Identification and monitoring of effector and regulatory T cells during experimental arthritis based on differential expression of CD25 and CD134.

Major problems in the analysis of CD4+ effector cell and regulatory T cell (Treg) populations in an activated immune system are caused by the facts that both cell types can express CD25 and that the discriminatory marker forkhead box p3 can only be analyzed in nonviable (permeabilized) cells. Here, we show that CD134 (OX40) can be used as a discriminatory marker combined with CD25 to isolate and characterize viable CD4+ effector cells and Tregs. Before and during adjuvant arthritis in rats, coexpression of CD134 and CD25 identified activated Tregs consistently, as these T cells proliferated poorly to disease-associated antigens and were suppressive in vitro and in vivo. Depending on the time of isolation and location, CD4+ T cell populations expressing CD134 or CD25 contained effector/memory T cells. Analysis of the function, phenotype, and amount of the CD4+ T cell subsets in different lymph node stations revealed spatiotemporal differences in effector cell and Treg compartments during experimental arthritis.

CD134 as target for specific drug delivery to auto-aggressive CD4+ T cells in adjuvant arthritis.

T cells have an important role during the development of autoimmune diseases. In adjuvant arthritis, a model for rheumatoid arthritis, we found that the percentage of CD4+ T cells expressing the activation marker CD134 (OX40 antigen) was elevated before disease onset. Moreover, these CD134+ T cells showed a specific proliferative response to the disease-associated epitope of mycobacterial heat shock protein 60, indicating that this subset contains auto-aggressive T cells. We studied the usefulness of CD134 as a molecular target for immune intervention in arthritis by using liposomes coated with a CD134-directed monoclonal antibody as a drug targeting system. Injection of anti-CD134 liposomes subcutaneously in the hind paws of pre-arthritic rats resulted in targeting of the majority of CD4+CD134+ T cells in the popliteal lymph nodes. Furthermore, we showed that anti-CD134 liposomes bound to activated T cells were not internalized. However, drug delivery by these liposomes could be established by loading anti-CD134 liposomes with the dipalmitate-derivatized cytostatic agent 5'-fluorodeoxyuridine. These liposomes specifically inhibited the proliferation of activated CD134+ T cells in vitro, and treatment with anti-CD134 liposomes containing 5'-fluorodeoxyuridine resulted in the amelioration of adjuvant arthritis. Thus, CD134 can be used as a marker for auto-aggressive CD4+ T cells early in arthritis, and specific liposomal targeting of drugs to these cells via CD134 can be employed to downregulate disease development.

Mechanisms of macrophage cytotoxicity in IL-2 and IL-12 mediated tumour regression.

IL-2 and IL-12 are promising anti-tumour agents. However, little attention has been paid to the role of macrophages during IL-2/IL-12 mediated tumour rejection. We studied the role of macrophages during IL-2/IL-12 mediated tumour rejection in DBA/2 mice bearing syngeneic SL2 lymphoma. Local treatment with IL-2 and IL-12 cured 85% of mice with severe metastasised tumour load. In vivo depletion studies showed that macrophages were required for the anti-tumour effect of IL-2 and IL-12. Macrophages could kill tumour cells both non-specifically and by antibody-dependent cellular cytotoxicity (ADCC). Treatment with IL-2, IL-12 or IL-2/IL-12 enhanced production of specific IgG1 immunoglobulins, while treatment with IL-12 and IL-2/IL-12 additionally induced IgG2a production. FcgammaRII and/or III were essential for ADCC expression after treatment with IL-2 and IL-12. These data show for the first time the essential role of macrophages during IL-2/IL-12 mediated tumour rejection and also suggest that IL-2 and IL-12 act via different mechanisms.

Liposomal meningococcal B vaccination: role of dendritic cell targeting in the development of a protective immune response.

The effect of targeting strategies for improving the interaction of liposomal PorA with dendritic cells (DC) on the immunogenicity of PorA was investigated. PorA, a major antigen of Neisseria meningitidis, was purified and reconstituted in different types of (targeted) liposomes, i.e., by using mannose or phosphatidylserine as targeting moieties, or with positively charged liposomes. We studied the efficiency of liposome uptake and its effect on the maturation of and interleukin 12 (IL-12) production by murine DC. Moreover, mice were immunized subcutaneously to study the localization and immunogenicity of PorA liposomes. Uptake of liposomes by DC was significantly increased for targeted liposomes and resulted in the maturation of DC, but to various degrees. Maturation markers (i.e., CD80, CD86, major histocompatibility complex class II, and CD40) showed enhanced expression on DC incubated with targeted PorA liposomes relative to those incubated with nontargeted PorA liposomes. Moreover, only the uptake of targeted PorA liposomes induced production of IL-12 by DC, with levels similar to those produced by lipopolysaccharide (LPS)-pulsed DC. Mannose-targeted PorA liposomes administered subcutaneously had an increased localization in draining lymph nodes compared to nontargeted PorA liposomes. Liposomes in draining lymph nodes interacted preferentially with antigen-presenting cells, an effect that was enhanced with targeted PorA liposomes. Immunization studies showed an improvement of the bactericidal antibody response (i.e., increased number of responders) generated by targeted PorA liposomes compared to that generated by nontargeted ones or LPS-containing outer membrane vesicles. In conclusion, the use of targeted PorA liposomes results in an improved uptake by and activation of DC and an increased localization in draining lymph nodes. These effects correlate with an enhanced immune response toward the vaccine.