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Keywords:

  • Cell differentiation;
  • Costimulation;
  • Cytokine receptors;
  • Graft versus host disease;
  • Tolerance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Naive, CD4+ T cells proliferate extensively but fail to differentiate when they are transferred into unirradiated recipients that express alloantigen or transgenic antigen on all MHC class II+ cells. Addition of an agonist antibody to OX40 (CD134), a costimulatory TNF receptor family member expressed on activated CD4+ T cells, enables the proliferating T cells to accumulate as differentiated effector cells and kill the host animals. The donor T cells from anti-OX40-treated animals express high levels of IL-2Rα (CD25) and acquire the ability to secrete IFN-γ when stimulated with IL-12 and IL-18. OX40 promotes differentiation by 48 h in T cell priming, before changes in Bcl-2 expression or cell recovery become apparent. We found that a Bcl-2 transgene or deficiency in Fas or TNFR1 failed to influence accumulation of differentiated donor cells, and found larger changes in expression of cytokine and cytokine receptor genes than in survival genes. Accumulation of differentiated CD4+ effector T cells is initiated directly through OX40, but some OX40-deficient donor cells can gain effector function as bystanders to OX40+/+ cells. Taken together, these data suggest that CD4+ T cell differentiation to effector function is an important effect of OX40 engagement under conditions of ubiquitous antigen presentation.

Abbreviation:
B6:

C57BL/6

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Optimal CD4+ T cell activation requires recognition of cognate antigen together with costimulatory signals. However, costimulatory signals are not limiting for initial T cell proliferation in vivo, even with resting APC under conditions of tolerance induction 1, 2. In vivo, additional costimulatory signals from activated APC are necessary to promote CD4+ T cell survival and acquisition of effector function. Without these additional costimulatory signals, proliferating CD4+ T cells fail to accumulate, differentiate into effector T cells, or generate long-lived memory T cells, and the surviving tolerant T cells are hyporesponsive upon subsequent engagement of cognate antigen 3, 4. Several costimulatory receptors that belong to the TNFR superfamily, including CD27, 4–1BB, and OX40, can enhance CD4+ T cell responses upon antigen recognition in vivo, allowing responding T cells to survive as well as to acquire differentiated effector functions 5.

OX40 is a costimulatory receptor expressed predominately on CD4+ T cells that appears on the cell surface 24–48 h after antigen recognition 6, and engages OX40L expressed on the surface of activated APC 7. Mice deficient in OX40 or OX40L have diminished antigen specific clonal expansion and memory T cell populations 813. Blocking OX40/OX40L interactions in vivo decreases the severity of inflammatory diseases such as GVH disease 14, EAE 15, and asthma 10, 16. In contrast, agonist OX40 antibody increases clonal expansion 9, promotes robust memory T cell populations 17, 18, and enhances anti-tumor immunity 1921. In addition, a signal through OX40 has been reported to reverse previously established peripheral tolerance 22.

Although OX40 costimulation can promote acquisition of effector function, the mechanism by which this occurs is not clear. Previous reports demonstrate that OX40 promotes T cell longevity, which can lead to a robust memory T cell population 9, 17, 18, 2325. More specifically, OX40 signaling results in enhanced Akt activity and Bcl-2, Bcl-xL, and survivin expression, leading to survival of activated T cells and enlarging the memory T cell populations 2325. OX40 engagement has also been shown to enhance secretion of IL-4 and IL-5 10 and germinal center formation 26 in Th2 responses, and IL-2 and IFN-γ production in Th1 responses 27, 28. Here, we examine whether OX40 engagement enhances effector function by promoting survival of differentiating cells, or by driving T cell differentiation independently of survival.

To determine the role of OX40 signaling in acquisition of effector function, we used two similar models of ubiquitous antigen presentation. We had previously established that agonist anti-OX40 promoted accumulation of differentiated effector CD4+ T cells when TCR-transgenic T cells are transferred into unirradiated, antigen transgenic hosts 27. In this study, we examine the polyclonal responses of alloreactive C57BL/6 (B6) CD4+ T cells, and show that agonist anti-OX40 antibody can promote the accumulation of differentiated effector CD4+ T cells in unirradiated recipients. To determine the role of OX40 costimulation in a polyclonal population of CD4+ T cells, we use Bcl-2-transgenic or death receptor- and OX40-deficient mice available on the common B6 background in the alloantigen model. We use the TCR-transgenic model to investigate the effects of OX40 signaling in the initial phases of T cell priming. The data herein suggest that OX40 directs differentiation to effector CD4+ T cells under conditions of ubiquitous antigen presentation.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

OX40 engagement drives CD4+T cells to accumulate and differentiate to effector cells in unirradiated allogeneic recipients

