Spontaneous tumor rejection by cbl-b-deficient CD8+ T cells

The concept of tumor surveillance implies that specific and nonspecific components of the immune system eliminate tumors in the early phase of malignancy. Understanding the biochemical mechanisms of tumor immunosurveillance is of paramount significance because it might allow one to specifically modulate spontaneous antitumor activity. We report that inactivation of the E3 ligase Casitas B cell lymphoma-b (Cbl-b) confers spontaneous in vivo rejection of tumor cells that express human papilloma virus antigens. Moreover, cbl-b(-/-) mice develop significantly fewer ultraviolet B (UVB)-induced skin malignancies and reject UVB-induced skin tumors. CD8(+) T cells were identified as key players in the spontaneous tumor rejection response. Loss of Cbl-b not only enhances antitumor reactivity of CD8(+) T cells but also occurs in the absence of CD4(+) T cells. Mechanistically, cbl-b(-/-) CD8(+) T cells are resistant to T regulatory cell-mediated suppression and exhibit enhanced activation and rapid tumor infiltration. Importantly, therapeutic transfer of naive cbl-b(-/-) CD8(+) T cells is sufficient to mediate rejection of established tumors. Even up to 1 yr after the first encounter with the tumor cells, cbl-b(-/-) mice carry an "anticancer memory." These data identify Cbl-b as a key signaling molecule that controls spontaneous antitumor activity of cytotoxic T cells in different cancer models. Inhibition of Cbl-b is a novel approach to stimulate long-lasting immunity against cancer.     

OX40 engagement and chemotherapy combination provides potent antitumor immunity with concomitant regulatory T cell apoptosis

