Christopher D. Rudulier

The Number of Responding CD4 T Cells and the Dose of Antigen Conjointly Determine the Th1/Th2 Phenotype by Modulating B7/CD28 Interactions

Our previous in vivo studies show that both the amount of Ag and the number of available naive CD4 T cells affect the Th1/Th2 phenotype of the effector CD4 T cells generated. We examined how the number of OVA-specific CD4 TCR transgenic T cells affects the Th1/Th2 phenotype of anti-SRBC CD4 T cells generated in vivo upon immunization with different amounts of OVA-SRBC. Our observations show that a greater number of Ag-dependent CD4 T cell interactions are required to generate Th2 than Th1 cells. We established an in vitro system that recapitulates our main in vivo findings to more readily analyze the underlying mechanism. The in vitro generation of Th2 cells depends, as in vivo, upon both the number of responding CD4 T cells and the amount of Ag. We demonstrate, using agonostic/antagonistic Abs to various costimulatory molecules or their receptors, that the greater number of CD4 T cell interactions, required to generate Th2 over Th1 cells, does not involve CD40, OX40, or ICOS costimulation, but does involve B7/CD28 interactions. A comparison of the level of expression of B7 molecules by APC and CD4 T cells, under different conditions resulting in the substantial generation of Th1 and Th2 cells, leads us to propose that the critical CD28/B7 interactions, required to generate Th2 cells, may directly occur between CD4 T cells engaged with the same B cell acting as an APC.

Direct demonstration of CD4 T cell cooperation in the primary in vivo generation of CD4 effector T cells

Many observations bear upon the cellular and molecular requirements for CD4 T cell activation. The interaction of CD4 T cells with dendritic cells (DC), central to the induction of most immune responses, is the most studied. However, leukocytes other than DC can dramatically affect the induction and differentiation of CD4 T cells into effector cells. We recently provided indirect evidence that in vivo CD4 T cooperation facilitates the activation of CD4 T cells. Here, we demonstrate that the activation of CD4 T cells, specific for the hen egg lysozyme (HEL)(105) (-120) peptide, is optimally achieved when BALB/c mice are immunized with additional MHC class II-binding HEL peptides in incomplete Freunds adjuvant. This cooperation cannot be mimicked by the coadministration of LPS or of an agonistic antibody to CD40, at the time of immunization. In contrast, OX40-OX40L interactions are necessary for CD4 T cell cooperation in that an OX40 agonistic antibody can replace, and an OX40L-blocking antibody can abrogate, CD4 T cell cooperation in situations where such cooperation would otherwise enhance the activation of CD4 T cells.

An Update on Lymphocyte Subtypes in Asthma and Airway Disease

&NA; Inflammation is a hallmark of many airway diseases. Improved understanding of the cellular and molecular mechanisms of airway disease will facilitate the transition in our understanding from phenotypes to endotypes, thereby improving our ability to target treatments based on pathophysiologic characteristics. For example, allergic asthma has long been considered to be driven by an allergen‐specific T helper 2 response. However, clinical and mechanistic studies have begun to shed light on the role of other cell subsets in the pathogenesis and regulation of lung inflammation. In this review, we discuss the importance of different lymphocyte subsets to asthma and other airway diseases, while highlighting the growing evidence that asthma is a syndrome that incorporates many immune phenotypes.

Treatment with anti-cytokine monoclonal antibodies can potentiate the target cytokine rather than neutralize its activity.

