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Originally published online as doi:10.1189/jlb.1103586 on March 2, 2004

Published online before print March 2, 2004
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(Journal of Leukocyte Biology. 2004;75:962-972.)
© 2004 by Society for Leukocyte Biology

The generation of T cell memory: a review describing the molecular and cellular events following OX40 (CD134) engagement

Andrew D. Weinberg*,1, Dean E. Evans*, Colin Thalhofer*, Tom Shi* and Rodney A. Prell{dagger}

* Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, Providence Portland Medical Center, Oregon; and
{dagger} Cell Genesys, Foster City, California

1 Correspondence: Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, Providence Portland Medical Center, 4805 N.E. Glisan, Portland, OR 97213. E-mail: andrew.weinberg{at}providence.org


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ABSTRACT
 
OX40 (CD134), a membrane-bound member of the tumor necrosis factor-receptor superfamily, is expressed primarily on activated CD4+ T cells. Following engagement on the cell surface, OX40 delivers a costimulatory signal that leads to potent, proinflammatory effects. Engagement of OX40 during antigen (Ag)-specific stimulation of T cells leads to increased production of memory T cells, increased migration of Ag-specific T cells, enhanced cytokine production by effector T cells, and the ability to break peripheral T cell tolerance in vivo. Therefore, OX40 engagement in vivo could have important ramifications for the enhancement of vaccine strategies and inhibition of unwanted inflammation. This review summarizes the molecular and cellular events that occur following OX40 engagement during Ag-specific T cell activation.

Key Words: TNF-R • antigen-specific T cells • autoimmunity • tumor immunology


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INTRODUCTION
 
Effector CD4+ T cells are notorious for their production of large amounts of cytokines upon antigen (Ag) recognition and their death soon thereafter [1 ]. Memory T cells arise after Ag activation and are a product of a transition in which activated effector T cells return to a quiescent state [2 ]. The number of memory T cells that is generated is limited by the susceptibility of effector T cells to apoptosis and/or other mechanisms of T cell death [3 , 4 ]. Recently, it has been shown that engagement of OX40 on activated T cells during an inflammatory response can rescue effector T cells from peripheral deletion and leads to increased numbers of functional, memory T cells [5 6 7 ]. Blockade of OX40 engagement in vivo results in a down-regulation of inflammation in animal models for autoimmune disease, and increased OX40 engagement can be therapeutic in tumor-bearing mice [8 ]. When OX40/OX40 ligand interaction is blocked during clinical episodes of autoimmunity, inflammation is reduced, and clinical signs of disease improve [9 10 11 12 13 14 15 16 ]. Engagement of OX40, via an antibody (Ab) or soluble OX40 ligand (OX40L) in tumor-bearing mice, increases the number of animals that survive without evidence of cancer [8 , 17 18 19 20 21 ]. Identification of the cellular and molecular events that lead to the potent, biologic activity generated by OX40 signaling will help elucidate the mechanisms that lead to productive CD4+ T cell immunity and may allow for manipulation of T cells for future clinical benefit.

The original Ab to OX40 was produced at Oxford University (UK) by injecting mice with activated rat T cell blasts [22 ]. Subsequently, the rat OX40 cDNA was cloned and shown to be a member of the tumor necrosis factor-receptor (TNF-R) superfamily [23 ]. OX40 has a unique pattern of expression, which is for the most part restricted to lymphoid tissue [24 ] and in particular, activated but not resting CD4+ T cells [22 ]. OX40 expression on naïve T cells peaks 24–48 h after T cell receptor (TCR) engagement and returns to baseline 120 h later [25 ]. This transient expression of OX40 is observed in vitro and in vivo [25 , 26 ]. Effector T cells up-regulate OX40 expression more rapidly than naïve T cells; the majority expresses OX40 within 4 h of Ag stimulation [25 ]. OX40 is expressed by T cells found at the site of inflammation in autoimmunity and tumor animal models, and the OX40-expressing cells are enriched for the recently stimulated auto- or tumor Ag-specific T cells [18 , 27 , 28 ]. T cell expression of OX40 has also been described in a number of inflammatory conditions in humans [14 , 28 29 30 31 32 33 ], suggesting that manipulation of OX40 signaling in humans could have clinical benefits.

The OX40L, a member of the TNF family, is expressed on activated Ag-presenting cells (APC) including B cells, macrophages, endothelial cells, and dendritic cells (DCs) and is the only ligand known to bind OX40 [9 , 34 35 36 ]. The most noteworthy function of OX40L is engagement of OX40 on Ag-activated T cells, which leads to enhanced cytokine production and increased survival of Ag-specific memory T cells [5 , 7 , 25 ]. Besides its effect on T cells, engagement of OX40L also delivers a signal to the APC, which results in increased production of interleukin (IL)-12 by DCs and enhanced B cell differentiation [36 , 37 ]. As OX40L is expressed on APC at the site of inflammation during an immune response, reagents that bind OX40L and block OX40/OX40L interaction exhibit potent anti-inflammatory activity in vivo [9 10 11 12 13 14 15 16 ]. A recent article has also shown that blocking OX40/OX40L interaction in response to influenza virus greatly reduces lung inflammation while not affecting immune clearance of the virus [38 ]. This review will focus on the cellular and molecular events that result from OX40 engagement with its native ligand (OX40L) or an agonist anti-OX40 Ab and include speculation as to what intracellular signaling pathways are crucial for the proinflammatory effects that follow OX40 engagement.


