Role Of CD74 Research Articles

Abstract 085: The Role of CD70 in Vascular Function

We have recently shown that CD70 is important in formation of effector memory T cells and that these cells seem to enhance the hypertensive response and end organ damage caused by repeated hypertensive stimuli. CD70 has traditionally been identified on antigen presenting cells and is thought to interact with CD27 on T cells, promoting proliferation and memory cell formation. The precise cell types that express CD70 and the role of CD70 on non-immune cells has not previously been investigated. In our prior study, we showed that hypertension is associated with a marked increase in CD70 surface expression on dendritic cells (DCs) and macrophages. To examine the specific effect of DC CD70, we performed adoptive transfer of DCs from hypertensive WT mice to normotensive CD70 -/- mice and from hypertensive CD70 -/- mice to normotensive WT mice. The recipient mice were treated with a normally subpressor dose of ang II (140 ng/kg/min). Interestingly the CD70 -/- recipients were protected from the development of hypertension, despite receiving WT DCs from hypertensive donors. In contrast, the WT recipeints that received CD70 -/- DCs developed modest hypertension, indicating that non-DC sources of CD70 likely contribute to the hypertensive phenotype. Studies of mesenteric vascular reactivity showed that CD70 -/- mice have markedly impaired endothelium-dependent vasodilatation to acetylcholine compared to WT mice (43±10 vs 25 ± 3 %) at baseline. In contrast, there were no differences in relaxation responses to sodium nitroprusside. In additional experiments, we showed that human umbillical vein endothelial cells express CD70 mRNA and that this is increased by > 30 fold by laminar shear stress (15 dynes/cm2) compared to oscillatory shear. Finally, using immunohistochemistry, we identified CD70 protein localized to resistance vessels of the kidney of ang II-treated mice. These data identify a new role of CD70 in modulating vascular function independent of its role on antigen presenting cells in memory T cell formation.

Abstract 2320: CD70 immune checkpoint ligand is associated with the epithelial-to-mesenchymal transition in non-small cell lung cancer

Abstract Non-small-cell lung cancers (NSCLC) account for 85% of lung cancers and are mostly diagnosed at advanced stage. Drugs interrupting immune checkpoints, such as anti-CTLA-4, anti-PD-1, anti-PD-L1, have proven to unleash anti-tumor immunity and mediate durable cancer regressions in a portion of patients with NSCLC. Epithelial-to-mesenchymal transition (EMT) is a developmental process that enables reprogramming of polarized epithelial cells towards a mesenchymal phenotype with migratory and invasive properties. EMT has been involved in escape from oncogenic-induced senescence, resistance to chemotherapy and development of metastatic disease. Moreover, EMT promotes cancer cell plasticity and favors their adaptability to selective pressures. Here we made use of in silico approaches and further in vivo validation to evaluate the potential role of EMT as a major escape mechanism to tumor immunosurveillance in NSCLC. We performed an initial in silico analysis to assess the expression of immune checkpoint ligands (ICPLs) in 129 NSCLC cell lines (CCLE), in relation to their EMT status defined by a 72-gene EMT signature that was previously reported in NSCLC. Unsupervised hierarchical clustering analysis revealed that the expression of selected ICPLs was associated with the mesenchymal or epithelial phenotype. In particular, CD70, a member of the tumor necrosis factor superfamily, was significantly associated with the mesenchymal status (p < 0.001). We identified an E-box sequence (CANNTG) in CD70 gene promoter thus suggesting the possible regulation of CD70 by ZEB1 and/or SNAI. We extended the in silico analysis to a set of 488 adenocarcinomas (ADC) and 501 squamous cell carcinomas (SCC) from the TCGA and confirmed that CD70 expression was associated with a mesenchymal status (q-value <0.001). In a set of E-like (H441 and H1650) and M-like (H23 and HCC44) NSCLC cell lines we validated the association between an increased level of CD70 by flow cytometry and the mesenchymal status. Expression of CD70 by immunohistochemistry was observed in 5/51 lung ADC (10%), 6/45 (13%) lung SCC, 16/26 (63%) pulmonary sarcomatoid carcinomas and 2/10 (20%) small cell lung carcinomas. Interestingly, in pulmonary sarcomatoid carcinomas, CD70 expression was closely associated with the mesenchymal component. Our results suggest an association between the expression of CD70 and the mesenchymal phenotype, thus shedding light on the potential role of CD70 in immune escape of a subset of NSCLC. Citation Format: Sandra Ortiz-Cuaran, Aurélie Swalduz, Maria A. Albaret, Christine Menetrier-Caux, Véronique Haddad, Arnaud Paré, Geneviève De Souza, Anne P. Morel, Maurice Pérol, Christophe Caux, Sylvie Lantuejoul, Alain Puisieux, Pierre Saintigny. CD70 immune checkpoint ligand is associated with the epithelial-to-mesenchymal transition in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2320.