Expansion and recruitment of CD4⁺ Foxp3⁺ regulatory T (T reg) cells are mechanisms used by growing tumors to evade immune elimination. In addition to expansion of effector T cells, successful therapeutic interventions may require reduction of T reg cells within the tumor microenvironment. We report that the combined use of the alkylating agent cyclophosphamide (CTX) and an agonist antibody targeting the co-stimulatory receptor OX40 (OX86) provides potent antitumor immunity capable of regressing established, poorly immunogenic B16 melanoma tumors. CTX administration resulted in tumor antigen release, which after OX86 treatment significantly enhanced the antitumor T cell response. We demonstrated that T reg cells are an important cellular target of the combination therapy. Paradoxically, the combination therapy led to an expansion of T reg cells in the periphery. In the tumor, however, the combination therapy induced a profound T reg cell depletion that was accompanied by an influx of effector CD8⁺ T cells leading to a favorable T effector/T reg cell ratio. Closer examination revealed that diminished intratumoral T reg cell levels resulted from hyperactivation and T reg cell–specific apoptosis. Thus, we propose that CTX and OX40 engagement represents a novel and rational chemoimmunotherapy.Clinically relevant cancers must establish efficient mechanisms of escaping destruction by the immune system. One of the most successful strategies of tumor-induced immune evasion is the recruitment, expansion, and activation of CD4⁺ Foxp3⁺ T reg cells (1, 2). T reg cells can potently inhibit immune responses and constitute a major barrier for eliciting effective antitumor immunity (3). Cancer patients suffering from a variety of malignancies have elevated numbers of tumor-associated T reg cells, and increased T reg cell levels correlate with poor prognosis (4–10).Not surprisingly, the inactivation or depletion of T reg cells has been actively pursued as means to develop more potent immune therapies. Depletion of T reg cells has been accomplished with alkylating agents such as cyclophosphamide (CTX) (11–17). As a chemotherapeutic drug, CTX is administered clinically at doses that directly kill tumor cells. However, immunologists have known for decades that CTX has significant effects on the immune system that can promote a favorable antitumor immune response (14, 18).In addition to specific effects of CTX on T reg cells, lymphocytes recovering from CTX-induced lymphopenia undergo homeostatic proliferation that can activate tumor-specific T cells (19). Reconstitution from CTX-induced lymphopenia is associated with elevated levels of numerous proinflammatory cytokines, which favors an antitumor immune response (20, 21). Moreover, direct tumor cell death can be associated with tissue necrosis and the release of danger signals that enhance tumor antigen cross-presentation and cross-priming (22, 23). To increase antitumor immune responses, CTX has been successfully combined with vaccination and adoptive immunotherapy strategies (chemoimmunotherapy) in multiple modalities and tumor models (24, 25).OX40 is a co-stimulatory molecule and member of the TNFR family constitutively expressed on T reg cells and inducibly expressed by effector CD4⁺ T cells upon activation (26). Signaling through OX40 up-regulates the expression of antiapoptotic Bcl-2 family members, Bcl-2 and Bcl-X_L, leading to increased clonal expansion and memory responses (17). OX40 signaling can also provide co-stimulation to activated CD8⁺ T, NK, and NKT cells (27–30).Although the function of OX40 has been established for effector CD4⁺ and CD8⁺ T cells, the biological role that OX40 plays on T reg cells is still controversial. Early reports have shown that OX40 ligation is crucial for T reg cell homeostasis (31). Young mice deficient in OX40 have reduced levels of CD4⁺ CD25⁺ T reg cells, and mice overexpressing OX40L possess elevated levels in the spleen and thymus. However, engaging OX40 can abrogate the T reg cell suppressive function and down-regulate Foxp3 expression (31–34). Additionally, it has been reported that OX40 ligation prevents the conversion of naive CD4⁺ T cells into T reg cells induced by TGF-β and antigen (33, 35).Given that OX40 engagement can potently stimulate T cells and potentially inhibit T reg cells, it has been successfully used in the treatment of a variety of transplantable tumors in mice (34, 36). Overexpression of OX40L in tumor cell lines and dendritic cells induced substantial antitumor immunity (37–39). The triggering of OX40 with recombinant soluble OX40L or the anti-OX40 agonist monoclonal antibody OX86 are additional strategies proven to be effective in treating immunogenic tumors (40–45). However, targeting OX40 alone or in combination with other therapeutic approaches has only marginal effects on less immunogenic, more clinically relevant mouse tumors.We hypothesized that OX40 ligation after administration of CTX would provide strong antitumor immunity. Indeed, we found that OX86 in combination with CTX synergized to mediate the regression of established, B16 mouse melanoma tumors. We demonstrate that CTX treatment resulted in the release of tumor antigens capable of priming effector CD4⁺ and CD8⁺ T cells, and that OX40 engagement amplified these responses. Furthermore, we show that the combination therapy targets T reg cells, inducing previously uncharacterized changes in this population. Unexpectedly, OX86 administration increased the number of T reg cells in the periphery, an effect that was exacerbated when OX86 was administered with CTX. In contrast, the combination therapy significantly reduced the number of T reg cells infiltrating the tumor, with a substantial, increased infiltration of effector CD8⁺ T cells. Closer mechanistic examination revealed that the combination therapy resulted in T reg cell–specific apoptosis, which accounted for the reduced intratumoral infiltration. Thus, we report a potent, clinically translatable chemoimmunotherapy and provide insight into a novel mechanism that involves elimination of T reg cells in the tumor microenvironment.     

Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells

Although CD8 T cell-mediated immunosuppression has been a well-known phenomenon during the last three decades, the nature of primary CD8 T suppressor cells and the mechanism underlying their generation remain enigmatic. We demonstrated that naive CD8 T cells primed with allogeneic CD40 ligand-activated plasmacytoid dendritic cells (DC)2 differentiated into CD8 T cells that displayed poor secondary proliferative and cytolytic responses. By contrast, naive CD8 T cells primed with allogeneic CD40 ligand-activated monocyte-derived DCs (DC1) differentiated into CD8 T cells, which proliferated to secondary stimulation and killed allogeneic target cells. Unlike DC1-primed CD8 T cells that produced large amounts of interferon (IFN)-gamma upon restimulation, DC2-primed CD8 T cells produced significant amounts of interleukin (IL)-10, low IFN-gamma, and no IL-4, IL-5, nor transforming growth factor (TGF)-beta. The addition of anti-IL-10-neutralizing monoclonal antibodies during DC2 and CD8 T cell coculture, completely blocked the generation of IL-10-producing anergic CD8 T cells. IL-10-producing CD8 T cells strongly inhibit the allospecific proliferation of naive CD8 T cells to monocytes, and mature and immature DCs. This inhibition was mediated by IL-10, but not by TGF-beta. IL-10-producing CD8 T cells could inhibit the bystander proliferation of naive CD8 T cells, provided that they were restimulated nearby to produce IL-10. IL-10-producing CD8 T cells could not inhibit the proliferation of DC1-preactivated effector T cells. This study demonstrates that IL-10-producing CD8 T cells are regulatory T cells, which provides a cellular basis for the phenomenon of CD8 T cell-mediated immunosuppression and suggests a role for plasmacytoid DC2 in immunological tolerance.     