To examine the role of OX40 costimulation, we employed a murine model of GVH, in which we transferred B6 splenocytes into unirradiated, MHC class II disparate (B6 × bm12)F1 recipients. A population of donor B6 CD4+ T cells mount an alloreactive immune response to the MHC class II I-Abm12 molecule, which differs from I-Ab due to mutations in the peptide-binding domain 29, 30. In the majority of GVH models, the recipient must be irradiated prior to transfer of donor T cells in order to promote effector cell development of donor B6 CD4+ T cells 31. In this report, we examined the effects of OX40 costimulation in unirradiated recipients by injecting an agonist antibody against OX40 or control IgG at the time of donor CD4+ cell transfer. In Fig. 1, 4.5 × 106 B6 CD4+ splenocytes were transferred with control IgG into unirradiated F1 recipients in the absence of exogenous costimulation or adjuvant. Five days later, the donor CD4+ T cells had proliferated extensively, as measured by CFSE dilution (Fig. 1A, Control IgG), and accumulated (Table 1, Control IgG, B6 group), while CD4+ T cells did not proliferate in control syngeneic recipients (data not shown). Consistent with the rapid expansion, on day 5 control IgG-treated donor CD4+ T cells expressed uniformly increased CD44 levels and heterogeneous levels of CD62L, but did not express the activation marker IL-2Rα (CD25), while undivided CD4+ donor cells remained CD25, CD44low, and CD62Lhigh (Fig. 1A, Control IgG). The alloreactive CD4+ cells developed poor effector T cell function measured as IFN-γ production when stimulated with PMA and ionomycin or recombinant IL-12 and IL-18, a cytokine combination that has been shown to stimulate IFN-γ production from effector T cells independently of the TCR 32 (Fig. 1B, Control IgG).With agonist anti-OX40 treatment, the donor CD4+ cells proliferated and accumulated to larger numbers compared to the control IgG-treated donor T cells by day 5 (Table 1, anti-OX40, B6 group). These alloreactive CD4+ T cells were larger and more granular than control cells (data not shown), and approximately 90% of divided cells had very high levels of CD25 expression (Fig. 1A, OX40). Upon stimulation in vitro with PMA and ionomycin, approximately 40% of the divided donor CD4+ cells produced IFN-γ, and stimulation with recombinant IL-12 and IL-18 drove more than 70% of the donor cells to produce IFN-γ (Fig. 1B, OX40). These data show that costimulation in the form of anti-OX40 allows accumulation of donor CD4+ T cells that have gained effector function in an unirradiated recipient.

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Figure 1. Anti-OX40 promotes acquisition of effector function in donor CD4+ alloreactive T cells. CD4+ T cells (4.5 × 106) in a B6 spleen cell suspension were transferred with 50 μg anti-OX40 or rat IgG i.v. into unirradiated (B6.CD45.1 × bm12)F1 recipients, and spleens were harvested 5 days (A and B) or 11 days (C) later. (A) Percent of CD45.1 negative donor CD4+ T cells in a representative sample is shown in the left panel. Among the CD4+ donor cells, T cell activation surface markers CD25, CD44, and CD62L, on CFSElo, divided donor cells are compared to CFSEhi, undivided, donor cells directly ex vivo, as shown in the right panels. (B) Percent IFN-γ production by donor CD4+ T cells restimulated in vitro for 5 h with nil, 20 ng PMA and 500 ng ionomycin (PMA/Iono), or 10 ng IL-12 and 100 ng IL-18 (IL-12/18). C) Recipient spleens were harvested 11 days after T cell transfer. Percent of CD45.1 negative donor CD4+ T cells is shown in the left panel. Percent of fresh CD25+ donor CD4+ T cells and IFN-γ+ donor cells after stimulation with IL-12 and IL-18 is shown in the right panels.

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Table 1. Donor CD4+ cell recoveries from spleena)
Donor CD4+Input (× 106)Donor CD4+Recovered ± SD (× 106)
Anti-OX40Rat IgG control
  1. a) CD4+ T cells in a B6 or TCR Tg spleen cell suspension were transferred with anti-OX40 or control IgG i.v. into unirradiated (B6.Ly5.1 × bm12)F1 or antigen transgenic recipients, and spleens were harvested at the indicated day. The number of donor CD4+ cells recovered was determined by multiplying the percent CD4+ donor cells determined by flow cytometry by the total cells recovered from the spleen. n equals the number of animals in each treatment group, combining data from one to four experiments.b) All six OX40-treated mice were moribund and euthanized.c) OX40–/– donor cells transferred alone.d) OX40–/– and OX40+/+ were mixed together and injected into a single recipient.