Airway diseases such as allergic rhinitis and allergic asthma are due to a Th2 response to innocuous environmental antigens. Animal models have shown that the antibody-mediated neutralization of Th2 cytokines can greatly diminish airway inflammation (1–3); these results lead to the development and clinical investigation of humanized anti-Th2 cytokine antibodies for the amelioration of asthma (4–8). Unfortunately, clinical trials of these biologics have not been a resounding success, with inabilities to significantly improve clinical symptoms being common and successful trials requiring strict patient stratification (9). These failures have spawned numerous plausible explanations, such as a requirement to neutralize more than one Th2 cytokine to achieve a diseasemodifying effect or that antibodies with a greater affinity for the target cytokine may be required. Alternatively, a welldocumented phenomenon may be hampering clinical efficacy, namely the potentiation of cytokines through the formation of cytokine/anti-cytokine immune complexes. The formation of cytokine/anti-cytokine immune complexes does not guarantee cytokine neutralization, as these complexes can actually increase the potency of a cytokine. Due to their bivalent nature, antibodies form immune complexes when neither the antibody nor the antigen is in excess. Murine studies have shown that cytokine/anti-cytokine immune complexes can potentiate the activity of numerous cytokines. For instance, IL-4/anti-IL-4 complexes are much more efficient than IL-4 alone at stimulating B cells, increasing their production of IgE and inducing the proliferation of CD8 T cells (10–12). Similarly, IL-2/anti-IL-2 complexes are much more potent in their ability to expand memory CD8 T cells, NK cells or regulatory T cells (Tregs) than IL-2 alone (12–15). An increase in the potency and bioactivity of IL-3, IL-6 and IL-7, when complexed with their respective antibodies, has also been reported (11, 16–22). Furthermore, recent studies have demonstrated the ability of IL-2/anti-IL-2 complexes to cause type 2 innate lymphoid cells (ILC2s) to expand, produce IL-5 and induce airway eosinophilia (23, 24). Together, these studies establish that the formation of immune complexes can potentiate the effects of the cytokine rather than neutralize its activity. The ability of immune complexes to increase the potency of the cytokine has also been described in humans. Stein et al. reported the presence of IL-5/anti-IL-5 complexes in the circulation of subjects treated with mepolizumab and that these immune complexes facilitated increased production of IL-5 by CD4 T cells (25). Indirect evidence that immune complexes can worsen disease comes from a dose-finding study using lebrikizumab, an anti-IL-13 antibody, in subjects with moderate to severe asthma in which there was a direct correlation between the exacerbation rate and the dose of lebrikizumab in periostin-high subjects (26). Although the presence of IL-13/lebrikizumab complexes was not assessed, the seemingly paradoxical dose response of lebrikizumab is readily explained by the hypothesis that higher doses of lebrikizumab led to increased complex formation; this is also in line with the observations from the murine studies outlined above. The potentiation of cytokine activity is not restricted to antibodies but also extends to decoy receptors. For instance, when subjects with ankylosing spondylitis were treated with etanercept, which is a dimerized TNF receptor/Fc fusion protein designed to bind to and neutralize soluble TNF-a, a significant increase in the percentage of CD4 and CD8 T cells producing TNF-a and IFN-c was reported (27). Collectively, these data extend the findings of murine studies to humans and emphasize that attempting to neutralize free cytokine with antibodies or decoy receptors may be an inefficient approach, as complexes can potentiate cytokine activity rather than neutralize it. Treatment of subjects with neutralizing antibodies can not only increase the activity of the cytokine but may also increase its levels in the circulation. For example, subjects treated with mepolizumab displayed greater levels of IL-5 in the circulation with the majority of it bound in IL-5/anti-IL5 complexes (17), while the treatment of asthmatic subjects with anti-IL-13 antibodies directly increased the levels of IL13 in the serum (7). Similarly, the treatment of patients with metastatic breast cancer with etanercept increased TNF-a levels in the plasma, demonstrating that this effect is not restricted to Th2 cytokines and diseases (28). Studies in mice using antibodies to IL-2, 4, 6 and 7 have paralleled these clinical findings as greater bioactive levels of the targeted cytokine have been found following antibody treatment (12, 14, 16). Despite these observations, there appears to be little importance placed upon the formation of cytokine/anti-cytokine complexes in subjects treated with monoclonal antibodies as it is largely assumed that cytokines present in immune complexes are neutralized. However, as we have discussed, studies have demonstrated that the neutralization of a cytokine by its binding to antibody is not a foregone conclusion and further studies in humans are required. The formation of immune complexes is prevented when the neutralizing antibody is present in excess. This concept is very clearly exhibited in early studies on the stimulatory potential of cytokine/anti-cytokine complexes where an

Distinct roles of dendritic and B cells in the activation of naive CD4+ T cells

Peter A Bretscher Author for correspondence: Department of Microbiology & Immunology, University of Saskatchewan, A305–107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada Tel.: +1 306 966 4322 Fax: +1 306 966 4298 peter.bretscher@usask.ca Naive CD4 T cells are activated by antigenpresenting dendritic cells (DCs) when these DCs express sufficient and appropriate costimulatory molecules. Classical studies have led to the idea that activated DCs, presenting the appropriate antigen, are sufficient for the robust activation of naive CD4 T cells. However, recent studies have demonstrated that in addition to activated DCs, B cells are also required for optimal CD4 T-cell responses. Herein, we discuss such studies and speculate on the importance of B cells in limiting the activation of autoreactive CD4 T cells.

The activation, by antigen, of naïve TCR transgenic CD4 T cells cultured at physiological, rather than artificially high, frequencies more accurately reflects the in vivo activation of normal numbers of naïve CD4(+) T cells.

The majority of in vitro studies investigating the activation of naïve TCR transgenic T cells routinely employ an artificially high frequency of such cells. To assess whether employing high frequencies of TCR transgenic cells in vitro accurately reflects the in vivo activation of a normal number of T cells, we cultured between 300 and 3×10(6) Rag2(-/-) DO11.10 T cells per well under otherwise identical conditions. We find that those T cells cultured at low frequencies proliferate more and are more potently activated, as assessed by the expression of CD44 and CD62L, each giving rise to a much larger number of cytokine producing cells, comparable to the number generated in vivo when a normal number of CD4(+) T cells are activated. The effect of T cell frequency on the level of their activation was not due to differences in MHCII or CD80/86 expression by B cells, the major APC population present, nor to increased death of B cells in high frequency cultures. Taken together, our observations illustrate the necessity of culturing naïve TCR transgenic CD4(+) T cells at a physiological frequency if one is to more accurately recapitulate the in vivo activation of naïve CD4(+) T cells.