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THE BIOLOGIC CONSEQUENCES OF OX40 ENGAGEMENT
 
The original OX40 monoclonal Ab (mAb) augmented T cell proliferation when it was present during the later stages of in vitro stimulation [22 ]. At the time of the original description of the biologic effects of the anti-OX40 Ab, the field of costimulation was in its infancy. During the late 1980s, the first costimulatory molecule described on T cells, CD28, was first shown to augment T cell stimulation when engaged in combination with a TCR signal [39 40 41 ]. B7.1 (CD80) and B7.2 (CD86), both of which are expressed on APC, were identified as the ligands for CD28 and were shown to stimulate T cells through CD28. The CD28/B7 interaction is required to achieve optimal activation of naïve T cells. A signal delivered through the TCR in the absence of CD28 ligation results in T cell anergy or premature T cell death [41 ]. Additional cell-surface molecules such as intercellular adhesion molecule (ICAM), 4-1BB (CD137), OX40, and others have been shown to be important for the costimulation of activated, effector T cells [42 ].

In vitro assays were developed to assess the costimulatory properties of agents that interact with T cell surface molecules in conjunction with TCR (anti-CD3) stimulation. Submitogenic doses of anti-CD3 were combined with increasing amounts of Ab to T cell proteins, e.g., CD28 or CD49d [43 , 44 ], and a dose-related increase in T cell proliferation was taken as evidence of costimulation. Another readout for this assay was an increase in IL-2 production [39 ]. An Ab to OX40 was compared directly to anti-CD28 in the costimulation assay described above. Anti-OX40 and anti-CD28 caused a significant increase in proliferation from an in vitro-cultured, Ag-specific T cell line that had been exposed to submitogenic doses of anti-CD3 [45 ]. A dose response analysis indicated that the CD28 and OX40 antibodies stimulated proliferation and increased IL-2 production with similar potency [45 ]. Similar data were obtained when OX40L was transfected into cell lines and combined with submitogenic levels of T cell stimulation [24 ]. Costimulation assays have since been refined to measure Ag-specific T cell responses when incubated in the presence of fibroblasts that express major histocompatibility complex (MHC) class II. Class II+ fibroblasts were transfected with one or more costimulatory ligands and were tested for their ability to activate naïve and effector T cells [46 ]. In one study, class II+ fibroblasts were transfected with the cDNAs for B7.1, OX40L, or both, and the ability of the different transfectants to stimulate naïve and effector T cells was compared [25 ]. B7.1-transfected but not OX40L-transfected fibroblasts were able to costimulate naïve T cells. However, fibroblasts that expressed B7.1 and OX40L provided the best costimulation of naïve T cells. Suprisingly, the OX40L-transfected fibroblasts were more efficient stimulators of effector T cells compared with B7.1+ fibroblasts, and the combination of B7.1 and OX40L expression was only slightly better than OX40L alone. It has been suggested that engagement of OX40 directs T cells toward a T helper cell type 2 (Th2) phenotype [47 48 49 ]. However, we and others have shown that OX40L-specific costimulation enhanced proliferative responses for Th1 and Th2 effector T cells, as well as enhanced production of Th1 and Th2 cytokines [25 , 50 ]. The data indicate that OX40-specific costimulation is important for the stimulation of Th1 and Th2 effector T cells and acts in concert with CD28 stimulation of naïve T cells. The costimulation of naive T cells appears to rely heavily on B7/CD28 interaction and OX40/OX40L expression is also dependent on prior engagement of CD28 [51 , 52 ].

The control point for OX40-dependent stimulation of T cells during an immune response appears to be at the level of OX40L expression on APC. OX40 is expressed on all CD4+ T cells after TCR engagement. The expression of OX40L, however, is more tightly regulated. When T cell activation via TCR engagement with peptide/MHC occurs in the absence of a strong adjuvant, the local expression of OX40L is minimal (D. E. Evans and A. D. Weinberg, unpublished observation). Therefore, in the absence of adjuvant, the Ag-stimulated T cells express OX40, but as OX40L-bearing APC are limiting. Thus, in this scenario the majority of OX40+ T cells will most likely never be engaged by the natural ligand. Evidence in support of this theory derives from two transgenic mouse models in which mice overexpress the OX40L [52 53 54 ]. OX40L transgenic mice immunized with Ag in alum exhibited increased numbers of CD4+ T cells within B cell follicles, and germinal centers in these mice contained threefold more T cells than germinal centers in wild-type (WT) mice [52 , 54 ]. This group concluded that increased expression of OX40L in vivo led to the expansion and survival of germinal center CD4+ T cells through engagement of OX40. The second group produced three separate OX40L transgenic mouse lines, each with a different level of expression. The transgenic mouse strains that expressed higher levels of OX40L had increased numbers of CD4 splenic lymphocytes and markedly enhanced Ag-specific recall responses [53 ]. In fact, the two transgenic mouse lines with the highest level of OX40L expression spontaneously developed autoimmune disease. All the data are consistent with the hypothesis that OX40 signaling in vivo is dependent on the level of OX40L available for binding to OX40.