B6 (n = 12)Day 54.519 ± 5.64.9 ± 1.6
B6 (n = 3)Day114.51.5 ± 12.2 ± 0.9
B6 (n = 3)Day 51042 ± 7.54.6 ± 1.4
B6 (n = 6)Day 7b)108.6 ± 0.743.2 ± 0.24
OX40–/– (n = 6)c)Day 54.52.9 ± 1.43.4 ± 0.5
OX40–/– (n = 6)d)Day 54.56.9 ± 0.94.1 ± 0.3
OX40+/+ (n = 6)d)Day 54.518.2 ± 3.11.8 ± 0.5
Bcl-2 Tg (n = 6)Day 54.518.6 ± 3.22.9 ± 0.31
B6math image (n = 6)Day 54.59.2 ± 0.952.8 ± 0.22
TNFR1–/– (n = 6)Day 54.510.5 ± 0.11.8 ± 0.67
TCR Tg (n = 6)Day 13.50.33± 0.340.15 ± 0.02
TCR Tg (n = 6)Day 23.51.1 ± 0.671.56 ± 0.67
TCR Tg (n = 9)Day 33.58.5 ± 4.2 10.8 ± 6 .5
TCR Tg (n = 9)Day 53.596.7 ± 7.643.8 ± 4.2

Five days after donor cell transfer, the mice appeared outwardly healthy with anti-OX40 treatment even though the alloreactive CD4+ T cells had acquired robust effector T cell function (Fig. 1A and B, OX40). By day 11, fewer donor CD4+ T cells were recovered (Table 1, anti-OX40, B6 group), and the percent of cells expressing CD25 had decreased from 89 to 40%, and IFN-γ producing cells following stimulation with IL-12 and IL-18 also decreased from 76 to 42% (Fig. 1C, OX40). These experiments showed that although anti-OX40 treatment resulted in accumulation of effector T cells in an otherwise tolerizing environment, the effect was transient and did not lead to overt disease.

Next, we wished to test the hypothesis that transfer of more donor cells would result in greater accumulation of effector cells and cause disease, as we reported earlier in a similar, TCR-transgenic model 27. When we transferred 107 CD4+ T cells, donor cell recovery on day 5 doubled (Table 1, anti-OX40, B6 group), acquisition of effector function was similar to cells recovered at 5 days (data not shown), and the mice appeared outwardly healthy. However, at day 7, six of six mice became moribund and were euthanized. Fewer donor CD4+ T cells were recovered from day-7 spleens (Table 1, anti-OX40 day 7), but these cells still expressed very high levels of CD25 and produced IFN-γ upon stimulation with PMA and ionomycin or with recombinant IL-12 and IL-18 (data not shown). As in our previously published experiments using TCR-transgenic T cells and antigen transgenic mice, providing a single injection of anti-OX40 allowed accumulation of donor cells with a Th1 differentiated phenotype that led to lethal disease in the recipient 27.

Anti-OX40 directly activates donor CD4+ T cells bearing the OX40 receptor

To determine whether anti-OX40 is acting directly on donor CD4+ T cells, and not on cells in the recipient, we transferred OX40-deficient (OX40–/–) B6 CD4+ T cells with agonist anti-OX40 antibody into unirradiated F1 recipients. The OX40–/– donor CD4+ cells accumulated (Table 1, OX40–/–) but did not differentiate to effector T cells, as measured by CD25 expression and IFN-γ production upon stimulation with IL-12 and IL-18 (Fig. 2A). OX40–/– donor CD4+ T cells treated with or without anti-OX40 are phenotypically the same as control IgG OX40+ donor cells as shown in Fig. 1A and B, control IgG panels (Fig. 2A). These data demonstrate that anti-OX40 is exerting its costimulatory effect on the donor T cells to allow accumulation of differentiated effector T cells, rather than acting on cells in the recipient.

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Figure 2. OX40 acts directly and indirectly on donor T cells to promote effector function. All graphs are gated on CD4+, CD45.1 donor lymphocytes. B6.Thy1.1 CD4+ T cells (4 × 106) and B6 OX40–/– CD4+ T cells (4 × 106) in a spleen cell mixture or 4 × 106 OX40–/– alone were transferred with 50 μg anti-OX40 or rat IgG into an unirradiated (B6.ly5.1 × bm12)F1 recipient for 5 days. (A) Top row represents mice treated with anti-OX40, bottom row represents mice treated with rat IgG. CD25 expression of donor CD4+ cells versus side scatter is shown in the middle panel. Percent of IFN-γ+ cells after stimulation with IL-12 and IL-18 for 5 h is shown on the right. (B) Top two rows represent mice treated with anti-OX40, bottom two rows represent mice treated with rat IgG. Percent of donor B6 (Thy1.1+) and donor OX40–/– (Thy1.1) is shown in the left panels. CD25 expression of each donor population is shown in center. Percent IFN-γ+ cells after stimulation with IL-12 and IL-18 for 5 h is shown on the right.

