Subtypes of type I IFN differentially enhance cytokine expression by suboptimally stimulated CD4+ T cells

Human type I interferons (IFNs) include IFN‐β and 12 subtypes of IFN‐α. During viral infection, infiltrating memory CD4 ⁺ T cells are exposed to IFNs, but their impact on memory T‐cell function is poorly understood. To address this, we pretreated PBMCs with different IFNs for 16 h before stimulation with Staphylococcus aureus enterotoxin B and measured cytokine expression by flow cytometry. IFN‐α8 and ‐α10 most potently enhanced expression of IFN‐γ, IL‐2, and IL‐4. Potency among the subtypes differed most at doses between 10 and 100 U/mL. While enhancement of IL‐2 and IL‐4 correlated with the time of preincubation with type I IFN, IFN‐γ production was enhanced best when IFN‐α was added immediately preceding or simultaneously with T‐cell stimulation. Comparison of T‐cell responses to multiple doses of Staphylococcus aureus enterotoxin B and to peptide libraries from RSV or CMV demonstrated that IFN‐α best enhanced cytokine expression when CD4 ⁺ T cells were suboptimally stimulated. We conclude that type I IFNs enhance Th1 and Th2 function with dose dependency and subtype specificity, and best when T‐cell stimulation is suboptimal. While type I IFNs may beneficially enhance CD4 ⁺ T‐cell memory responses to vaccines or viral pathogens, they may also enhance the function of resident Th2 cells and exacerbate allergic inflammation.     

Differential Expression of CD30 on CD3 T Lymphocytes in Patients with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune systemic disease caused as a result of an imbalance of Th1‐/Th2‐type cytokines. The soluble form of CD30 (CD30s) released from peripheral blood cells has been described as a marker of active disease in Th2‐type immune response as in SLE. However, the expression of CD30 on CD3 T lymphocytes from patients with SLE has not been studied yet. Therefore, we have addressed our study to attempt this issue, studying CD30 expression by flow cytometry on CD3 T lymphocytes and CD4/CD8 subsets in samples from SLE patients mainly with lupus nephritis. In parallel, we have determined the production of the cytokines IL‐4 (Th2), IFNγ (Th1), IL‐10 and TGFβ by intracellular staining. Differences between positive CD30 T cells in healthy controls and patients with SLE were found, with a higher percentage of CD30‐expressing T cells in patients with SLE (P = 0.001). In contrast to healthy controls, CD30 was mainly expressed on CD8 T cells from patients with SLE. The intracellular cytokine staining showed that TGFβ is the main cytokine expressed in CD3 T cells from patients with SLE. In addition to this, we have found a positive correlation between CD30‐expressing T cells and IL‐4, IFNγ, and immunosuppressive cytokines (IL‐10 and TGFβ) (P < 0.05). These results suggest that CD30 could play a role in the pathogenesis of SLE and its expression on CD3 T lymphocytes is not restricted only to Th2‐type response.     

DX5+CD4+ T cells modulate cytokine production by CD4+ T cells towards IL‐10 via the production of IL‐4

CD4⁺ Th cells play a critical role in orchestrating the adaptive immune response. Uncontrolled Th1 responses are implicated in the pathogenesis of autoimmune diseases. T cells with immune‐modulatory properties are beneficial for inhibiting such inflammatory responses. Previously we demonstrated that repetitive injections of immature DC induce expansion of DX5⁺CD4⁺ T cells, which upon adoptive transfer show potent regulatory properties in murine collagen‐induced arthritis as well as in delayed‐hypersensitivity models. However, their regulatory mechanism remains to be defined. Here, we analyzed the effect of DX5⁺CD4⁺ T cells on other CD4⁺ T cells in vitro. Although proliferation of naïve CD4⁺ T cells upon antigenic triggering was not altered in the presence of DX5⁺CD4⁺ T cells, there was a striking difference in cytokine production. In the presence of DX5⁺CD4⁺ T cells, an IL‐10‐producing CD4⁺ T‐cell response was induced instead of a predominant IFN‐γ‐producing Th1 response. This modulation did not require cell–cell contact. Instead, IL‐4 produced by DX5⁺CD4⁺ T cells was primarily involved in the inhibition of IFN‐γ and promotion of IL‐10 production by CD4⁺ T cells. Together, our data indicate that DX5⁺CD4⁺ T cells modulate the outcome of Th‐responses by diverting Th1‐induction into Th responses characterized by the production of IL‐10.     