Sustained suppression by Foxp3+ regulatory T cells is vital for infectious transplantation tolerance

A paradigm shift in immunology has been the recent discovery of regulatory T cells (T reg cells), of which CD4(+)Foxp3(+) cells are proven as essential to self-tolerance. Using transgenic B6.Foxp3(hCD2) mice to isolate and ablate Foxp3(+) T reg cells with an anti-hCD2 antibody, we show for the first time that CD4(+)Foxp3(+) cells are crucial for infectious tolerance induced by nonablative anti-T cell antibodies. In tolerant animals, Foxp3(+) T reg cells are constantly required to suppress effector T cells still capable of causing tissue damage. Tolerated tissue contains T cells that are capable of rejecting it, but are prevented from doing so by therapeutically induced Foxp3(+) T reg cells. Finally, Foxp3(+) cells have been confirmed as the critical missing link through which infectious tolerance operates in vivo. Peripherally induced Foxp3(+) cells sustain tolerance by converting naive T cells into the next generation of Foxp3(+) cells. Empowering Foxp3(+) regulatory T cells in vivo offers a tractable route to avoid and correct tissue immunopathology.     

TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand

We recently showed that dendritic cells (DCs) activated by thymic stromal lymphopoietin (TSLP) prime naive CD4(+) T cells to differentiate into T helper type 2 (Th2) cells that produced high amounts of tumor necrosis factor-alpha (TNF-alpha), but no interleukin (IL)-10. Here we report that TSLP induced human DCs to express OX40 ligand (OX40L) but not IL-12. TSLP-induced OX40L on DCs was required for triggering naive CD4(+) T cells to produce IL-4, -5, and -13. We further revealed the following three novel functional properties of OX40L: (a) OX40L selectively promoted TNF-alpha, but inhibited IL-10 production in developing Th2 cells; (b) OX40L lost the ability to polarize Th2 cells in the presence of IL-12; and (c) OX40L exacerbated IL-12-induced Th1 cell inflammation by promoting TNF-alpha, while inhibiting IL-10. We conclude that OX40L on TSLP-activated DCs triggers Th2 cell polarization in the absence of IL-12, and propose that OX40L can switch IL-10-producing regulatory Th cell responses into TNF-alpha-producing inflammatory Th cell responses.     

Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression

The study of T regulatory cells (T reg cells) has been limited by the lack of specific surface markers and an inability to define mechanisms of suppression. We show that the expression of CD39/ENTPD1 in concert with CD73/ecto-5'-nucleotidase distinguishes CD4(+)/CD25(+)/Foxp3(+) T reg cells from other T cells. These ectoenzymes generate pericellular adenosine from extracellular nucleotides. The coordinated expression of CD39/CD73 on T reg cells and the adenosine A2A receptor on activated T effector cells generates immunosuppressive loops, indicating roles in the inhibitory function of T reg cells. Consequently, T reg cells from Cd39-null mice show impaired suppressive properties in vitro and fail to block allograft rejection in vivo. We conclude that CD39 and CD73 are surface markers of T reg cells that impart a specific biochemical signature characterized by adenosine generation that has functional relevance for cellular immunoregulation.     