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To determine whether OX40 engagement on activated donor cells is capable of eliciting costimulatory functions in the recipient that then act independently of OX40, we mixed 4.5 × 106 CD4+ B6 cells congenic for the Thy1.1 marker with 4.5 × 106 CD4+ OX40–/– cells and transferred them into unirradiated F1 recipients with anti-OX40 or control IgG. After 5 days, both donor CD4+ populations accumulated in the spleen (Table 1, OX40–/– and Fig. 2B). In control IgG-treated animals, the OX40–/– donor cells accumulated more than OX40+ donor cells (Table 1), but both populations expressed low levels of CD25 and produced little IFN-γ (Fig. 2B, bottom panels). The enhanced accumulation of OX40–/– T cells compared to their normal counterparts has been reported before in other systems 33, 34. With anti-OX40, CD4+ cells bearing the OX40 receptor differentiated to effector T cells as before (Fig. 2B, top panels). In the presence of OX40+ donor cells, a portion of OX40–/– CD4+ T cells reproducibly expressed CD25 and produced IFN-γ upon IL-12 and IL-18 stimulation in six of six mice (Fig. 2B, top panels). It must be noted that fewer OX40–/– donor T cells were recovered, and of those, a smaller proportion acquired the effector cell phenotype (Fig. 2B). These data show that anti-OX40 must act on OX40+ donor cells to initiate acquisition of effector function. However, bystander differentiation does occur in OX40-deficient donor T cells, implying that the OX40+ donor cells can induce OX40-independent effector function in bystander T cells, perhaps by eliciting other costimulatory signals in the recipients.

CD4 T cells that have a transgene or mutation that enhances survival do not acquire effector T cell function without OX40 engagement

We have shown that anti-OX40 acts directly through OX40 on the alloreactive donor CD4+ T cells (Fig. 2), however, the mechanism of OX40-mediated accumulation of effector CD4+ T cells is unknown. Triggering OX40 could promote the differentiation of proliferating donor cells or, alternatively, OX40 could promote survival and accumulation of differentiated donor cells. It is known that over expression of proteins that promote survival directly rescue OX40–/– defects in accumulation of activated T cells and memory cell generation in other systems 2325. To test the hypothesis that OX40 engagement enhances T cell survival in our system of ubiquitous antigen presentation, we transferred 4.5 × 106 splenic CD4+ cells from B6 mice transgenic for the anti-apoptotic protein Bcl-2 into unirradiated F1 recipients. Although Bcl-2 over expression was shown to promote survival in a previous report 23, the transgenic T cells failed to accumulate to larger numbers than WT donor CD4+ cells in our model (Table 1, Bcl-2 and B6). Donor cell recovery, CD25 expression, and IFN-γ production upon stimulation with IL-12 and IL-18 from Bcl-2-transgenic CD4+ cells were similar to WT in the presence or absence of anti-OX40 (Fig. 3A and Table 1). Enhanced expression of Bcl-2 did not increase the numbers of IFN-γ-secreting effector cells in the absence of anti-OX40, showing that Bcl-2 overexpression does not mimic anti-OX40 treatment in this system.

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Figure 3. Gain of effector function is not influenced by the Bcl-2 transgene or mutations in death receptors. (A) Bcl-2 transgenic, (B) B6lpr, and (C) TNFR1-/- mutant donor CD4+ T cells (thick line) are compared to individual wildtype donor CD4+ T cells (thin line). All histograms represent spleen cells gated on CD4+, CD45.1 donor T cells. CD4+ T cells (4 × 106) were transferred in a spleen cell suspension into (B6 × bm12)F1 hosts for 5 days with 50 μg anti-OX40 or rat IgG. CD25 expression on freshly isolated cells and IFN-γ production of donor cells restimulated with 10 ng IL-12 and 100 ng IL-18 for 5 h are shown.

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It is possible that Bcl-2 failed to protect differentiating donor cells from activation-induced T cell death owing to Fas (CD95) or TNFR1 in our model. To test this hypothesis, we transferred donor B6 splenocytes with a natural mutation in CD95 (B6lpr), or splenocytes deficient in TNFR1, into unirradiated F1 hosts with or without anti-OX40. After 5 days, alloreactive CD4+ T cell accumulation, CD25 expression, and IFN-γ production in response to recombinant IL-12 and IL-18 was similar to WT responses with or without anti-OX40 (Table 1 and Fig. 3B and C). The most straightforward explanation of these results is that OX40 engagement promotes differentiation to effector function in this model of ubiquitous antigen presentation, rather than to prevent death of differentiating CD4+ T cells.

OX40 signaling leads to increased cytokine and cytokine receptor expression

To further explore the consequences of OX40 signaling, we compared changes in gene expression profiles between anti-OX40 and control IgG treated antigen-specific donor cells using Affymetrix oligonucleotide arrays. To obtain a homogenous population of antigen-specific donor CD4+ T cells, we employed our previously published model in which TCR- transgenic T cells are transferred into antigen-transgenic mice 27. This model yields results similar to the parent into F1 model described in this report. Data from two independent experiments using the MG-U74Av2 chip were analyzed for comparison of RNA levels in donor T cells from animals treated with anti-OX40 or control IgG for 3.5 days. We looked for genes encoding cytokines and cytokine receptors as measures of T cell differentiation, as well as for genes known to be involved in apoptosis and survival.