Relationship of CD146 expression to secretion of interleukin (IL)‐17, IL‐22 and interferon‐γ by CD4+ T cells in patients with inflammatory arthritis

Expression of the adhesion molecule, CD146/MCAM/MelCAM, on T cells has been associated with recent activation, memory subsets and T helper type 17 (Th17) effector function, and is elevated in inflammatory arthritis. Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) and spondyloarthritides (SpA). Here, we compared the expression of CD146 on CD4⁺ T cells between healthy donors (HD) and patients with RA and SpA [ankylosing spondylitis (AS) or psoriatic arthritis (PsA)] and examined correlations with surface markers and cytokine secretion. Peripheral blood mononuclear cells (PBMC) were obtained from patients and controls, and synovial fluid mononuclear cells (SFMC) from patients. Cytokine production [elicited by phorbol myristate acetate (PMA)/ionomycin] and surface phenotypes were evaluated by flow cytometry. CD146⁺CD4⁺ and interleukin (IL)‐17⁺CD4⁺ T cell frequencies were increased in PBMC of PsA patients, compared with HD, and in SFMC compared with PBMC. CD146⁺CD4⁺ T cells were enriched for secretion of IL‐17 [alone or with IL‐22 or interferon (IFN)‐γ] and for some putative Th17‐associated surface markers (CD161 and CCR6), but not others (CD26 and IL‐23 receptor). CD4⁺ T cells producing IL‐22 or IFN‐γ without IL‐17 were also present in the CD146⁺ subset, although their enrichment was less marked. Moreover, a majority of cells secreting these cytokines lacked CD146. Thus, CD146 is not a sensitive or specific marker of Th17 cells, but rather correlates with heterogeneous cytokine secretion by subsets of CD4⁺ helper T cells.     

High Functional CD70 Expression on α‐Type 1‐Polarized Dendritic Cells from Patients with Chronic Lymphocytic Leukaemia

Antigen‐loaded dendritic cells (DCs) used as anticancer vaccine holds promise for therapy, but needs to be optimized. The most frequently described DC vaccine is being matured with a cocktail containing prostaglandin E₂ (PGE₂DC). However, even though PGE₂DCs express both costimulatory and migratory receptors, their IL‐12p70‐prodcution is low, leading to an insufficient Th1 immune response. As an alternative, α‐type‐1 polarized DCs (αDC1s) have shown a superior production of IL‐12p70 and subsequent activation of effector cells. From chronic lymphocytic leukaemia (CLL) patients, αDC1s can be generated to induce a functional Th1‐immune response. Yet, another costimulatory receptor, CD70, appears to be essential for optimal DC function by promotion of T cell survival and function. So far, PGE₂ is suggested as one of the most important factors for the induction of CD70 expression on DCs. Therefore, we wanted to investigate whether αDC1s have the ability to express functional CD70. We found that CD70 expression on αDC1s could be upregulated in the same manner as PGE₂DCs. In an allogeneic mixed leucocyte reaction, we found that antibody‐blocking of CD70 on αDC1s from controls reduced effector cell proliferation although this could not be found when using CLL αDC1s. Nevertheless, CD70‐blocking of αDC1s from both controls and patients with CLL had a negative influence on the production of both IL‐12p70 and the Th1 cytokine IFN‐γ, while the production of the Th2 cytokine IL‐5 was enhanced. Together, this study further suggests that αDC1s should be considered as a suitable candidate for clinical antitumour vaccine strategies in patients with CLL.     

Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues

Expression of the lymph node homing and CC‐chemokine receptor 7 (CCR7), with L‐selectin (CD62L), has been shown to divide human memory T cells into two functionally distinct subsets. We generated a polyclonal antibody against murine CCR7 and used this antibody to study CCR7 expression on murine T‐cell subsets. Using flow cytometric staining of T cells for visualisation expression of CCR7 in association with CD62L and CD44, a major population of CD4 or CD8 T cells expressing CCR7 were found to be CD62L^high CD44^low, which would suggest a naïve cell phenotype. By analogy with human studies, memory cells could be subdivided into CCR7^high CD62L^high CD44^high (central memory) and CCR7^low CD62L^low CD44^high (effector memory). The proportions of these populations were different in lymph node, blood and spleen. Functional, short‐term in vitro polyclonal stimulation of blood, spleen and lymph node cells from naive mice demonstrated that CCR7^high CD4 T cells produced predominantly interleukin (IL)‐2, whereas CCR7^low CD4 T cells produced both IL‐2 and interferon‐γ (IFN‐γ). However, in contrast to previously published reports, the CCR7^high CD8 T‐cell subpopulation produced both IFN‐γ and IL‐2. Analysis of effector T cells, induced by immunization in vivo, showed that a proportion of activated naïve CD4 T cells down‐regulated CCR7 only after multiple cell divisions, and this coincided with the down‐regulation of CD62L and production of IL‐4 and IFN‐γ. Finally, analysis of effector T cells during the phase of maximal clonal expansion of secondary immune responses in vivo indicated that the vast majority of both IL‐2‐ and IFN‐γ‐producing cells are CCR7^low, while few cytokine‐expressing CCR7^high T cells were detected. Our results support the hypothesis, developed from studies with human cells, that CCR7 may separate functionally different murine memory T‐cell subpopulations, but indicate additional complexity in that CCR7^high CD8 T cells also may produce IFN‐γ.     

Regulation by cytokines (IL‐12, IL‐15, IL‐4 and IL‐10) of the Vγ9Vδ2 T cell response to mycobacterial phosphoantigens in responder and anergic HIV‐infected persons

Human Vγ9Vδ2 T cells contribute to immunity against intracellular pathogens and recognize nonpeptidic antigens, such as the mycobacterial phosphoantigen TUBAg. HIV infection is associated with a polyclonal decrease of peripheral Vγ9Vδ2 T cells and we previously reported that the remaining cells show a proliferative anergy to stimulation with Mycobacterium tuberculosis in 60 % of patients. Because of alterations in the Th1/Th2 cytokine balance reported in HIV infection, we analyzed, at the single‐cell level, the influence of exogenous IL‐4, IL‐10, IL‐12 and IL‐15 on the response to mycobacterial phosphoantigens of γ δ T cells from HIV‐infected patients and healthy donors. We report that the strong γ δ T cell response to TUBAg is characterized by the rapid and selective production of the Th1/proinflammatory cytokines IFN‐γ and TNF‐α in responder HIV‐infected donors. In addition, a positive regulation by IL‐12 and IL‐15 of the production of these cytokines by Vγ9Vδ2 T cells in response to nonpeptidic ligands was observed, whereas IL‐4 and IL‐10 had no effect. In contrast, Vγ9Vδ2 T cells from the anergic HIV‐infected donors had lost the ability to produce Th1 cytokines and were not shifted towards a Th2 profile. Furthermore, neither IL‐12 nor IL‐15 could reverse this functional anergy. The consequences of these observations are discussed in the context of HIV pathogenesis.     