TMOD-07DIFFERENCES IN TUMOR GRAFT ACCEPTANCE OF 261 GLIOBLASTOMA MULTITFORME (GBM) CELL LINE BETWEEN WILD TYPE C57BL6, CD74 DEFICIENT, AND GAMMA DELTA TCR DEFICIENT MICE

BACKGROUND AND SIGNIFICANCE: Glioblastoma Multiforme (GBM) is a deadly and highly aggressive brain tumor with grim prognosis. At present none of the treatment strategies extend the life of most patients beyond a year. A contribution from the immune system in the control and/or growth of tumors is currently under intense investigation. However, the strategies GBM cells use to hide from the immune system are poorly understood. EXPERIMENTAL APPROACH: To address this issue, we compared the tumor graft acceptance of the mouse GBM 261 neurosphere (mouse glioma cell line/derived from a tumor from a C57Bl6 strain background) into C57Bl6 mice with three genetic modifications (CD74, invariant chain deficient mice; gamma delta knockout mice; and control, wild type C57Bl6 mice). Neurospheres were made from 261 cells by plating 10^5 cells/mL in DMEM/F12 media supplemented with 20 ng/mL EGF and 10 ng/mL bFGF, 0.002% heparin and B27 supplement without retinol. Cells started showing neurosphere morphology at around 6 days. The 261 neurospheres were injected stereotactically in the caudate nucleus and mice were observed for 4-6 weeks after neurosphere injection. RESULTS: The data showed that the tumor acceptance in C57Bl6 mice was higher than acceptance in either CD74 deficient or Gamma Delta TCR deficient mice. In addition, C57Bl6 mice showed rapid tumor growth with robust neurological deficit as early as 4 weeks. No neurological deficit was observed in invariant chain deficient mice or gamma delta knockout mice. CONCLUSIONS: These data suggest a contribution from CD74 as well as gamma delta T cells in tumor graft acceptance. Further experiments are ongoing to determine the potential role of CD74 and/or gamma delta T cells in the progression of GBM.

Role of CD74 in Mesenchymal Stem Cell Dysfunction in Patients with Acute Myeloid Leukemia

Abstract 4344Mesenchymal stem cell (MSC) dysfunction has been implicated in the biology of the acute myeloid leukemia (AML). Preliminary studies from our laboratory have demonstrated that MSCs derived from AML patients have diminished T cell immunosuppressive capacity, and these MSCs demonstrate signs of transformation. In the current study, we seek to explore the mechanisms of MSC dysfunction in patients with AML. Recent reports have implicated migration inhibitory factor (MIF) and its receptor CD74 in cancer progression. CD74 is a cell surface glycoprotein that also serves as the invariant chain for the class II major histocompatibility complex (MHC-II). The significance of these studies is two-fold: (1) CD74 participates in intracellular signal transduction via NF-kappaB, ERK, and PI(3)K pathways, thereby promoting cell proliferation, and (2) MHC-II invariant chain expression in MSCs may prevent tumor antigen loading and presentation to T cells, allowing for immune subversion. Our results show increased expression of CD74 on MSCs derived from AML patients. The readily targetable phenotype and the availability of anti-CD74 antibody milatuzumab merits further investigation into the role of CD74 in AML biology. Disclosures:No relevant conflicts of interest to declare.

