OX40 Is Expressed on Memory Th2 Cells.
Characteristic features of allergic asthma are produced when mice are sensitized with OVA, and subsequently challenged several weeks later by inhalation of OVA (1
). Using OX40-deficient mice, we previously showed the importance of OX40 in controlling initial development of asthma symptoms (22
). However, as these mice are defective in generating a primary T cell response (10
), no conclusions could be drawn regarding any requirement for OX40 in the secondary response of the memory T cell that ultimately induces lung inflammation.
We initially looked to see if memory CD4 cells express OX40. By staining T cells from mice primed 4 wk prior with OVA in alum, we visualized a significant number of CD44hi
memory CD4 cells in lymph nodes that expressed OX40 at low/moderate levels. In contrast, as shown previously (9
naive CD4 cells did not express OX40 without antigen exposure ( A). Unimmunized mice also contained a proportion of CD44hi
CD4 cells that expressed OX40 at low levels (unpublished data), suggesting that OX40 can be readily available to some memory T cells. OX40 levels were up-regulated on responding CD44hi memory T cells in lung draining lymph nodes after challenge with aerosolized antigen ( A), and the absolute number of OX40 positive CD44hi
CD4 cells increased markedly ( C, and see below). As this large increase in the number of OX40-expressing CD44hi
cells was not seen in unprimed mice challenged with aerosolized antigen, the latter directly reflects the response of memory T cells and accumulation of memory effector cells.
Figure 1. OX40 is expressed on memory and memory effector T cells. Groups of four C57BL/6 mice were immunized i.p. with OVA adsorbed to alum (primed). 25 d later mice were challenged by inhalation of nebulized OVA on 4 consecutive days (primed/challenged). (A) (more ...)
Few CD4 cells were present in the lungs of primed but unchallenged mice, and OX40 was not detected on either CD44hi or CD44lo cells ( B). However, after antigen challenge, a large number of OX40-expressing CD4/CD44hi cells were seen in the lung ( B, and see below). Analysis of lung sections revealed the presence of OX40+ cells in peribronchial and perivascular areas, sites previously shown to contain the majority of antigen-responding memory effector T cells ( D). These data show that OX40 is expressed on memory/memory effector T cells and suggest that it is available to play a role during the secondary response that occurs after reencounter with antigen.
Preventing OX40/OX40L Interaction Impairs Development of Airway Hyperreactivity and Eosinophilia.
To examine the contribution of OX40/OX40L interactions to lung inflammation, we administered a blocking anti-OX40L mAb to OVA-sensitized mice at the time of rechallenge with aerosolized antigen ( A). OVA-immunized mice treated with the isotype control developed increases in AHR whereas administration of anti-OX40L dramatically reduced the degree of AHR ( B). Sensitized mice treated with control Ab during aerosol challenge (Alum-OVA/IgG-OVA) responded with an increase in the total number of cells in BAL ( C), which was evident as early as 48 h and was mostly eosinophils ( D). In striking contrast, administration of anti-OX40L during the challenge period (Alum-OVA/RM134L-OVA) virtually eliminated the increase in total leukocytes ( C) and eosinophils ( D). As no cell infiltration was seen in unprimed but challenged animals, this directly shows that OX40/OX40L interactions were essential to the recall response.
Figure 2. Anti-OX40L suppresses memory T cell induced AHR and airway inflammation. (A) Experimental protocol for anti-OX40L administration. Unprimed control mice were injected i.p. with alum alone (Alum), while primed mice were sensitized with OVA adsorbed to alum (more ...)
Blocking OX40/OX40L Interactions Inhibits the Development of Airway Tissue Eosinophilia, Goblet–Cell Hyperplasia, and Mucus Production.
To support the findings from lung lavages we conducted histological evaluations on lung sections. Mice receiving control Ab developed inflammatory lesions, characterized by a predominance of eosinophils and lymphocytes () , together with hyperplasia of the mucus-secreting bronchial epithelial cells ( A). In contrast, lungs from anti-OX40L treated animals had almost normal bronchial epithelium, and a minimum of infiltrating cells around the bronchioles and blood vessels (). Many PAS+ (mucus-secreting) cells were detected in the airway of mice that were sensitized and challenged and received the control Ab (), whereas treatment with anti-OX40L markedly reduced the number of PAS+ cells ().