T‐cell‐derived,but not B‐cell‐derived,IL‐10 suppresses antigen‐specific T‐cell responses in Litomosoides sigmodontis‐infected mice

IL‐10, a cytokine with pleiotropic functions is produced by many different cells. Although IL‐10 may be crucial for initiating protective Th2 responses to helminth infection, it may also function as a suppressive cytokine preventing immune pathology or even contributing to helminth‐induced immune evasion. Here, we show that B cells and T cells produce IL‐10 during murine Litomosoides sigmodontis infection. IL‐10‐deficient mice produced increased amounts of L. sigmodontis‐specific IFN‐γ and IL‐13 suggesting a suppressive role for IL‐10 in the initiation of the T‐cell response to infection. Using cell type‐specific IL‐10‐deficient mice, we dissected different functions of T‐cell‐ and B‐cell‐derived IL‐10. Litomosoides sigmodontis‐specific IFN‐γ, IL‐5, and IL‐13 production increased in the absence of T‐cell‐derived IL‐10 at early and late time points of infection. In contrast, B‐cell‐specific IL‐10 deficiency did not lead to significant changes in L. sigmodontis‐specific cytokine production compared to WT mice. Our results suggest that the initiation of Ag‐specific cellular responses during L. sigmodontis infection is suppressed by T‐cell‐derived IL‐10 and not by B‐cell‐derived IL‐10.     

Interleukin‐10‐secreting T cells define a suppressive subset within the HIV‐1‐specific T‐cell population

Recent studies have indicated that Treg contribute to the HIV type 1 (HIV‐1)‐related immune pathogenesis. However, it is not clear whether T cells with suppressive properties reside within the HIV‐1‐specific T‐cell population. Here, PBMC from HIV‐1‐infected individuals were stimulated with a 15‐mer Gag peptide pool, and HIV‐1‐specific T cells were enriched by virtue of their secretion of IL‐10 or IFN‐γ using immunomagnetic cell‐sorting. Neither the IL‐10‐secreting cells nor the IFN‐γ‐secreting cells expressed the Treg marker FOXP3, yet the IL‐10‐secreting cells potently suppressed anti‐CD3/CD28‐induced CD4⁺ as well as CD8⁺ T‐cell proliferative responses. As shown by intracellular cytokine staining, IL‐10‐ and IFN‐γ‐producing T cells represent distinct subsets of the HIV‐1‐specific T cells. Our data collectively suggest that functionally defined HIV‐1‐specific T‐cell subsets harbor potent immunoregulatory properties that may contribute to HIV‐1‐associated T‐cell dysfunction.     

Regulatory effects of IFN‐β on production of osteopontin and IL‐17 by CD4+ T Cells in MS

IFN‐β currently serves as one of the major treatments for MS. Its anti‐inflammatory mechanism has been reported as involving a shift in cytokine balance from Th1 to Th2 in the T‐cell response against elements of the myelin sheath. In addition to the Th1 and Th2 groups, two other important pro‐inflammatory cytokines, IL‐17 and osteopontin (OPN), are believed to play important roles in CNS inflammation in the pathogenesis of MS. In this study, we examined the potential effects of IFN‐β on the regulation of OPN and IL‐17 in MS patients. We found that IFN‐β used in vitro at 0.5–3 ng/mL significantly inhibited the production of OPN in primary T cells derived from PBMC. The inhibition of OPN was determined to occur at the CD4⁺ T‐cell level. In addition, IFN‐β inhibited the production of IL‐17 and IL‐21 in CD4⁺ T cells. It has been described that IFN‐β suppresses IL‐17 production through the inhibition of a monocytic cytokine, the intracellular translational isoform of OPN. Our further investigation demonstrated that IFN‐β also acted directly on the CD4⁺ T cells to regulate OPN and IL‐17 expression through the type I IFN receptor‐mediated activation of STAT1 and suppression of STAT3 activity. Administration of IFN‐β to EAE mice ameliorated the disease severity. Furthermore, spinal cord infiltration of OPN⁺ and IL‐17⁺ cells decreased in IFN‐β‐treated EAE mice along with decreases in serum levels of OPN and IL‐21. Importantly, decreased OPN production by IFN‐β treatment contributes to the reduced migratory activity of T cells. Taken together, the results from both in vitro and in vivo experiments indicate that IFN‐β treatment can down‐regulate the OPN and IL‐17 production in MS. This study provides new insights into the mechanism of action of IFN‐β in the treatment of MS.     