The endogenous expression of OX40 and OX40L is important for the generation of long-lived memory T cells in vivo [7 , 50 ]. OX40 knockout (KO) mice develop fewer Ag-specific CD4 cells late in a primary response in vivo and consequently have ten- to 20-fold lower frequencies of surviving long-term, memory T cells [7 ]. OX40 KO mice also have a four- to five-fold reduction in the number of interferon-{gamma} (IFN-{gamma})-secreting CD4 T cells following lymphocytic choriomeningitis virus (LCMV) infection [55 ]. Mice that lacked OX40L developed lower frequencies of Ag-specific Th1 and Th2 recall responses following immunization, and delayed-type hypersensitivity responses were also severely diminished [50 , 56 ]. Defective Th2 responses in OX40L-deficient mice could be restored by the addition of OX40L-expressing B cells [57 ].

Ag-specific stimulation of T cells in vivo results in an early (days 3–5) increase in the number of Ag-specific cells, which is then followed by a sharp decline in the numbers of T cells, suggesting that most Ag-stimulated T cells do not survive the initial activation event [58 ]. In vitro data have shown that OX40 KO T cells are more susceptible to cell death during the later stages of proliferation following TCR engagement compared with their OX40-expressing WT T cell counterparts [51 , 59 ]. We and others hypothesized that OX40 stimulation might prolong T cell survival beyond the effector T cell stage and thereby serve to increase the number of memory T cells by inhibiting effector T cell death [60 ]. Initial in vivo experiments showed that OX40 stimulation [achieved with an agonist Ab or OX40L:immunoglobulin (Ig) fusion protein] in conjunction with a "danger" signal [lipopolysaccharide (LPS)] saved super-Ag-stimulated CD4+ T cells from peripheral deletion in vivo [5 ]. It has recently been reported that OX40 engagement can render T cell leukemias resistant to Fas-induced apoptosis [61 ], and this mechanism may be responsible for the survival of the super-Ag-stimulated T cells described above.

The effects of OX40 engagement on memory T cell survival in vivo have been evaluated following stimulation of T cells with soluble Ag (in the absence of adjuvant) to transgenic CD4+ T cells (DO11.10 model) [5 ]. The DO11.10 TCR transgenic model was used, as Ag (OVA)-specific memory T cells could be followed with the anti-idiotypic TCR Ab, KJ1-26. The number of Ag-specific T cells that survive in the spleen long-term (60 days postimmunization) was increased 15-fold in mice that received Ag plus anti-OX40 and 60-fold in mice receiving Ag, anti-OX40, and LPS compared with mice that received Ag alone [5 ]. OX40 engagement in vivo (via a mAb) also enhanced the generation of T cell memory in the presence of two different adjuvants (complete Freund’s adjuvant or alum with pertussis [7 ]). OX40 stimulation also induces a substantial increase in the number of Ag-specific T cells detected in the peripheral blood and other peripheral organs following immunization, suggesting that OX40 engagement increases peripheral migration [6 ]. This increase in circulating Ag-specific T cells was long-lived, as elevated levels of these peripheral blood T cells were maintained 196 days after immunization and OX40 treatment [62 ]. The long-lived memory T cells, generated as a consequence of OX40 engagement, were functionally competent, as evidenced by their production of cytokines ex vivo [6 ]. The transgenic T cell adoptive-transfer model also showed that OX40 engagement increased the levels of Ag-specific Ab produced after immunization [6 ]. These data suggest that engagement of OX40 alone could be a potent adjuvant for CD4 T cell survival, enhanced peripheral migration, and increased Ab production. The OX40-specific T cell effects following OX40 engagement described thus far are summarized in Figure 1 .



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  		Figure 1. OX40-specific function in CD4 T cell differentiation and survival. Naïve T cells require prior stimulation through CD28 to respond to OX40 engagement. Once T cells reach the effector T cell stage, they are more responsive to OX40 costimulation. If OX40 is not engaged, the effector T cells make cytokines, and the majority undergoes cell death (leaving behind a few surviving memory T cells). When OX40 is engaged, the effector T cells produce increased levels of cytokines (Th1 and Th2), undergo enhanced proliferation, increase the number of Ag-specific T cells that migrate throughout the body, and increase the number of memory T cells that survive long-term.