Figure 3. Anti-OX40L inhibits lung infiltration, goblet cell hyperplasia, mucus, serum IgE, and BAL Th2 cytokine production. Groups of mice were immunized and challenged as described in . 24 h after the final OVA aerosol challenge, lung tissue was stained (more ...)
Allergen-induced IgE and Th2 Cytokine Production Are Impaired in Anti-OX40L–treated Mice.
As an indirect test of whether OX40 signals were controlling the memory Th2 response driving the asthmatic reaction, we measured cytokine levels in the BAL and production of the Th2-associated antibody IgE in the serum. Treatment with anti-OX40L prevented the increase in IgE that resulted from the recall response ( G) and all Th2 cytokines () associated with the secondary response. IFN-γ was either not present or at only low levels in all groups (unpublished data), suggesting that there was not a switch to a Th1 response.
OX40 Signals Control Memory Effector T Cell Accumulation in Secondary Lymphoid Organs.
To determine whether functional antigen-reactive T cells were present in mice where OX40 signals were blocked, OVA-specific proliferation and production of Th2 cytokines was measured in vitro. Strong responses were seen in lung cell cultures from OVA-primed and challenged mice but not in unprimed challenged mice ( , compare grp B and grp A), again reflecting the memory effector response and that a functional primary response did not result from exposure to airborne antigen alone. Those animals treated with anti-OX40L during the recall response showed little or no OVA-specific T cell reactivity in the lungs (, grp C). To test whether recall responses were absent at other sites, spleen and lung draining lymph nodes were examined. Greatly reduced OVA-reactivity was found in secondary lymphoid organs after anti-OX40L treatment (). These results suggest that the effect of OX40/OX40L blockade is not tissue specific, but, rather, that OX40 signaling is critical for the development of functional memory effector cells after memory T cells reencounter antigen.
Figure 4. OX40 signals control memory T cell responses in secondary lymphoid organs. Mice were immunized and challenged as in . (A–D) 1 d after the last OVA challenge, lung (A and B), and lymph node cells (C and D) were cultured in medium alone or (more ...)
As OX40 is only present on antigen-responding or antigen-experienced T cells, we tracked the accumulation of OX40-expressing CD4 cells over time. As shown in (and ), before antigen challenge, only a low number of T cells expressed OX40. After OVA challenge, an large increase was observed in the number of CD4+
cells in the bronchial LN ( E), lung ( F), and BAL ( G) in primed and challenged mice, far greater than in unprimed challenged mice, reflecting the population of responding memory effector T cells. The number of OX40+
cells in the LN peaked at day 1, whereas the number in the BAL rose progressively from day 2 over the 4-d aerosol exposure. This suggests that the initial memory T cell response developed in the secondary lymphoid organs, and within 24 h the T cells started to migrate to the lung. This correlates with other published data (31
). Significantly, treatment of mice with anti-OX40L reduced the number of CD4+
cells visualized in the LN at the peak of response at day 1, and subsequently. Consequently, few activated OX40+
T cells were then observed in the lung and BAL. These results suggest that OX40/OX40L interactions are required early in the response of a memory T cell and control the ability to expand in numbers and survive in order to form a large population of memory effector T cells.
As further evidence for this, we performed kinetic blocking experiments where mice received anti-OX40L 1, 2, or 3 d after the initial aerosol (). Although delaying anti-OX40L treatment by 1 or 2 d reduced the response by 50% and 30%, the most dramatic effect was seen when OX40/OX40L interactions were inhibited at the time of initial encounter with recall antigen.
Primed OX40-deficient OVA-specific Th2 Cells Do Not Survive Efficiently in Recall Responses.
To further investigate the requirement for OX40 in recall responses of antigen-primed Th2 cells, OVA-specific OX40-deficient CD4 cells were produced by crossing OX40-knockout mice to OT-II TCR transgenic mice. Naive CD4 cells from wild-type (OX40+/+
) or OX40-deficient (OX40−/−
) OT-II mice were cultured in vitro with peptide and APCs under Th2 (IL-4, anti-IL-12, and anti-IFN-γ) polarizing conditions for a time period that allows primary T cell expansion and contraction to proceed normally. Previous data have shown that such in vitro–stimulated cells mimic in vivo generated memory cells (32
) and can also be used in adoptive transfer experiments to directly induce lung inflammation (34
Restimulation of primed OX40+/+ Th2 cells in vitro with OX40L-sufficient APCs resulted in proliferation, expansion, survival, and production of Th2 cytokines () . Primed OX40−/− T cells secreted Th2 cytokines normally, suggesting that there is not a role for OX40 in this T cell activity, and OX40−/− T cells also initially proliferated comparably with wt T cells. However, far fewer T cells survived in the secondary response in the absence of OX40 signals, which was also reflected in weak proliferation late in culture ().