Species‐dependent role of type I IFNs and IL‐12 in the CTL response induced by humanized CpG complexed with β‐glucan

CpG oligodeoxynucleotide (ODN) is one of promising nucleic acid‐based adjuvants. We recently improved its ability to enhance CD8⁺ T‐cell responses to coadministered protein antigen without conjugation or emulsion, by forming a nanoparticulate complex between CpG ODN (K3) and mushroom‐derived β‐glucan schizophyllan (SPG), namely K3‐SPG. Here, we sought to elucidate the cellular immunological mechanisms by which K3‐SPG induce such potent CD8⁺ T‐cell responses to coadministered antigen. By focusing on two DC subsets, plasmacytoid DCs and CD8α⁺ DCs, as well as the secreted cytokines, IFN‐α and IL‐12, we found that K3‐SPG strongly activates mouse plasmacytoid DCs to secrete IFN‐α and CD8α⁺ DCs to secrete IL‐12, respectively. Although a single cytokine deficiency had no impact on adjuvant effects, the lack of both type I IFN and IL‐12 in mice resulted in a significant reduction of Th1 type immune responses and CD8⁺ T‐cell responses elicited by protein vaccine model. By sharp contrast, type I IFN, but not IL‐12, was required for the production of IFN‐γ by human PBMCs as well as antigen‐specific CD8⁺ T‐cell proliferation. Taken together, K3‐SPG may overcome the species barrier for CpG ODN to enhance antigen‐specific CD8⁺ T‐cell responses despite the differential role of IL‐12 between human and mice.     

M2 polarization of murine peritoneal macrophages induces regulatory cytokine production and suppresses T‐cell proliferation

Bone‐marrow‐derived macrophages are divided into two phenotypically and functionally distinct subsets, M1 and M2 macrophages. Recently, it was shown that adoptive transfer of M2‐polarized peritoneal macrophages reduced the severity of experimental colitis in mice. However, it is still unclear whether peritoneal macrophages possess the same ability to be polarized to cells with functionally different phenotypes and cytokine production patterns as bone‐marrow‐derived macrophages. To address this question, we examined the ability of peritoneal macrophages to be polarized to the M1 and M2 phenotypes and determined the specific cytokine profiles of cells with each phenotype. We showed that peritoneal macrophages, as well as bone‐marrow‐derived macrophages, were differentiated into M1 and M2 phenotypes following stimulation with interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4)/IL‐13, respectively. Following in vitro stimulation with lipopolysaccharide, M2‐polarized peritoneal macrophages predominantly expressed T helper type 2 (Th2) cytokines and regulatory cytokines, including IL‐4, IL‐13, transforming growth factor‐β and IL‐10, whereas M1‐polarized peritoneal macrophages expressed negligible amounts of Th1 and pro‐inflammatory cytokines. ELISA showed that M2‐polarized peritoneal macrophages produced significantly more IL‐10 than M1‐polarized peritoneal macrophages. Notably, M2‐polarized peritoneal macrophages contributed more to the suppression of T‐cell proliferation than did M1‐polarized peritoneal macrophages. The mRNA expression of Th2 cytokines, including IL‐4 and IL‐13, increased in T‐cells co‐cultured with M2‐polarized macrophages. Hence, our findings showed that M2 polarization of peritoneal macrophages induced regulatory cytokine production and suppressed T‐cell proliferation in vitro, and that resident peritoneal macrophages could be used as a new adoptive transfer therapy for autoimmune/inflammatory diseases after polarization to the regulatory phenotype ex vivo.     