Consistent with the flow cytometry data, OX40 ligation induced expression of mRNA for CD25 (Table 2). Two other cytokine receptors acting through the common gamma chain, IL-15Rα and IL-7R, were also induced (Table 2). Like IL-2R, the IL-15 and IL-7 receptors have been implicated in survival and differentiation of T cells 3537.

Table 2. Effects of the OX40 signal on cytokine, cytokine receptor, and survival gene expression in donor T cellsa)
Gene or protein nameAccession numberFold change
  1. a) AND TCR transgenic T cells were injected into antigen transgenic mice with or without anti-OX40. After 3.5 days, transgenic T cells were enriched and total RNA was purified for hybridization on an Affymetrix MG-U74Av2 gene chip as described in the Materials and methods. Fold change numbers in parenthesesrepresent genes with absent detection calls for control treatment in genes that increase, and for anti-OX40 treatment in genes that decrease. Additional data from this analysis are presented in Table 1–3 in the online supplement.

Genes that increase
IL-2RαNM_008367(18.2)
IL-7RNM_0083725.5
IL-15RαNM_133836(4.4)
IL-12Rβ2NM_008354(15.4)
IFN-γNM_0083372.9
Lymphotoxin-αNM_010735(4.0)
GM-CSFNM_009969(19.5)
IL-3NM_010556(3.9)
OX40NM_0116594.0
4–1BBNM_0116126.1
Bcl-xNM_0097431.8
Hexokinase-2NM_0138205.0
Glut-1NM_011400(3.5)
Genes that decrease
IL-4NM_021283(−20.0)
IL-16NM_010551−3.2
gp-130NM_010560−8.9
CD27XM_284241−3.6
CD30LNM_009403(−13.5)
Genes that do not change
Bcl-2NM_1774101.1
Mcl-1NM_008562-1.5
BimNM_0097540.0
FasNM_0079871.3
TNFR1NM_0116090.1
DR6NM_178589(-2.0)

Notably, anti-OX40 also induced mRNA for IL-12Rβ2 (Table 2), consistent with the strong response to stimulation with IL-12 and IL-18 in the anti-OX40-treated T cells. In addition to IFN-γ production measured by intracellular cytokine staining (Fig. 1B, OX40), the OX40 signal increased expression of mRNA for several other pro-inflammatory T cell cytokines: GM-CSF, lymphotoxin-α (also known as TNFβ), and IL-3 (Table 2 and Supplementary Table 1). Cytokines and receptors that decreased with OX40 costimulation included IL-4, IL-16, and gp130 (the signal transducer subunit of IL-6R family members). CD153 (CD30 ligand) and CD27 were also decreased with anti-OX40 administration, while OX40 and 4–1BB expression increased (Table 2 and Supplementary Table 2).

Because the OX40 signal has been shown to induce expression of Bcl-2 and Bcl-xL in a model of transient antigen exposure 23, 24, we were surprised to find that Bcl-2 and some other Bcl-2 family members (Mcl-1 and Bim) scored as "no change" with anti-OX40 treatment (Table 2). However, we did see a 1.8-fold increase in Bcl-xL expression, which was below our analysis threshold of 2.9-fold change in expression (Table 2). Two genes involved in glucose transport and metabolism, Glut-1 and hexokinase-2, which are reported to decrease in T cells upon growth factor withdrawal 38, were increased greater than 2.9-fold with OX40 engagement (Table 2). Messages for the death receptors Fas, TNFRI, and DR6 (Tnfrsf21) also scored as "no change" with anti-OX40, as did message for the negative regulatory molecules, CTLA-4 and PD-1 (Supplementary Table 3). Overall, these data support the idea that OX40 drives the differentiation of proliferating T cells to Th1 effector cells in this model.