OX40 (CD134) controls memory T helper 2 cells that drive lung inflammation

Asthma is caused by memory Th2 cells that often arise early in life and persist after repeated encounters with allergen. Although much is known regarding how Th2 cells develop, there is little information about the molecules that regulate memory Th2 cells after they have formed. Here we show that the costimulatory molecule OX40 is expressed on memory CD4 cells. In already sensitized animals, blocking OX40-OX40L interactions at the time of inhalation of aerosolized antigen suppressed memory effector accumulation in lung draining lymph nodes and lung, and prevented eosinophilia, airway hyperreactivity, mucus secretion, and Th2 cyto-kine production. Demonstrating that OX40 signals directly regulate memory T cells, antigen-experienced OX40-deficient T cells were found to divide initially but could not survive and accumulate in large numbers after antigen rechallenge. Thus, OX40-OX40L interactions are pivotal to the efficiency of recall responses regulated by memory Th2 cells.     

The human OX40/gp34 system directly mediates adhesion of activated T cells to vascular endothelial cells

Fresh leukemic cells from patients with adult T cell leukemia (ATL) and some ATL-derived T cell lines show adhesion to human umbilical vein endothelial cells (HUVECs) mainly through E-selectin, but a proportion of this binding remains unaffected by the addition of combinations of antibodies against known adhesion molecules. By immunizing mice with one of such cell lines, we established monoclonal antibodies (mAbs), termed 131 and 315, that recognize a single cell surface antigen (Ag) and inhibit the remaining pathway of the adhesion. These mAbs did not react with normal resting peripheral blood mononuclear cells (PBMC) or most of the cell lines tested except for two other human T cell leukemia virus type I (HTLV-I)-infected T cell lines. After stimulation with phytohemagglutinin (PHA), PBMC expressed Ag 131/315 transiently, indicating that these mAbs define a T cell activation Ag. Western blotting and immunoprecipitation revealed that Ag 131/315 has an apparent molecular mass of 50 kD. Expression cloning was done by transient expression in COS-7 cells and immunological selection to isolate a cDNA clone encoding Ag 131/315. Sequence analysis of the cDNA indicated that it is identical to human OX40, a member of the tumor necrosis factor/nerve growth factor receptor family. We then found that gp34, the ligand of OX40, was expressed on HUVECs and other types of vascular endothelial cells. Furthermore, it was shown that the adhesion of CD4+ cells of PHA-stimulated PBMC to unstimulated HUVECs was considerably inhibited by either 131 or 315. Finally, OX40 transfectants of Kit 225, a human interleukin 2-dependent T cell line, were bound specifically to gp34 transfectants of MMCE, a mouse epithelial cell line, and this binding was blocked by either 315 or 5A8, an anti-gp34 mAb. These results indicate that the OX40/gp34 system directly mediates adhesion of activated T cells or OX40+-transformed T cells to vascular endothelial cells.     

Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells

Concomitant tumor immunity describes immune responses in a host with a progressive tumor that rejects the same tumor at a remote site. In this work, concomitant tumor immunity was investigated in mice bearing poorly immunogenic B16 melanoma. Progression of B16 tumors did not spontaneously elicit concomitant immunity. However, depletion of CD4(+) T cells in tumor-bearing mice resulted in CD8(+) T cell-mediated rejection of challenge tumors given on day 6. Concomitant immunity was also elicited by treatment with cyclophosphamide or DTA-1 monoclonal antibody against the glucocorticoid-induced tumor necrosis factor receptor. Immunity elicited by B16 melanoma cross-reacted with a distinct syngeneic melanoma, but not with nonmelanoma tumors. Furthermore, CD8(+) T cells from mice with concomitant immunity specifically responded to major histocompatibility complex class I-restricted epitopes of two melanocyte differentiation antigens. RAG1(-/-) mice adoptively transferred with CD8(+) and CD4(+) T cells lacking the CD4(+)CD25(+) compartment mounted robust concomitant immunity, which was suppressed by readdition of CD4(+)CD25(+) cells. Naturally occurring CD4(+)CD25(+) T cells efficiently suppressed concomitant immunity mediated by previously activated CD8(+) T cells, demonstrating that precursor regulatory T cells in naive hosts give rise to effective suppressors. These results show that regulatory T cells are the major regulators of concomitant tumor immunity against this weakly immunogenic tumor.     