Figure 5. Primed OX40−/− T cells do not survive efficiently in recall responses in vitro. OVA-specific Th2 memory cells were generated in vitro from wild-type (OX40+/+, closed symbols) or OX40-deficient (OX40−/−, open symbols) OT-II (more ...)
To determine if a similar requirement for OX40 was apparent in vivo during a recall response to aerosolized antigen, in vitro–primed T cells were labeled with CFSE, adoptively transferred into naive recipients, and then these mice were challenged intranasally with OVA () . Challenge with PBS did not result in division () or expansion () of either OX40+/+ or OX40−/− T cells in draining lymph nodes or lung. Challenge with OVA resulted in pronounced division of all OX40+/+ T cells and accumulation in numbers was observed in lymph nodes and particularly evident in the lung. Correlating with the in vitro data, recovered OX40−/− T cells displayed the same division profile as their wt counterparts (), but in total approximately fourfold fewer accumulated at the end of the antigen challenge period (). This directly mimicked the data obtained by tracking OX40-expressing T cells after OX40L blockade (). This shows that OX40 signals regulate the number of memory effector T cells that are generated after memory T cells reencounter antigen.
Figure 6. Primed OX40−/− T cells do not accumulate efficiently in recall responses in vivo. OVA-specific Th2 memory cells were generated in vitro as in , from wild-type (OX40+/+) or OX40-deficient (OX40−/−) OT-II TCR transgenic (more ...)
Primed OX40-deficient OVA-specific Th2 Cells Do Not Efficiently Promote Lung Inflammation.
To correlate a lack of expansion/survival of memory effector cells with reduced lung inflammation, eosinophilia, lung histology, and Th2 cytokines were analyzed after adoptive transfer of primed OX40−/− OT-II Th2 cells () . Transfer of wild-type cells followed by repeated intranasal OVA challenge induced profound inflammation accompanied by production of Th2 cytokines in BAL (). In contrast, mice receiving OX40-deficient cells showed only a small increase in the total numbers of eosinophils recovered from the BAL ( A), relatively normal lung histology (compare E to 7 C), and reduced Th2 cytokines ( F). These results clearly show that OX40 expressed on a memory Th2 cell is required for lung inflammation.
Figure 7. Primed OX40-deficient T cells cannot induce pronounced airway inflammation. OVA-specific Th2 memory cells were generated in vitro as in , from wild-type (OX40+/+) or OX40-deficient (OX40−/−) OT-II TCR transgenic mice. Primed T cells (more ...)
OX40/OX40L Interactions Control both Late Secondary and Tertiary Recall Responses to Inhaled Antigen.
As our initial studies addressed the role of OX40 signals in the recall response of what can been termed early memory T cells, persisting 4 wk after primary immunization, we determined whether OX40/OX40L interactions were also critical to the response of memory T cells that persist for longer periods of time and survive following a secondary response. In one set of experiments, mice were primed for 8 wk and anti-OX40L administered during the secondary response to aerosolized antigen ( A). Again, blocking OX40 signals strongly inhibited all aspects of lung inflammation, including total leukocyte ( B) and eosinophil ( C) infiltration, and production of Th2 cytokines (unpublished data). In further experiments, mice were primed for 4 wk, challenged with aerosolized antigen in a secondary response without blocking OX40L, rested for 13 wk, and then challenged again but with anti-OX40L blockade ( D). Inhibiting OX40 signals during this tertiary response again strongly inhibited lung inflammation (), with a greater than 50% reduction in cellular infiltration. As no lung inflammation was observed in unsensitized control mice that were repeatedly exposed to aerosolized antigen, ruling out any primary response (, grp A), these data demonstrate that blocking OX40/OX40L interactions severely limits the recall response of memory Th2 populations that can persist for extended periods of time.
Figure 8. OX40/OX40L interactions control both late secondary and tertiary recall responses to inhaled antigen. (A) Immunization protocol for secondary response of late memory T cells. Unprimed control mice were injected i.p. with alum alone (Alum), while primed (more ...)