OX40 drives differentiation early in T cell priming

We wished to test the hypothesis that OX40 signaling promotes acquisition of effector function early in T cell priming, during exponential expansion and before extensive death of proliferating T cells. Using the AND TCR-transgenic mice, we transferred 3.5 × x106 CFSE-labeled splenic AND Rag1–/– T cells with a single injection of anti-OX40 or control IgG into antigen-transgenic recipients 27 and harvested splenocytes 30, 48, 72, and 120 h after injection. Anti-OX40-treated donor CD4+ T cells expressed CD25 30 h after injection and maintained uniformly high CD25 expression through 120 h, while control cells initially expressed CD25, but became CD25 intermediate at 48 h, and CD25 low to negative by 72 h (Fig. 4A). IFN-γ production directly ex vivo or after stimulation with PMA and ionomycin or IL-12 and IL-18 was equal with or without anti-OX40 30 h after injection. However, at 48, 72, and 120-h time points, anti-OX40 treated donor cells produced markedly increased IFN-γ upon stimulation compared to control IgG-treated cells (Fig. 4B). Donor CD4+ T cells recovered from both anti-OX40 and control IgG populations uniformly proliferate with the same kinetics at each time point as shown by CFSE dilution (Fig. 4B, bottom). While donor cell recovery is similar if not greater in control IgG-treated animals in the first 72 h, greater accumulation of anti-OX40-treated donor cells over control cells occurred between 72 and 120 h (Table 1, TCR Tg group), consistent with previously published results 27. These data show that OX40 engagement induces differentiation early in T cell priming before effects of enhanced survival can be measured.To determine whether there is a correlation between differentiation and survival protein expression at early time points, we examined Bcl-2 by flow cytometry on donor cells 30, 48, 72, and 120 hours post-injection in anti-OX40- or control IgG-treated recipients. We also examined Bcl-xL protein expression at 72 h, a time at which Bcl-xL mRNA levels are increased in OX40- over control IgG-treated animals (Table 2). Consistent with increased mRNA, Bcl-xL protein expression at 72 h was significantly enhanced in OX40-treated recipients (Fig. 4C and Table 2). We found no difference in the mean fluorescence intensity (ΔMFI) of Bcl-2 protein expression between anti-OX40- and control IgG-treated cells at 30 and 48-h (Fig. 4C). We were surprised to find that Bcl-2 expression decreased significantly at 72 h in the control IgG group compared to OX40-treated cells, since the gene chip analysis at 3.5 days showed no difference in Bcl-2 RNA expression (Fig. 4C and Table 2). However, at 120 h, Bcl-2 expression was very low in both anti-OX40- and control IgG-treated cells (Fig. 4C). Acquisition of IFN-γ effector cytokine production is apparent at 48 h, before a difference in CFSE dilution, cell recovery, or Bcl-2 expression. These data suggest that the primary effect of OX40 signaling may be to promote cytokine receptor expression and differentiation to effector function, as these are the earliest responses we measured during T cell priming in vivo in our system.

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Figure 4. Anti-OX40 promotes acquisition of effector function early in T cell priming. AND TCR-transgenic T cells (3.5 × 106) in a spleen cell suspension were transferred with 50 μg anti-OX40 or control IgG i.v. into antigen-transgenic recipients, and spleens were harvested 30, 48, 72, or 120 h later. (A) Forward scatter and percent of CD25+ donor cells gated on CD4, Vα11, and Vβ3 for each time point is shown. (B) Percent IFN-γ production of donor CD4+ T cells stimulated for 5 h in vitro with nil, 20 ng PMA and 500 ng ionomycin (PMA/Iono), or 10 ng IL-12 and 100 ng IL-18 (IL-12/IL-18). For each condition, the bars show mean percent IFN-γ+ cells for donor cells from animals treated with anti-OX40, control IgG, naive, and day 5 or 6 in vitro primed TCR- transgenic cells. In the bottom eight panels, a representative plot shows cell division (CFSE) and IFN-γ production upon IL-12 and IL-18 stimulation for anti-OX40 and control IgG treated donor cells. (C) Mean fluorescence intensity (MFI) and representative histograms of Bcl-2 or Bcl-xL protein expression and isotype-matched staining controls in donor cells gated on CD4, Vα11, and Vβ3. In the bar graphs, ΔMFI represent the change in intensity between protein and isotype control MFI for each sample.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Recent literature on the role of OX40 costimulation indicates that a signal through OX40 enhances survival proteins to promote CD4+ T cell accumulation and memory 18, 2325. Data in this report clearly show that OX40 costimulation promotes CD4+ T cell differentiation, measured as the ability to produce IFN-γ, independently of survival. OX40 promotes differentiation at early time points in T cell priming, before differences in Bcl-2 expression or cell recovery can be measured. We have also shown that donor CD4+ T cells with genetic alterations that prolong survival are not able to differentiate without anti-OX40. In addition, we found that anti-OX40 acts directly through OX40 on responding CD4+ T cells to initiate acquisition of effector function, although OX40-deficient CD4+ T cells are able to acquire effector function as bystanders to OX40+ donor cells.

We show for the first time that OX40 engagement promotes enhanced effector function beginning at 48 h of T cell priming in vivo. Perhaps differentiation early in T cell priming leads first to cytokine and cytokine receptor expression and then to acquisition of effector function and enhanced survival of effector and memory cells as a secondary outcome. In fact, we saw much larger increases at 3.5 days in expression of cytokine and cytokine receptor genes than genes for anti-apoptotic proteins. In our experiment, only a small percentage of donor cells were able to make IFN-γ as early as 48 hours. In another report, differentiation to IFN-γ synthesis occurs early and is maintained in a large population of antigen-specific T cells in response to viral immunization 39. While effector cell differentiation directly mediated by OX40 may take longer to develop compared to viral priming, it is possible that OX40 engagement can promote only a small fraction of CD4 cells to become effectors, and that the ability to promote survival is also focused selectively on this small population. However, as there was no detectable advantage in the ability of Bcl-2 transgenic, Fas-deficient or TNFR1-deficient genetic mutants to accumulate or differentiate in our system, we prefer a simpler model in which effector cell differentiation driven by OX40 occurs over several days in most of the proliferating donor cells.