Another important biologic function is the ability of OX40 ligation to break peripheral T cell tolerance in vivo and in vitro [63 ]. T cells presented with Ag in the context of MHC without "proper" costimulation are rendered unresponsive/anergic upon subsequent encounter with Ag [41 , 64 ]. An example of this unresponsiveness can be seen when a host encounters Ag in a noninflammatory environment, such as intravenous administration of high-dose peptide (Ag) in saline [58 ]. Engagement of OX40 during the induction of T cell tolerance can prevent anergy. OX40 signaling can also break an existing state of tolerance in the CD4+ T cell compartment [63 ]. Another way to induce T cell tolerance is to expose T cells to Ag that are expressed in peripheral organs [65 , 66 ]. A study presented at the FASEB 2001 Autoimmunity Conference showed that engagement of OX40 overcomes tolerance induced by global expression of Ag (David C. Parker, Oregon Health Science University, Portland, OR, unpublished results). This is similar to a form of tolerance that has been described in cancer patients and murine tumor models, in which T cells from tumor-bearing hosts are hyporesponsive when they are stimulated by tumor Ag ex vivo [67 , 68 ]. Recently, we and others have reported that engagement of OX40 in tumor-bearing mice augments antitumor T cell responses, resulting in beneficial antitumor effects [17 18 19 20 21 , 69 ]. Hypothetically, the engagement of OX40 in tumor-bearing mice could lead to a reversal of the tolerizing effects that tumors have on T cells. Table 1 shows the OX40-specific, therapeutic effects in a variety of murine tumor models, which lead to improved, tumor-free survival [17 18 19 20 21 , 69 ]. Another study showed that engagement of OX40 enhanced eradication of lung and brain metastases following the adoptive transfer of tumor Ag-specific T cells [20 ]. All of the studies described thus far suggest that OX40 signaling can have a profound effect on the function and survival of CD4+ T cells, which in turn can have a positive influence on the function and longevity of Ag-specific CD8+ T cells [19 , 70 ]. The remainder of the review will attempt to dissect the intracellular pathways that mediate these OX40-specific biologic functions.


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  		Table 1. OX40-Mediated Tumor Therapy


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MOLECULAR MEDIATORS OF OX40 SIGNALING [TNF-R-ASSOCIATED FACTORS (TRAFs)]
 
As with most TNF-R family members that lack an intracytoplasmic death domain, OX40 signals through adaptor proteins referred to as TRAFs, which comprise a family of six genetically conserved adaptor proteins in mammals (TRAF1–6) [71 72 73 ]. Homologues of these proteins can be found in other multicellular organisms including Drosophila, Caenorhabditis elegans, and Dictyostelium discoideum. TRAFs have emerged as the major signal transducer/adaptor proteins for the TNF-R family members and also transduce signals for the IL-1R/Toll-like receptor superfamily [71 , 72 ]. The TRAF proteins are characterized by the presence of a novel TRAF domain at the C terminus, which consists of an {alpha}-helix domain followed by a conserved TRAF-C domain that is present in all six members. The "TRAF domain" plays an important role in the interactions with the cytoplasmic regions of the cell-surface receptors and also permits hetero- and homo-TRAF associations. The N-terminal portion of most TRAF proteins (except TRAF1) contains a RING finger and several zinc finger motifs, which are essential for downstream signaling of the TRAF molecules. Many of the downstream biologic effects appear to be mediated through the activation of the nuclear factor (NF)-{kappa}B and activated protein-1 transcription factor families [71 ], although there do appear to be additional TRAF-specific pathways that elicit different biologic functions that are still undefined.