Antigen dose‐dependent suppression of murine IgE responses is mediated by CD4−CD8− double‐negative T cells

Background The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. Objective The aim of the study was to identify immune cells that are involved in antigen dose‐dependent regulation of IgE formation. Methods Wild‐type mice as well as T helper (Th)1‐deficient IL‐12p40^?/? and IFN‐γ^?/? mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen‐dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets among splenocytes from K01 and K100 mice was compared using fluorocytometry and RT‐PCR analysis. Splenocytes or T cell subpopulations were transferred into naïve mice and the effect of lymphocyte transfer on IgE production after priming of recipients with low antigen doses was determined. Results Specific IgE production was considerably impaired in K100 mice. Antigenic restimulation revealed hypoproliferation of K100 splenocytes and reduced production of Th2 cytokines IL‐4, IL‐5 and IL‐13, but no induction of IFN‐γ production. Moreover, lymphocytes from K01 and K100 mice did not show significant differences in the expression of molecules associated with the phenotype or activity of conventional regulatory T cells. Transfer of splenocytes or purified T cells from K100 mice substantially suppressed the induction of IgE production in the recipients in an antigen‐ and isotype‐specific manner. Neither CD4⁺ nor CD8⁺ T cells from K100 mice were able to inhibit IgE formation; instead, we identified CD4^?CD8^? double‐negative T cells (dnT cells) as the principal T cell population, which potently suppressed IgE production. Conclusion Our data demonstrate that CD4^?CD8^? dnT cells play a major role in the regulation of IgE responses induced by high antigen doses.     

RORγt antagonist suppresses M3 muscarinic acetylcholine receptor‐induced Sjögren's syndrome‐like sialadenitis

We showed recently that M3 muscarinic acetylcholine receptor (M3R)‐reactive CD3⁺ T cells play a pathogenic role in the development of murine autoimmune sialadenitis (MIS), which mimics Sjögren's syndrome (SS). The aim of this study was to determine the effectiveness and mechanism of action of retinoic acid‐related orphan receptor‐gamma t (RORγt) antagonist (A213) in MIS. Splenocytes from M3R knockout (M3R^–/–) mice immunized with murine M3R peptide mixture were inoculated into recombination‐activating gene 1 knockout (Rag‐1^–/–) mice (M3R^–/–→Rag‐1^–/–) with MIS. Immunized M3R^–/– mice (pretransfer treatment) and M3R^–/–→Rag‐1^–/– mice (post‐transfer treatment) were treated with A213 every 3 days. Salivary volume, severity of sialadenitis and cytokine production from M3R peptide‐stimulated splenocytes and lymph node cells were examined. Effects of A213 on cytokine production were analysed by enzyme‐linked immunosorbent assay (ELISA) and on T helper type 1 (Th1), Th17 and Th2 differentiation from CD4⁺ T cells by flow cytometry. Pretransfer A213 treatment maintained salivary volume, improved MIS and reduced interferon (IFN)‐γ and interleukin (IL)‐17 production significantly compared with phosphate‐buffered saline (PBS) (P < 0·05). These suppressive effects involved CD4⁺ T cells rather than CD11c⁺ cells. Post‐transfer treatment with A213 increased salivary volume (P < 0·05), suppressed MIS (P < 0·005) and reduced IFN‐γ and IL‐17 production (P < 0·05). In vitro, A213 suppressed IFN‐γ and IL‐17 production from M3R‐stimulated splenocytes and CD4⁺ T cells of immunized M3R^–/– mice (P < 0·05). In contrast with M3R specific responses, A213 suppressed only IL‐17 production from Th17 differentiated CD4⁺ T cells without any effect on Th1 and Th2 differentiation in vitro. Our findings suggested that RORγt antagonism is potentially suitable treatment strategy for SS‐like sialadenitis through suppression of IL‐17 and IFN‐γ production by M3R‐specific T cells.     