The outcome of OX40 costimulation could favor acquisition of effector function or enhanced survival depending on the context of T cell activation, such as ubiquitous versus localized antigen, or persistent versus transient antigen presentation. While it is clear that a signal through OX40 promotes T cell survival and memory upon transient or localized antigen stimulation in vitro as well as in vivo 6, 9, 17, 18, 2325, 40, 41, data in this report indicate that differentiation may be the primary effect of OX40 engagement upon persistent and ubiquitous antigen presentation. Croft and co-workers 23 reported that OX40 costimulation promotes Bcl-2 and Bcl-xL expression upon stimulation in vitro and that Bcl-2 or Bcl-xL overexpression in CD4+ donor cells enhances survival and memory T cell generation in OX40-deficient CD4+ T cells 23, 24. When we tested this possibility in our system, we discovered that Bcl-2 overexpression did not promote differentiation or significantly enhance cell recovery compared to control CD4+ T cells in unirradiated F1 recipients without OX40 costimulation. However, expression of the anti-apoptotic Bcl-2 protein rescues growth factor withdrawal mediated apoptosis but has little effect on apoptosis through the death receptor pathway 42. We considered that triggering OX40 could prevent apoptosis mediated by death receptors rather than growth factor withdrawal in vivo, and thus allow accumulation of differentiated antigen specific donor cells. However, when we transferred CD4+ T cells that were deficient in the death receptors Fas or TNFR1, they did not acquire effector function in unirradiated F1 recipients without anti-OX40, and we observed no differences with anti-OX40.

In addition to data in this paper, there are other examples in which differentiation may be the primary result of OX40 signaling 22, 27, 4345. Administration of agonist OX40 antibody after the onset of tolerance in vivo can reverse the tolerant phenotype 22. Recently, OX40 signaling has been shown to prolong IL-2, IL-3, and IFN-γ expression by promoting stabilization of the cytokine mRNA in human CD4+ memory cells 45.

Others and we have shown that OX40 induces sustained expression of IL-2R 17, IL-7R, and IL-15R ( 27 and this report). Perhaps both differentiation to IFN-γ synthesis and the increase in T cell survival and memory cell generation are results of cytokine receptor signaling rather than a direct effect of OX40, as the common gamma chain receptors are known to enhance both survival and effector function 35, 37, 44. OX40 signaling may drive expression of common gamma chain receptors such as IL-2 receptor, whose signals would then promote differentiation or survival, depending on the context of T cell activation. A recent report has shown that OX40 costimulation activates Akt to promote T cell longevity by enhancing anti-apoptotic protein expression 24. Akt activity has also been reported to be downstream of common gamma chain receptors such as IL-2, IL-7, and IL-15 46, 47. Thus, increased T cell longevity following OX40 ligation may depend on signaling through this family of cytokine receptors.

Notably, our data indicate that OX40 costimulation is not the only signal capable of permitting accumulation of differentiated effector donor T cells in unirradiated recipients. OX40-deficient CD4+ T cells have a modest ability to acquire effector function when mixed with OX40+/+ donor cells in the presence of agonist anti-OX40. Differentiation seen in OX40–/– CD4+ T cells mixed with OX40+/+ depends on OX40-mediated T cell differentiation. Differentiating OX40+ cells in this mixing experiment may initiate a positive feedback loop by licensing APC to promote expression of other costimulatory molecules and cytokines that could indirectly induce effector function in OX40–/– CD4+ T cells. In concordance with this idea, one report has shown cytokine-driven bystander activation of CD4+ T cells mediated in this way 48.

In summary, we provide evidence that T cell differentiation is an important effect of OX40 ligation. We show that anti-OX40 promotes accumulation of CD4+ T cells with effector function in a model that otherwise promotes tolerance, and can result in fatal GVHD in unirradiated recipients. We also found that other signaling pathways could drive differentiation of proliferating T cells, because a portion of OX40 deficient donor CD4+ T cells can differentiate into effector cells when mixed with normal donor T cells in our model. Thus, both T cell survival and acquisition of effector functions must be considered when costimulatory signals are manipulated in approaches to treating or preventing chronic infections, autoimmune diseases, GVH, allogeneic bone marrow graft rejection, and cancer.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Mice and adoptive transfers