T regulatory cell chemokine production mediates pathogenic T cell attraction and suppression

T regulatory cells (Tregs) control immune homeostasis by preventing inappropriate responses to self and nonharmful foreign antigens. Tregs use multiple mechanisms to control immune responses, all of which require these cells to be near their targets of suppression; however, it is not known how Treg-to-target proximity is controlled. Here, we found that Tregs attract CD4⁺ and CD8⁺ T cells by producing chemokines. Specifically, Tregs produced both CCL3 and CCL4 in response to stimulation, and production of these chemokines was critical for migration of target T cells, as Tregs from Ccl3^–/– mice, which are also deficient for CCL4 production, did not promote migration. Moreover, CCR5 expression by target T cells was required for migration of these cells to supernatants conditioned by Tregs. Tregs deficient for expression of CCL3 and CCL4 were impaired in their ability to suppress experimental autoimmune encephalomyelitis or islet allograft rejection in murine models. Moreover, Tregs from subjects with established type 1 diabetes were impaired in their ability to produce CCL3 and CCL4. Together, these results demonstrate a previously unappreciated facet of Treg function and suggest that chemokine secretion by Tregs is a fundamental aspect of their therapeutic effect in autoimmunity and transplantation.     

The T cell-B cell interaction via OX40-OX40L is necessary for the T cell-dependent humoral immune response

Recent in vitro studies have established that activated B cells express OX40 ligand (L), a member of the tumor necrosis factor/nerve growth factor family of cytokines, and become stimulated to proliferate and secrete immunoglobulin (Ig) after cross-linking of OX40L by its counterreceptor OX40, which is expressed on activated T cells. In the present study we investigated the in vivo role of this receptor-ligand pair for the interaction of T and B cells in the course of the T- dependent B cell response against 2,4,6 trinitro-phenyl-keyhole limpet hemocyanin. First, we showed that OX40 is maximally expressed by T cells in the periarteriolar lymphoid sheath (PALS) 3 d after primary immunization. These OX40+ cells are located in close proximity to antigen-specific, activated B cells. Second, we demonstrated that blocking of OX40-OX40L interaction with polyclonal anti-OX40 antibody or with antibodies against certain peptide sequences within its extracellular domain resulted in a profound decrease of the anti-hapten IgG response, whereas the antihapten IgM response was grossly unchanged. Third, we showed that this antibody treatment leads to an inhibition of the development of PALS-associated B cell foci, whereas the formation of germinal centers remained intact. Finally, our data suggest that, whereas B cell memory development was not impaired by anti-OX40 administration, OX40-OX40L interaction seems to be crucial in the secondary immune response. We conclude from these data that the OX40-OX40L interaction in vivo is necessary for the differentiation of activated B cells into highly Ig-producing cells, but is not involved in other pathways of antigen-driven B cell differentiation such as memory cell development in the germinal centers.     

Control of homeostatic proliferation by regulatory T cells

Homeostatic proliferation of T cells leads to the generation of effector/memory cells, which have the potential to cause harm to the host. The role of Tregs in the control of homeostatic proliferation is unclear. In this study we utilized mice that either harbor or lack Tregs as recipients of monoclonal or polyclonal T cells. We observed that while Tregs completely prevented cell division of T cells displaying low affinity for self ligands, they had a less marked, albeit significant, effect on cell cycle entry of T cells displaying higher affinity. The presence of Tregs resulted in a lower accumulation of T cells, enhanced apoptosis, and impaired differentiation to a cytokine-producing state. We conclude that Tregs play a major role in the control of homeostatic proliferation.     