Innate and adaptive stimulation of murine diverse NKT cells result in distinct cellular responses

Natural killer T (NKT) cells recognize glycolipids presented on CD1d. They share features of adaptive T lymphocytes and innate NK cells, and mediate immunoregulatory functions via rapid production of cytokines. Invariant (iNKT) and diverse (dNKT) NKT cell subsets are defined by their TCR. The immunological role of dNKT cells, that do not express the invariant TCRα‐chain used by iNKT cells, is less well explored than that of iNKT cells. Here, we investigated signals driving Toll‐like receptor (TLR) ligand activation of TCR‐transgenic murine dNKT cells. IFN‐γ production by dNKT cells required dendritic cells (DC), cell‐to‐cell contact and presence of TLR ligands. TLR‐stimulated DC activated dNKT cells to secrete IFN‐γ in a CD1d‐, CD80/86‐ and type I IFN‐independent manner. In contrast, a requirement for IL‐12p40, and a TLR ligand‐selective dependence on IL‐18 or IL‐15 was observed. TLR ligand/DC stimulation provoked early secretion of pro‐inflammatory cytokines by both CD62L⁺ and CD62L^? dNKT cells. However, proliferation was limited. In contrast, TCR/co‐receptor‐mediated activation resulted in proliferation and delayed production of a broader cytokine spectrum preferentially in CD62L^? dNKT cells. Thus, innate (TLR ligand/DC) and adaptive (TCR/co‐receptor) stimulation of dNKT cells resulted in distinct cellular responses that may contribute differently to the formation of immune memory.     

Allergen‐specific T cell responses to immunotherapy monitored by CD154 and intracellular cytokine expression

Background A sensitive measurement of low numbers of intracellular cytokine‐expressing antigen‐specific T cells from peripheral blood mononuclear cells (PBMC) is possible using CD154 as a marker of recently activated T cells. This technique may have potential for monitoring peripheral blood T cell responses to immunotherapy. Objective To evaluate the applicability of this method for measuring changes in cytokine production by allergen‐specific T cells in a clinical trial setting. Methods Ex vivo ragweed‐specific CD154 and intracellular cytokine expression were evaluated using a subset of subjects in an environmental chamber study of allergic rhinitis immunotherapy. PBMC were collected and cryopreserved from Amb a 1‐immunostimulatory oligodeoxynucleotide conjugate (AIC)‐treated (n=17) and placebo‐treated (n=15) ragweed‐allergic subjects both after pre‐ and post‐treatment ragweed exposures. In vitro allergen‐stimulated CD3⁺CD4⁺CD154⁺ T cell intracellular IL‐4, IL‐5, IL‐13, and IFN‐γ expression were evaluated by flow cytometry. Results Compared with the T helper type 2 (Th2) cytokine expression measured after pre‐treatment ragweed exposures, placebo‐treated subjects demonstrated a significantly elevated ragweed‐ and Amb a 1‐specific T cell IL‐4 and IL‐13 co‐expression (P=0.005 and P=0.022, respectively) and a significantly elevated ragweed‐specific IL‐5 expression (P<0.001) following post‐treatment ragweed exposures. In contrast, AIC‐treated subjects demonstrated no increases in allergen‐specific Th2 cytokine expression following post‐treatment ragweed exposures. IFN‐γ expression remained low and un‐changed in both groups. Subject reported total nasal symptom scores demonstrated modest but significant correlations with Amb a 1‐ and ragweed‐stimulated intracellular Th2 cytokine responses. Conclusion Combined CD154 and intracellular cytokine staining in PBMC can be used to sensitively monitor changes in antigen‐specific T cell subset frequencies in clinical studies. Antigen‐specific cytokine expression moderately correlated with the reported levels of allergic symptoms. Trial Registration NCT00537355 Cite this as: J. D. Campbell, P. Buchmann, S. Kesting, C. R. Cunningham, R. L. Coffman and E. M. Hessel, Clinical & Experimental Allergy, 2010 (40) 1025–1035.