The anti-CD74 humanized monoclonal antibody, milatuzumab, which targets the invariant chain of MHC II complexes, alters B-cell proliferation, migration, and adhesion molecule expression

IntroductionTargeting CD74 as the invariant chain of major histocompatibility complexes (MHC) became possible by the availability of a specific humanized monoclonal antibody, milatuzumab, which is under investigation in patients with hematological neoplasms. CD74 has been reported to regulate chemo-attractant migration of macrophages and dendritic cells, while the role of CD74 on peripheral naïve and memory B cells also expressing CD74 remains unknown. Therefore, the current study addressed the influence of milatuzumab on B-cell proliferation, chemo-attractant migration, and adhesion molecule expression.MethodsSurface expression of CD74 on CD27- naïve and CD27+ memory B cells as well as other peripheral blood mononuclear cells (PBMCs) obtained from normals, including the co-expression of CD44, CXCR4, and the adhesion molecules CD62L, β7-integrin, β1-integrin and CD9 were studied after binding of milatuzumab using multicolor flow cytometry. The influence of the antibody on B-cell proliferation and migration was analyzed in vitro in detail.ResultsIn addition to monocytes, milatuzumab also specifically bound to human peripheral B cells, with a higher intensity on CD27+ memory versus CD27- naïve B cells. The antibody reduced B-cell proliferation significantly but moderately, induced enhanced spontaneous and CXCL12-dependent migration together with changes in the expression of adhesion molecules, CD44, β7-integrin and CD62L, mainly of CD27- naïve B cells. This was independent of macrophage migration-inhibitory factor as a ligand of CD74/CD44 complexes.ConclusionsMilatuzumab leads to modestly reduced proliferation, alterations in migration, and adhesion molecule expression preferentially of CD27- naïve B cells. It thus may be a candidate antibody for the autoimmune disease therapy by modifying B cell functions.

Role for CD74 and CXCR4 in clathrin-dependent endocytosis of the cytokine MIF

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that plays a role in innate and adaptive immunity. Depending on the cellular context and disease state, MIF signaling is mediated by its receptors CXCR2, CXCR4 and/or CD74. Although it is known that MIF is endocytosed, the exact mechanism has remained unknown. In exploring the mechanism of MIF endocytosis with biologically active Alexa546MIF, pathway-specific inhibitors (monodansylcadaverine, MDC; chlorpromazine, CPZ; dynasore; dominant-negative dynamin, bafilomycin, nocodazole) and receptor overexpression and blockade approaches, we identified a clathrin/dynamin-dependent endocytosis pathway as the main track for MIF internalization. MIF endocytosis was rapid and colocalization with both early and late endosomal vesicles in a microtubule- and acidification-dependent manner was observed. LDL endocytosis (which is clathrin-mediated) served as a control and was similarly inhibited by MDC or dynasore. When MIF endocytosis was compared to that of transferrin, acetylated LDL, and choleratoxin B (the latter internalized by a clathrin-independent pathway) by colocalization studies, the MIF internalization pathway clearly resembled that of LDL but also shared early trafficking with transferrin, whereas no colocalization with choleratoxin was noted. To identify the receptors involved in MIF endocytosis, we focused on CD74 and CXCR4 which form a heteromeric complex. Ectopic overexpression of CD74 in HEK293 and HeLa cells, which do not endogenously express CD74, led to a marked acceleration of MIF endocytosis while pharmacological blockade of CXCR4, which is endogenously expressed on these cells, with AMD3100 led to a 20% reduction of MIF endocytosis in HEK293-CD74 transfectants, whereas in untransfected cells, a blockade of 40% was observed. Of note, both CD74 and CXCR4 strongly colocalize with Alexa546MIF both on the plasma membrane and in endosomal compartments. Moreover, MIF-stimulated AKT signaling, which was previously shown to involve both CD74 and CXCR4, was reduced by endocytosis inhibitors, indicating that MIF signaling is at least in part due to endosomal signaling mechanisms. Thus, MIF uptake follows a rapid LDL-like, clathrin- and dynamin-dependent endocytosis pathway, which is dependent on the receptors CD74 and CXCR4 and leads to the initiation of endosomal signaling responses.

Macrophage Migration Inhibitory Factor and CD74 Regulate Macrophage Chemotactic Responses via MAPK and Rho GTPase