Mice were housed under specific pathogen-free conditions at the Oregon Health & Science University animal facility. (B6 × bm12) F1 mice were made by crossing female B6.Ly5.1 (C57BL/6 congenic for CD45.1), obtained from the National Cancer Institute (Frederick, MD), to B6.C-H2bm12/KhEg mice, obtained from the Jackson Laboratory (Bar Harbor, ME). C57BL/6J, TNFR1–/– (C57BL/6-tnfrsf1a), B6lpr, Thy1.1 congenic (C57BL/6J-IghaThy1aGpia), and Bcl-2 transgenic (C57BL/6-TgN(Bcl2)36wehi) were obtained from the Jackson Laboratory. OX40–/– mice were kindly provided by Nigel Kileen 13. Pigeon cytochrome C (PCC)-specific AND TCR-transgenic, Rag-1-deficient mice, and antigen-transgenic mice expressing I-Ek and covalently associated PCC with the amino acid T102S substitution on the C57BL/6 background have been described previously 27. Donor splenocytes for each experiment were prepared for intravenous injection as described 27. In some experiments, donor cells were labeled with 2 μM CFSE in 0.1% BSA in PBS for 10 min at 37°C and washed in Hanks Buffered Saline Solution (HBSS) with 2% serum. Cells were injected i.v. with 50 μg anti-OX40 or control IgG in HBSS without serum into unirradiated (B6 × bm12)F1 recipients or into antigen-transgenic recipients.

Antibodies

PerCP-anti-CD4 (RM4–5), biotin-anti-CD25 (7D4), allophycocyanin-anti-IL-2 (JES6–5H4), PE-anti-Bcl-2 (3F11), and labeled isotype controls were purchased from BD PharMingen (San Diego, CA). Allophycocyanin-anti-IFN-γ (XMG1.2), FITC-anti-CD90.1 (HIS51), PE-anti-CD45.1 (A20), and appropriate isotype controls were purchased from eBiosciences (San Diego, CA). PE-anti-Bcl-xL (7B2.5) and labeled isotype control were purchased from Southern Biotechnology (Birmingham, AL). Anti-OX40 antibody from clone OX86 (European Cell Culture Collection, Porton Down, UK) was produced and purified for i.v. injection. Rat IgG was purchased from Cappel, ICN Pharmaceuticals (Costa Mesa, CA). Recombinant mouse IL-12 was purchased from Cell Sciences (Norwood, MA), and recombinant mouse IL-18 was purchased from R & D Systems (Minneapolis, MN).

Cell culture and flow cytometry

Spleen cell suspensions were prepared for intracellular cytokine staining as described 27. Labeled cells were analyzed on a FACSCalibur flow cytometer (BD Immunocytometry, San Jose, CA) and analyzed using FlowJo (Tree Star, San Jose, CA).

Microarrays

AND Rag1–/– TCR-transgenic T cells (5 × 106, first experiment or 3.5 × 106, second experiment) were transferred into antigen transgenic recipients with 50 μg of anti-OX40 or control IgG, as previously described 27. The TCR-transgenic T cells were enriched from antigen transgenic spleen cell suspensions on day 3.5 by labeling cells with anti-Vβ3 biotinylated antibodies (BD PharMingen) followed by anti-biotin magnetic beads (Miltenyi Biotec, Auburn, CA) and separation with an autoMACS magnetic column (Miltenyi Biotec). We found the anti-OX40-treated T cells difficult to isolate, perhaps because they are very large and granular and tend to aggregate. Thus, we obtained 90% Vβ3+ cells in the enriched population from control IgG-treated animals, but only 75% Vβ3+ cells from anti-OX40-treated animals in two independent experiments. RNA was prepared from the enriched cells using the RNeasy kit from Qiagen (Valencia, CA). Isolated total RNA was checked for quality and used at 1 μg/μL to prepare labeled cDNA, which was hybridized to the murine genome MG-U74Av2 array (12 488 genes) according to manufacture's guidelines (Affymetrix, Santa Clara, CA) at the OHSU Gene Microarray Shared Resource. Data generated from the duplicate gene chip experiments were analyzed using Affymetrix Microarray Suite 5.0 (MAS 5.0) software, and the gene documentation and probe match software are available on the Affymetrix website, http://www.affymetrix.com/. Briefly, we excluded all genes with absent detection calls on all four chips and genes that showed no change in both comparisons. Of the remaining 3653 genes, we selected genes that increased or decreased at least 2.9-fold (mean fold change derived from signal log2 ratio) and had p-values for the present detection call less than 0.1 in anti-OX40 increased or control IgG decreased in either experiment. The genes that increased or decreased according to these criteria are listed in Supplemental Table 1 (96 genes) and Supplemental Table 2 (88 genes). Some genes reported to affect T cell survival or differentiation that did not meet these criteria for robust change, but did not have an absent detection call on all four chips, are listed in Supplemental Table 3.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

This work was supported by Grant AI50823 (to D.C.P.) and CA81383 (to A.D.W.) from the National Institutes of Health. C.A.H. is supported as a trainee in the Molecular Hematology Training Program, Grant T32-HL007781.

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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2040/2006/35637_s.pdf or from the author.

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