Two manuscripts published in 1998 reported TRAF-specific binding to the cytoplasmic tail of OX40 [74 , 75 ]. The human OX40 cytoplasmic sequence bound TRAF1, -2, -3, and -5 [75 ], and murine OX40 bound TRAF1, -2, and -3 [74 ]. The OX40 consensus sequence for TRAF binding and signal transmission was reported to be amino acids 256–263 in the human and amino acids 256–262 in the mouse sequence. Both reports confirmed that TRAF2 binding to OX40 led to NF-{kappa}B activation, and TRAF3 binding appeared to inhibit NF-{kappa}B activation. A dominant-negative form of TRAF2 (TRAF2 DN), which does not express the RING finger domain (signaling domain), was shown to inhibit OX40-associated NF-{kappa}B activation [74 , 75 ]. Both studies described above used nonlymphoid cells for in vitro analysis. As OX40 expression is restricted primarily to T cells [76 ], our group performed additional studies in TRAF2 DN transgenic T cells to determine the in vivo significance of TRAF2 expressed in T cells stimulated via OX40 [62 ]. Mice that express the TRAF2 DN protein produce suboptimal CD4 and CD8 T cell responses after infection with the influenza virus [77 ]. This group also showed that TRAF2 DN protein expression inhibited 4-1BB (CD137) costimulatory activity [78 ], which is another TNF-R family member expressed on activated T cells [79 ]. To address the functional consequence of OX40/TRAF2 interaction(s) in CD4+ T cells, our laboratory set out to determine if a DN form of TRAF2 could interrupt the potent, proinflammatory effects mediated by OX40 signaling in vivo. To investigate the physiologic role of TRAF2 in OX40-mediated generation of Ag-specific memory T cells, we mated the OVA-specific TCR transgenic mice (DO11.10) to TRAF2 DN transgenic mice. The in vitro Ag-specific responses by naïve and effector CD4+ T cells as well as the induction and expression of OX40 were normal in the TRAF2 DN mice. However, Figure 2A shows that the expected OX40-specific increase in the number of Ag-specific T cells in the blood was dramatically reduced when the TRAF2 DN protein was expressed. The expected OX40-specific enhancement of long-term memory was also diminished in T cells that expressed the TRAF2 DN protein (in the blood, LN, and spleen) [62 ]. Furthermore, Ag-specific, day-3 effector T cells from the TRAF2 DN mice injected with Ag plus anti-OX40 secreted lower amounts of IFN-{gamma} compared with WT T cells (Fig. 2B) . Figure 2C shows that proliferation of draining LN, Ag-specific T cells stimulated with anti-OX40 from the TRAF2 DN mice was similar compared with WT T cells up to day 3 after immunization. However, there was a large OX40-induced increase in proliferation between days 3 and 4 in WT T cells stimulated, which was completely negated in the TRAF2-deficient T cells. These data suggest that TRAF2-dependent downstream signaling is necessary to generate OX40-enhanced effector T cell function, LN proliferation, peripheral T cell migration, and memory T cell survival. Not all OX40-enhanced T cell functions were negated by TRAF2 DN protein expression. Figure 2D shows that OX40 engagement in vivo dramatically increased the cell size after immunization (day 3), and this increase in cell size was only slightly inhibited by TRAF2 DN protein expression. We hypothesize that engagement of OX40 involves the integration of complex signaling events and adaptor proteins, which work in concert to enhance T cell function in vivo, but TRAF2 appears to be a critical signaling component for several OX40-specific functions leading to enhanced memory T cell generalized survival.



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  		Figure 2. TRAF2 DN protein expression inhibits the generation, effector function, and survival of Ag-specific T cells after OX40 engagement. One million TRAF2 DN+ (T2DN) OVA-specific DO11.10 T cells (detected/quantitated with the TCR-specific Ab, KJ-126; T2DN, squares) or T cells from littermate controls (WT, circles) were transferred into BALB/c mice. Two days later, mice were immunized subcutaneously with OVA and injected with rat Ig (open symbols) or anti-OX40 (filled symbols). (A) On the indicated days, peripheral blood lymphocytes (PBL) were isolated and stained with anti-CD4 and the KJ-126 mAb and were analyzed by flow cytometry. Each number represents the mean ± SE of five mice. (B) Three days after Ag injection, lymph nodes (LN; popliteal axillary and lateral axillary) were removed, lymphocytes were normalized for the number of KJ-126+ cells and restimulated in vitro with OVA peptide, and IFN-{gamma} was measured 48 h later. (C) Two, 3, and 4 days following immunization, the draining LN were removed, the cells were pooled and stained, and the percentage of CD4+/KJ-126+ T cells was measured. (D) The forward-scatter plot was derived from LN T cells from the day-3 time-point in C. Lymphocytes were double-stained with anti-CD4 and the KJ-126 mAb and examined by flow cytometry. The forward-scatter plot (a measure of cell size), shown in D, represents cells from each group that were gated on Ag-specific T cells (CD4+ and KJ-126+; reproduced with permission from the Journal of Immunology [62 ]).


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DOWNSTREAM MOLECULES INVOLVED IN MEDIATING THE OX40-SPECIFIC, BIOLOGIC EFFECTS
 
The most dramatic effect of signaling through OX40 during an ongoing immune response is the prevention of T cell-specific death, particularly during the effector T cell stage [5 ]. This function has been shown in vivo and in vitro and verified in OX40 KO mice [7 , 59 ]. In vitro analysis of T cells from OX40 KO mice revealed that Ag-induced proliferation and survival were decreased compared with WT T cells [51 ]. This decrease occurred primarily during the later phase of proliferation (days 5–12). Consequently, the recovery of T cells from OX40 KO mice was decreased four- to fivefold on day 12 after activation. Impaired survival of OX40 KO T cells in culture could be reversed by the addition of peptide inhibitors of caspases [51 ]. This suggests that one consequence of signaling through OX40 is the inhibition of caspase-mediated apoptosis. The authors reported this finding and speculated that the death of OX40 KO T cells was a result of "neglect", more specifically, a lack of cytokines or costimulation rather than apoptosis mediated by death receptors such as Fas. Cell death from neglect is in part mediated by the interplay of the proapoptotic members of the Bcl-2 family (Bax, Bad, and Bim) and their antiapoptotic counterparts (Bcl-xL and Bcl-2) [80 ]. Rogers et al. [51 ] found that levels of Bcl-2 and Bcl-xL proteins were higher in WT T cells that survived Ag stimulation than they were in the surviving OX40 KO T cells. No difference in Bax expression was observed in the two sets of surviving T cells. OX40 KO T cells, transduced with Bcl-xl or Bcl-2, survived longer in vitro compared with OX40 KO T cells transduced with a control virus. The Bcl-xL-tranduced OX40 KO T cells increased in numbers from day 4 to 8, and the Bcl-2-transduced T cells remained constant over that time period. These data suggest that Bcl-xL may be more important for the survival of CD4 T cells during the proliferative phase and ultimately in long-term survival of CD4 T cell, as recently suggested by Zhang et al. [81 ]. The authors did not address whether the Bcl family members were directly up-regulated via OX40 signaling or indirectly through OX40-mediated enhancement of cytokines and/or cytokine receptors. However, they did point out that NF-{kappa}B activation has been shown to directly influence transcriptional activity of the Bcl-2 family members [82 ].