Macrophage migration inhibitory factor (MIF) promotes leukocyte recruitment to sites of inflammation. However, whether this stems from a direct effect on leukocyte migration is unknown. Furthermore, the role of the MIF-binding protein CD74 in this response has not been investigated. Therefore, the aim of this study was to examine the contributions of MIF and CD74 to chemokine-induced macrophage recruitment. Intravital microscopy studies demonstrated that CCL2-induced leukocyte adhesion and transmigration were reduced in MIF(-/-) and CD74(-/-) mice. MIF(-/-) and CD74(-/-) macrophages also exhibited reduced chemotaxis in vitro, although CD74(-/-) macrophages showed increased chemokinesis. Reduced CCL2-induced migration was associated with attenuated MAPK phosphorylation, RhoA GTPase activity, and actin polymerization in MIF(-/-) and CD74(-/-) macrophages. Furthermore, in MIF(-/-) macrophages, MAPK phosphatase-1 was expressed at elevated levels, providing a potential mechanism for the reduction in MAPK phosphorylation in MIF-deficient cells. No increase in MAPK phosphatase-1 expression was observed in CD74(-/-) macrophages. In in vivo experiments assessing the link between MIF and CD74, combined administration of MIF and CCL2 increased leukocyte adhesion in both MIF(-/-) and CD74(-/-) mice, showing that CD74 was not required for this MIF-induced response. Additionally, although leukocyte recruitment induced by administration of MIF alone was reduced in CD74(-/-) mice, consistent with a role for CD74 in leukocyte recruitment induced by MIF, MIF-treated CD74(-/-) mice displayed residual leukocyte recruitment. These data demonstrate that MIF and CD74 play previously unappreciated roles in CCL2-induced macrophage adhesion and migration, and they indicate that MIF and CD74 mediate this effect via both common and independent mechanisms.

Optimal stimulation for CD70 induction on human monocyte‐derived dendritic cells and the importance of CD70 in naive CD4+ T‐cell differentiation

Studies in mice have shown that CD70 on dendritic cells (DCs) is sufficient to convert T-cell tolerance into immunity and hence induce anti-tumour immune responses. Therefore, it is important to investigate (i) optimal stimuli to induce CD70 on human monocyte-derived DCs (MoDCs), which are widely used for tumour immunotherapy, and (ii) the role of CD70 in functional differentiation of naive CD4(+) and CD8(+) T cells stimulated with MoDCs. We show that interferon-alpha (IFN-alpha) is a key cytokine to differentiate monocytes into DCs with the capacity to express CD70 upon maturation. CD70 expression on IFN-alpha-induced MoDCs was elicited by different categories of maturation-inducing factors (Toll-like receptor ligands, CD40 ligand and pro-inflammatory mediators), among which prostaglandin E(2) was most effective. Naive T cells stimulated with MoDCs also expressed CD70. Stimulation with MoDCs promoted naive CD4(+) T cells to acquire the ability to produce T helper type 1 and 2 cytokines in a CD70-dependent manner. In contrast, the CD70-CD27 interaction diminished the production of an immunoregulatory cytokine IL-10. The CD27 signal did not play a dominant role in the induction of effector molecules in naive CD8(+) T cells during the stimulation with MoDCs. This study adds a novel function to the versatile cytokines, type I IFNs, that is, the induction of CD70 on MoDCs. CD70 promotes naive CD4(+) T cells to acquire immunostimulatory activity through the DC-T-cell and T-cell-T-cell interactions during the stimulation with MoDCs. Hence, the CD70-CD27 interaction may play an important role in inducing effective immune responses in DC-based immunotherapy.

Milatuzumab – a promising new immunotherapeutic agent

Milatuzumab is a new immunotherapeutic agent targeting CD74, a membrane protein preferentially expressed in hematopoietic cancers and some solid tumors. Broad expression and fast internalization makes CD74 an ideal target for cancer therapy. We reviewed published articles about CD74 and milatuzumab. We present a comprehensive review of CD74 functions and provide explanation of milatuzumab antitumor effects. This review describes CD74 protein biology with the emphasis on the role of CD74 in tumor survival and its new role in regulation of the Fas death receptor. The development of CD74 targeting therapies to induce tumor regression and cancer cell apoptosis is described and results of clinical trials are discussed. Milatuzumab shows selective binding and rapid internalization into CD74-positive cancer cells. Milatuzumab with and without conjugated toxins synergizes with other chemotherapeutic agents and elicits significant antitumor effects in mice. In a Phase I trial, milatuzumab showed no severe adverse effects in patients with relapsed/refractory multiple myeloma and it stabilized the disease in some patients for up to 12 weeks. Ongoing trials testing different treatment schedules of milatuzumab in chronic lymphocytic leukemia, non-Hodgkin's lymphoma and multiple myeloma indicate that milatuzumab shows no severe adverse effects in humans.

