Lewis rat, induced by the injection of spinal cord tissue in complete Freund's adjuvant (SC/CFA), was studied in vivo by treatment with liposomes containing central nervous tissue antigens, and in vitro by lymphocyte proliferation assays. Intracardiac administration of myelin basic protein (MBP) liposomes, galactocerebroside (GC) liposomes, or MBP + GC liposomes substantially reduced the clinical severity and/or delayed the onset of the initial phase of disease. Liposomes prepared from whole myelin provided even greater protection, and were effective at suppressing both the first disease episode and the relapses. These results indicate that while GC and MBP may play significant roles in the development of CR-EAE in the Lewis rat, immune responses to other antigens are probably also involved. Splenic and lymph node lymphocytes from MBP-GC liposome-treated rats, and splenic lymphocytes from cytochrome-GC (CYT-GC) liposome-treated rats, showed drastically reduced abilities to proliferate in response to MBP in culture. Spleen cells from both the MBP-GC- and CYT-GC-liposome-treated donors were able to actively suppress antigen-induced proliferation of MBP-primed lymphocytes. These findings suggest participation of both clonal anergy, and active suppressor cells in the liposome-mediated suppression of CR-EAE in the Lewis rat.

Immunophenotypic and cell cycle analysis of lymph node cells from dimethylbenzanthracene-treated mice

The chemical carcinogen 7, 12-dimethylbenz-(a)anthracene (DMBA) depletes Langerhans cells from murine epidermis. Application of contact sensitizers to DMBA-treated skin induces specific immunological tolerance due to a DMBA-resistant epidermal antigen presenting cell (APC) migrating to local lymph nodes where it presents antigen in a way which activates suppressor cells. As alterations in local lymph node lymphocytes may enhance the ability of the DMBA-resistant APC to activate suppressor cells, these cells were examined in DMBA-treated mice. Lymph nodes in DMBA-treated mice had normal morphology but were larger and contained increased numbers of lymphocytes. Cell cycle analysis revealed that these lymphocytes did not arise from division within the lymph node, suggesting alterations in homing properties of lymphocytes. Contact sensitizer applied to DMBA-treated skin did not increase lymphocyte division, possibly due to suppressor cell inhibition of the development of effector lymphocytes. DMBA treatment had no effect on B cells or Ia expression, but decreased levels of the T lymphocyte cell surface molecule Thy-1, and increased L3T4 and Lyt-2 as quantitated by flow cytofluorimetry. These changes could influence the development of immune responses as these T cell molecules are receptors involved in lymphocyte interactions.

Effect of Hemodialysis on T Lymphocyte Subsets, Con‐A‐Activated Suppressor Cell Activity, and Interleukin‐2 Receptor Expression on Lymphocytes in Chronic Uremic Patients

Effect of hemodialysis (HD) on some indices of immune response was studied in nine chronic uremics. Total lymphocyte, OKT4+, and OKT8+ cell numbers significantly decreased during the first 20 min of HD, and they were decreased till the third hour of the procedure, whereas the OKT4+/OKT8+ cell number ratio did not change significantly. Before HD, Con-A--activated suppressor cells exerted a stimulatory action on autologous responder cells measured in two-step culture. During HD, Con-A-activated suppressor cell activity transiently appeared, with its peak at 60 min after the start of HD. It was accompanied by a transient rise in lymphocyte count with spontaneous interleukin-2 (IL-2) receptor expression, whereas the number of cells expressing IL-2 receptor following phytohemaglutinin (PHA) stimulation was progressively decreased during HD. A significant correlation was found between the increment of Con-A-activated suppressor cell activity and the increment of spontaneous IL-2 receptor expression on lymphocytes during one single blood flow through the dialyzer. The results supply further evidence that HD may impose additional disturbances on immune regulation in chronic uremics.

Activated suppressor cell function in severely disabled patients with multiple sclerosis

Defective suppressor cell function has previously been demonstrated in patients with multiple sclerosis (MS) with progressive disease and moderate degrees of disability. In the present study activated suppressor cell function was assessed in patients with documented progressive disease who, at the time of study, had experienced severe disability (Kurtzke score greater than or equal to 6.5) for at least 2 years. We found that mean suppressor levels were significantly increased in this patient group compared with the suppressor levels in the MS patient group with progressive disease but only moderate disability (Kurtzke score of less than or equal to 6.0 within 2 years of study) (59 +/- 8% vs 19 +/- 7%, respectively, p less than 0.01). The mean value in the latter group was significantly reduced compared with the mean value for normal control subjects (47 +/- 4%, p less than 0.01), a finding consistent with previous reports. The results of this study indicate that suppressor cell function, as measured by our assay system, need not be defective in MS patients who have become severely disabled from the progressive form of the disease. Whether the patients who are now severely disabled from progressive MS passed through a phase of disease associated with the same suppressor defects as found in the progressive patients currently with moderate disability will remain speculative until long-term longitudinal studies are performed.

Dual labelling of circulating CD8 cells in patients with multiple sclerosis.

Peripheral blood T cell phenotypes have been analysed in serial samples from patients with multiple sclerosis, their unaffected relatives and controls using a panel of antibodies chosen to distinguish T suppressor and activated suppressor cells from other CD8 lymphocytes. Overall, the percentage of Leu 2a cells correlated with alterations in the Leu 2a/15 suppressor sub-population (r = 0.79, p less than 0.001). Fewer circulating Leu 2a and Leu 2a/15 positive cells were identified in multiple sclerosis patients than unaffected individuals but there was no alteration in percentage of activated (Leu 2a/DR) CD8 cells. These findings suggest that the fluctuations in CD8 cells, characteristic of patients with multiple sclerosis, are due to alterations in T suppressor phenotype; this may then lower the threshold for activation of other T cell subpopulations.

Antigen presented in the local lymph node by cells from dimethylbenzanthracene-treated murine epidermis activates suppressor cells

Application to skin depleted of LC by treatment with the chemical carcinogen DMBA of a dose of contact sensitizer optimal for inducing contact sensitivity activates transferrable suppressor cells. Excision of solvent- or DMBA-treated skin at various times following application of the contact sensitizer DNFB indicated that the fraction of antigen which leaves the skin within the first few hours induces tolerance. An initial signal inducing unresponsiveness, observed within 1 2 hr, was overturned 3–6 hr later. A more permanent tolerogenic signal in the DMBA-but not solvent-treated lymph node resulted from an epidermal cell from DMBA-treated skin presenting antigen to suppressor cells. Therefore it is likely that suppressor cells are activated in DMBA-treated mice by an epidermal cell which migrates to the local lymph node. Local lymph node cells from DMBA-treated mice also have a diminished ability to present antigen in vivo but they do not activate suppressor cells.