The results by Rogers et al. [51 ] are intriguing and suggest that engagement of OX40 enhanced the lifespan of T cells through neglect-specific, antiapoptotic molecules. However, their hypothesis does not explain other OX40-specific effects, such as enhanced T cell cytokine production, early enhancement of Ag-specific T cell proliferation, and blockade of activation-induced cell death in vivo in a model shown to be Fas ligand-dependent (i.e., staphylococcal enterotoxin B-induced peripheral T cell deletion) [83 ]. Therefore, our group developed an approach to ascertain the in vivo effects of OX40 engagement on gene expression in Ag-stimulated T cells. We hypothesized that the dramatic effects delivered to T cells through OX40 engagement would translate to differences observed at the transcriptional level. DNA microarray analysis (Affymetrix murine GeneChips) was performed to evaluate mRNA sequences that were up- or down-regulated in Ag-specific T cells after OX40 engagement in vivo. Ag-specific CD4+ T cells were isolated from LN (>90% purity) 3 days after stimulation in vivo with soluble Ag and rat Ig or Ag and {alpha}OX40 (T cells were isolated from the DO11.10 TCR transgenic model described earlier; Fig. 2 ). The day-3 time-point was chosen, as several differences were observed in Ag-specific T cell function 3 days after OX40 engagement in vivo (see Fig. 2B and 2D ) [6 ]. As the greatest differences occurred on a cellular level between OX40-treated and control mice on day 3, we postulated the differences in gene expression would give us the greatest insight into critical, downstream mediators of OX40 signaling.

Table 2 lists known genes whose expression was consistently increased or decreased in Ag-specific T cells 3 days after anti-OX40 administration in vivo in three separate experiments. The expression of 44 genes was consistently increased and that of 112 genes was decreased greater than twofold upon OX40 engagement in vivo. These results provide a starting point to assess OX40-specific downstream function via gene products that were altered and will hopefully be verified in future studies. At the present time, two of the gene product differences have been verified via protein expression on Ag-specific T cells (IL-2R {alpha}-chain [6 ] and CTLA-4 [62 ]). Several genes of immunologic interest were down-regulated upon OX40 engagement, including Mad4, CD27, L-selectin, Fas, and CTLA-4. A 13-fold decrease in the expression of the transcriptional repressor Mad4 was observed. Mad4 associates with the c-myc-interacting protein Max. The Mad/Max complex has been shown to suppress transcription initiated by c-myc [84 85 86 ]. Therefore, the OX40-stimulated T cells should have increased c-myc activity, which is associated with increased cell-cycle progression (proliferation) and an increase in cell size [86 ]. Both of these outcomes were observed when LN T cells were stimulated through OX40 (Fig. 2C and 2D) . Another consequence of c-myc-specific effects on transcription is an increase in apoptotic cell death, which is the opposite of what was observed in OX40-stimulated T cells [86 ]. The increase in T cell survival following OX40 engagement, however, may be explained in part by OX40-specific down-regulation of Fas mRNA, which is associated with apoptosis. At this early time point (day 3 after Ag stimulation), no changes in Bcl-2 or Bcl-xL transcript levels were observed; however, the later time points may show differences.


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  		Table 2. Genes Whose Expression Was Increased or Decreased Twofold or Greater by Anti-OX40 Versus Control (Rat Ig)

Another immune regulatory gene product that was consistently down-regulated in OX40-stimulated T cells was CTLA-4, a result that was confirmed by measuring protein surface expression (see Fig. 3A ). CTLA-4 is associated with putting the proliferative "brakes" on T cells through engagement of CD80 and/or CD86 [87 ]. Down-regulation of CTLA-4 on T cells following OX40 stimulation would be consistent with the proliferative burst of T cells observed early after OX40 engagement. Recently, it has been shown that limiting CTLA-4/B7 engagement can overcome peptide-induced tolerance [88 ]. As engagement of OX40 has been shown to overcome peptide-induced tolerance [63 ], it is interesting to speculate that OX40-mediated down-regulation of CTLA-4 might be involved in the initial steps of breaking T cell tolerance via OX40 engagement. Engagement of OX40 also led to down-regulation of mRNA for the cell-surface receptors L-selectin (CD62L) and CD27, which may influence the migratory patterns of recently stimulated effector T cells [89 ]. Most notably, the CD62L low and CD27 negative CD4+ T cells will home to nonlymphoid tissues including the lung, skin, and gut [89 ]. As discussed earlier, it has been shown that OX40 engagement can alter the migration of T cells, resulting in a large increase in Ag-specific T cells found in the blood and lungs [6 ]. Others have also shown an increase in the chemokine receptor CXCR5 on T cells isolated from mice that overexpress OX40L [52 ]; however, no increase in CXCR5 was observed in the gene array experiments.