CD74 in antigen presentation, inflammation, and cancers of the gastrointestinal tract

CD74 is a protein whose initial role in antigen presentation was recognized two decades ago. Recent studies have revealed that it has additional functions as a receptor for macrophage migration inhibitory factor and as a receptor for an important human pathogen, Helicobacter pylori (H pylori). The role of CD74 as a receptor is important because after binding of migration inhibitory factor or H pylori, NF-kappaB and Erk1/2 activation occurs, along with the induction of proinflammatory cytokine secretion. This review provides an up-to-date account of the functions of CD74 and how it might be involved in inflammation and cancer within the gastrointestinal tract.

The CD70/CD27 Pathway Is Critical for Stimulation of an Effective Cytotoxic T Cell Response against B Cell Precursor Acute Lymphoblastic Leukemia

For effective immunotherapy, maintaining the frequency and cytotoxic potential of effector cells is critical. In this context costimulation via the CD70/CD27 pathway has been proven essential. CD70 has been reported to be expressed to varying degrees on malignant B cells. However, in B cell precursor acute lymphoblastic leukemia, the most common childhood malignancy, the role of CD70 in stimulation of antileukemic T cell responses has so far not been delineated. Herein we demonstrate that in B cell precursor acute lymphoblastic leukemia expression of CD70 is low but can be induced upon blast activation via CD40. Both CD70 and CD80/CD86 up-regulated on CD40-stimulated blasts contribute to primary stimulation of T cell proliferation and cytokine production in an additive manner. These two signals also cooperate in the prevention of T cell anergy. In contrast to blockade of CD70 during the effector phase, inhibition of CD70-mediated costimulation during generation of antileukemic T cells prevents effector cell proliferation and reduces their cytotoxic capacity. Modulation of the CD70/CD27 pathway may thus represent a novel therapeutic approach for augmenting magnitude and quality of the antileukemic response in B cell precursor acute lymphoblastic leukemia.

CD74 Is a Regulator of Fas-Mediated Apoptotic Signaling

Resistance to Fas-mediated apoptosis in hematopoietic cancers interferes with the efficacy of currently available chemotherapy. Our prior studies of human herpesvirus 8 oncoprotein K1 showed that K1 binds to Fas and interferes with activation of Fas-mediated apoptotic signaling (Wang, W, et al, Blood 2007; 109:5455–62). Herpesvirus proteins often mimic host proteins or their functions, we thus searched for cellular proteins associated with inactive Fas in order to identify potential regulators of Fas signaling. We identified CD74, the invariant light chain of the major histocompatibility class II complex, associated exclusively with inactive/activation resistant Fas in B-cell lymphoma-derived BJAB cell line. Interestingly, overexpression of CD74 has been previously reported in ~ 90% of hematopoietic cancers and derived cell lines (Stein, R, et al., Ciln Cancer Res. 2007; 13: 5556s–63s), as well as in ~ 80% of non-small-cell lung carcinoma (Ioachim, H.L., Am J Surg Pathol. 1996; 20: 64–71). The role of CD74 in carcinogenesis was thus suspected. Through siRNA suppression of CD74 expression in BJAB cells we have determined that CD74 positive cells are more resistant to agonistic antibody CH-11-induced Fas-mediated apoptosis then the CD74 siRNA transfected cells (38 ± 7.8 % vs. 54 ± 9.8 %; P = 0.045). In addition, a challenge with agonistic anti-Fas antibody showed a significant survival advantage of the mice expressing CD74 in their livers over the vector-transfected mice (83% vs. 0 %; P < 0.016). We have mapped domain of CD74 required for its association with Fas and for significant protection of mice to a membrane-proximal extracellular region of CD74. Treatment of BJAB cells for 24 hours with humanized anti-CD74 antibody (hLL1 + crosslinking antibody), FasL-Fc, or anti-Fas antibody CH-11 induced apoptosis in 19%, 25% and 13% of cells, respectively. Combination of hLL1+crosslinking antibody with FasL-Fc or CH-11 increased apoptosis to ~ 45% of cells in both cases (P < 0.01), while combination of FasL-Fc or CH-11 with hLL1 + irrelevant antibody had no significant effect on the extent of apoptosis. Our results support the idea of an endogenous regulatory system of Fas-mediated apoptosis that utilizes transmembrane proteins interacting with Fas. We anticipate that specific blocking of the CD74-Fas interaction will sensitize CD74 overexpressing cancer cells to Fas-mediated apoptosis and thus will lead to a more effective chemotherapy for hematopoietic cancers.