CD4+CD25+ regulatory T cells suppress allograft rejection mediated by memory CD8+ T cells via a CD30-dependent mechanism

CD4(+)CD25(+) regulatory T (Treg) cells suppress naive T cell responses, prevent autoimmunity, and delay allograft rejection. It is not known, however, whether Treg cells suppress allograft rejection mediated by memory T cells, as the latter mount faster and stronger immune responses than their naive counterparts. Here we show that antigen-induced, but not naive, Treg cells suppress allograft rejection mediated by memory CD8(+) T cells. Suppression was allospecific, as Treg cells induced by third-party antigens did not delay allograft rejection. In vivo and in vitro analyses revealed that the apoptosis of allospecific memory CD8(+) T cells is significantly increased in the presence of antigen-induced Treg cells, while their proliferation remains unaffected. Importantly, neither suppression of allograft rejection nor enhanced apoptosis of memory CD8(+) T cells was observed when Treg cells lacked CD30 or when CD30 ligand-CD30 interaction was blocked with anti-CD30 ligand Ab. This study therefore provides direct evidence that pathogenic memory T cells are amenable to suppression in an antigen-specific manner and identifies CD30 as a molecule that is critical for the regulation of memory T cell responses.     

OX40 signaling directly triggers the antitumor effects of NKT cells

Pathways involving the costimulatory molecule OX40 and OX40 ligand (OX40L) enhance tumor rejection. It was presumed that this effect was mediated by changes in DCs and/or T cells. In this issue of the JCI, Zaini et al. report that, in mice, intratumoral injection of DCs genetically modified to express OX40L suppressed the growth of a preexisting melanoma by directly triggering an antitumor NKT cell response (see the related article beginning on page 3330). This work suggests that the intratumoral NKT cell population may be harnessed for cancer immunotherapy and that OX40 costimulation may be used as a unique trigger of the antitumor activity of these cells.     

小分子干扰RNA抑制293T细胞中外源基因OX40的表达

目的研究小分子干扰RNA（siRNA）抑制293T细胞中OX40基因表达的可行性。方法根据大鼠OX40 cDNA已知序列，设计并化学合成一条含19个核苷酸siRNA。将此siRNA包装入质粒载体后，转化质粒到大肠杆菌中，经克隆，扩增，纯化。应用Lipofectamine 2000将OX40基因表达载体与其相应siRNA（OX40-siRNA）质粒共转染293T细胞，观察siRNA载体对OX40基因的抑制效应。在转染后48h用实时定量PCR从基因转录水平检测OX40基因的表达率，并以western blot在蛋白水平检查293T细胞中OX40蛋白的表达水平。结果siRNA转染293T细胞28h后，OX40靶mRNA及OX40蛋白的表达水平明显下降；且siRNA所介导的抑制效应与干扰载体剂量大小有关。结论siRNA能抑制293T细胞中OX40基因表达，从而为siRNA作为一种可能的治疗手段提供了理论依据。     

Limited tumor infiltration by activated T effector cells restricts the therapeutic activity of regulatory T cell depletion against established melanoma

Interference with inhibitory immunological checkpoints controlling T cell activation provides new opportunities to augment cancer immunotherapies. Whereas cytotoxic T lymphocyte–associated antigen-4 blockade has shown promising preclinical and clinical results, therapeutic CD4⁺CD25⁺ T reg cell depletion has failed to consistently enhance immune-based therapies. Using B16/BL6, a transplantable murine melanoma model, we show a dichotomy between the effects of T reg cell depletion on tumor rejection dependent on whether depletion occurs before (prophylactic) or after (therapeutic) tumor engraftment. Failure to promote rejection with therapeutic depletion is not related to lack of T reg cell depletion, to elimination of CD25⁺ effector T cells, or to a failure to enhance systemic antitumor T cell responses, but correlates with failure of effector cells to infiltrate the tumor and increase the intratumor ratio of effector T cell/T reg cell. Finally, systemic antitumor responses generated upon therapeutic T reg cell depletion are significantly stronger than those generated in the presence of T reg cells, and are capable of eliciting rejection of established tumors after transfer into immunoablated recipients receiving combination immunotherapy. The data demonstrate a dissociation between measurable systemic responses and tumor rejection during CD25-directed T reg cell depletion, and suggest an alternative, clinically applicable strategy for the treatment of established tumors.