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  		Figure 3. Decreased CTLA-4 expression on Ag-specific T cells may play a role in OX40-enhanced T cell proliferation. (A) Normal BALB/c mice received 1 x 106 TRAF2 DN+ KJ1-26+ or control (WT) KJ1-26+ T cells. Two days later, mice were primed with Ag and given rat IgG or anti-OX40. The mice were sacrificed 3 days after immunization, and the draining LN lymphocytes were prepared for Ab staining. The cells were triple-stained for KJ1-26, CD4, and CTLA-4. Cells were gated on KJ+/CD4+ cells and assessed for CTLA-4 expression. The staining was performed on live cells to measure CTLA-4, which had appeared on the cell surface. The six histograms represent lymphocytes obtained from six individual mice treated as shown in the figure. The dashed lines represent staining with the isotype control Ab, and the solid red lines represent staining with anti-CTLA-4. (B) Normal BALB/c mice received 1 x 106 TRAF2 DN+ KJ1-26+ (TRAF2 DN) or control (WT) KJ1-26+ T cells. Two days later, mice were primed with Ag and given rat IgG, anti-OX40, anti-CTLA, or anti-OX40 and anti-CTLA-4. (Peripheral Blood) The upper graphs depict a time course experiment in which blood was analyzed in the four groups shown on days 4, 14, and 29 after immunization for WT and TRAF2 DN transfers. Lymphocytes were isolated, stained to detect the Ag-specific T cells, and analyzed by flow cytometry to obtain the percentage of Ag-specific T cells. Error bars represent SE of five mice/group. (Lymph Nodes) The lower graphs depict a time course experiment in which the draining LN were analyzed in the four groups shown on days 2, 4, and 7 after immunization for WT and TRAF2 DN T cell transfers. Lymphocytes were isolated, counted, stained to detect the Ag-specific T cells, and analyzed by flow cytometry to assess the number of Ag-specific T cells per mouse. Error bars represent SE of five mice/group. (Reproduced with permission from the Journal of Immunology [62 ].)

The mRNA for two cytokine receptors showed substantial changes in the day-3 samples. mRNA for the IL-6R was decreased in the OX40-treated group, and the IL-2R{alpha}-chain was increased (Table 2) . Signaling through IL-6 may be detrimental to the development of memory T cells, although currently, there are no data to support this hypothesis. Alternatively, the increase in IL-2R mRNA suggests that the OX40-stimulated T cells may be able to proliferate after Ag exposure for a longer duration of time and therefore yield greater numbers of Ag-specific cells. Further evidence to support this hypothesis is displayed in Table 2 , where a 24.6-fold increase was observed in the DNA polymerase "regulatory" subunit from OX40-stimulated T cells. Again, it suggests that OX40-stimulated T cells are proliferating with greater vigor through an increase DNA synthesis, and this may be a result of the combination of increased IL-2R expression, increased c-myc transcriptional signaling through down-regulation of Mad4, and down-regulation of CTLA-4.

As the TRAF2 DN T cells were not as responsive as WT T cells to OX40 stimulation in vivo, we attempted to determine which genes might be modulated by decreased TRAF2 signaling in response to OX40 engagement. A gene array experiment similar to the one described in Table 2 was performed to compare RNA isolated from TRAF2 DN or WT DO11.10 T cells stimulated with OVA and anti-OX40 (at the day-3 time-point). Of the 156 genes that were up- or down-regulated in WT T cells upon OX40 engagement, only 20 were altered in the TRAF2 DN T cells stimulated with anti-OX40. CTLA-4 and L-selectin were two immune-specific gene products whose mRNA levels were decreased upon OX40 engagement in WT T cells, and this OX40-specific decrease was not observed in RNA isolated from the anti-OX40-stimulated TRAF2 DN T cells.

The TRAF2-dependent decrease observed in CTLA-4 expression was intriguing because of the known capacity of CTLA-4 to down-regulate T cell function after Ag-specific T cell activation [90 ]. We hypothesized that a block in OX40 signaling via an aberrant TRAF2 adaptor molecule (in TRAF2 DN T cells) could lead to increased B7–CTLA-4 interaction and decreased T cell function. Therefore, to examine the biologic significance of the OX40-specific decrease in CTLA-4 expression, we attempted to reverse the OX40-specific TRAF2 DN defect by CTLA-4 blockade in vivo (Fig. 3B) . Although CTLA-4 blockade was able to partially overcome the early, OX40-specific proliferation defect attributed to TRAF2 DN protein expression, the effect was short-lived and not able to accentuate the long-term survival of Ag-specific T cells. The data suggest that OX40-specific down-regulation of CTLA-4 helps to enhance early T cell proliferation, but it does not contribute to OX40-enhanced, long-term T cell survival. OX40-mediated, long-term T cell survival is also dependent on TRAF2 signaling, although as of yet, the molecular mechanism involved with OX40/TRAF2-dependent T cell survival has not been identified.