Evidence for the role of CD74 in innate immunity, arthritis, and the action of migration inhibitory factor

Recent studies have identified CD74, the cell surface form of the class II-associated invariant chain, as a binding site for the cytokine macrophage migration inhibitory factor (MIF). MIF is implicated in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus, but a functional relationship between MIF and CD74 in inflammation has not been demonstrated. We used CD74-/- mice to examine the role of this molecule in cellular and in vivo inflammatory responses, using innate immune response-dependent models to avoid confounding by the role of MHC Ii in adaptive immunity. MIF has a demonstrated role in macrophage responses to endotoxin. LPS induced IL-1, IL-6, TNF and MIF release from WT bone-marrow macrophages. Bone-marrow macrophages lacking CD74 released significantly lower amounts of IL-1 and IL-6, but no difference in TNF release was evident. LPS-induced MIF release by WT and CD74-/- bone-marrow macrophages was also similar. Murine K/BxN serum-transfer arthritis is MIF dependent [1], and also requires the induction of IL-1. Compared with WT mice, arthritis severity (clinical score) was significantly lower in mice lacking CD74 (P < 0.05). These data imply but do not demonstrate a role of CD74 in MIF-dependent inflammatory responses. To confirm the role of CD74 in responses to MIF in vivo, we examined a recently described action of MIF – the induction of leukocyte trafficking [2]. rhMIF was injected into mouse cremaster tissue and leukocyte–endothelial interactions examined using intravital microscopy. MIF induced leukocyte adhesion and emigration in vivo in WT mice. In CD74-/- mice, in contrast, this response could not be elucidated. These data demonstrate for the first time the role of CD74 in innate immune responses and inflammatory arthritis, and demonstrate for the first time in vivo the requirement for CD74 for the action of MIF. A functional role for CD74 in MIF responses in inflammation is strongly supported.

Immune Escape for Renal Cell Carcinoma: CD70 Mediates Apoptosis in Lymphocytes

Tumors can escape immune recognition and destruction through the induction of apoptosis in lymphocytes. Although renal cell carcinoma (RCC) is able to prevent immune recognition, only a few genes (such as FasL) that are relevant for RCC immune escape have been identified so far. We have previously shown that some apoptosis-inducing genes are overexpressed in RCC. We hypothesized that these genes could be part of the immune-escape strategy of these tumors. Here we report that CD70, a cytokine overexpressed in RCC, promotes lymphocyte apoptosis through interaction with its receptor CD27 and with the intracellular receptor-binding protein SIVA. Apoptosis increased after cocultivating lymphocytes with the RCC cell lines A498 and CAKI2. The addition of recombinant soluble CD70 to both native lymphocytes and a T-cell cell line resulted in increased lymphocyte apoptosis as well. Furthermore, induced apoptosis could be partially blocked with anti-CD27 and anti-CD70 antibodies. Our results strongly indicate a role for CD70 and CD27 receptor in lymphocyte apoptosis within the tumor environment. Apoptosis mediated by exposure to the CD70 secreted by tumor cells may contribute to the failure of RCC patients to develop an effective lymphocyte-mediated antitumor response.

CD70 expression on T-cell subpopulations: study of normal individuals and patients with chronic immune activation

CD70, the ligand of CD27, is a member of the TNF family, which includes molecules essential in the regulation of lymphocyte growth and survival. It is absent on resting lymphocytes but can be induced in vitro with activating stimuli. To extend information about its expression by different T-cell subpopulations, and its regulation in normal and pathologic conditions, highly purified T-cell subpopulations were studied: CD70 expression depended both on the activating stimulus and on the T-cell subset analyzed. PMA + Ionomycin induced CD70 on the large majority of CD8 + cells, but only on a minority of CD4 + cells ( P < 0.002), and among these, preferentially on the CD45R0 + subset compared with the CD45RA +. The presence of CD4 + lymphocytes in cell cultures containing mixtures of T-cell subsets inhibited CD70 expression on CD8 + cells. On the contrary, stimulation with allogeneic cells induced CD70 expression also on CD4 + cells. Moreover, CD70 was found to be expressed by chronically in vivo activated T-cells, in conditions characterized by allogeneic and autoimmune reactions. These data suggest a possible role of CD70 in the pathogenesis of immune dysregulation; interestingly, this role may not be simply restricted to bind to, and signal through, CD27, since cross-linking of CD70 enhances the proliferative response induced by the stimuli used to elicit its expression.