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CONCLUSIONS
 
Engagement of OX40 increases the proinflammatory activity of Ag-activated CD4+ T cells and enhances their long-term survival. The downstream molecular events associated with these biological activities are in the process of being identified and are described in Figure 4 . This review describes the current knowledge of cellular and molecular downstream events that follow OX40 engagement.



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  		Figure 4. Molecular characterization of the downstream effects that occur within CD4+ T cells following engagement of OX40. When OX40 is engaged via the OX40 ligand or an agonist Ab, a number of molecular events occur that increase CD4 T cell function, resulting in enhanced immunity. The molecular events that occur following OX40 engagement within T cells include TRAF-dependent signals that are transmitted to the nucleus, most likely via NF-{kappa}B and/or c-jun NH2-terminal kinases (JNKs). These signals induce alterations in transcriptional activity and/or mRNA stability that can be detected via mRNA detection assays (e.g., gene array analysis).

The cellular consequences of OX40 engagement include increased survival of Ag-activated T cells (leading to greater numbers of memory T cells), enhanced T cell cytokine production, increased T cell proliferation, enhanced production of Ag-specific Ab, and the ability to overcome T cell tolerance/anergy. OX40 function has been attributed mainly to the enhanced function of CD4+ effector T cells, which are essential to provide "help" to the other cellular components of the immune system. Therefore, OX40 downstream signaling events are most likely at the center of immune regulation and involved in controlling the magnitude of an ongoing T cell-mediated response. The biologic functions of this TNF-R family member make OX40 a unique and important target for therapeutic intervention in several disease states.

Understanding the molecular consequences of OX40 engagement will help define intracellular pathways that form the basis of cellular immune function described in this review. Although the molecular data point to the importance of the TRAF adpator protein family members (in particular, TRAF2), there could also be important TRAF-independent events involved in the transmission of OX40-specific signals. Data show that T cells expressing the TRAF2 DN protein on a TCR transgenic background were defective in OX40 signaling. This led to a decrease in Ag-stimulated CD4+ T survival, decreased proliferation, and poor effector T cell function. There appear to be two distinct phases of OX40-mediated, enhanced generation of Ag-specific CD4 T cell memory. TRAF2 signaling appears to be essential for both phases of the OX40-enhanced, Ag-specific T cell response. OX40-mediated down-regulation of CTLA-4 likely contributes to the enhanced, early expansion of Ag-specific T cells but does not appear to be responsible for their improved survival. It is also likely that the down-regulation of the c-myc repressor, Mad4, and the up-regulation of the IL-2R ({alpha}-chain) contribute to OX40-enhanced, early proliferation (both are TRAF2-independent observations). The decreased survival of OX40 KO T cells appears to be linked to inadequate expression of the Bcl-2 and Bcl-xL survival proteins; however, this observation has not been linked to TRAF2 adaptor protein function.

Although our understanding of OX40 signaling has increased in the past few years, a number of questions still remain. What are the downstream signal-transduction pathways that account for the effects of OX40 engagement? Are cytokine and cytokine receptors ultimately responsible for the downstream OX40-specific effects? What are the early transcriptional differences observed in OX40-treated, Ag-specific T cells (days 1 and 2 post-OX40 engagement)? Do other TRAF family members other than TRAF2 play a critical role in transmitting or inhibiting the OX40 signal? What is/are the key TRAF2-dependent molecules involved in OX40-enhanced T cell survival?

OX40 signaling is likely a key point at which the life or death fate of CD4+ effector T cells is ultimately decided. Therefore, understanding the molecular and cellular events that occur downstream of OX40 signaling will be beneficial in devising schemes to increase or decrease the inflammatory process. The knowledge gained from the molecular studies described in this review provides a foundation for future strategies aimed at enhancing vaccination or blocking inflammation through manipulation of OX40 or its ligand.


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NOTE
 
A manuscript published after acceptance of this review shows that protein kinase B (PKB/Akt) activity is diminished over time in OX40 ko T cells [91 ]. The authors further showed that the addition of functional PKB signaling in OX40 ko T cells overcomes defects found in OX40 ko T cells. Others have shown that IL-2 signaling can promote T cell survival through activating PKB. Therefore, the increase in IL-2 receptor expression via OX40 signaling discussed in this review may lead to prolonged PKB activation, which in turn, may promote some of the OX40-specific proinflammatory effects described in this review.

Received November 24, 2003; revised January 22, 2004; accepted January 28, 2004.


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