Informing the Recommended Phase III Dose of Alnuctamab, a CD3 × BCMA T-Cell Engager, Using Population Pharmacokinetics and Exposure-Response Analysis.

Alnuctamab, a B-cell maturation antigen (BCMA)-targeting T-cell engager, has demonstrated encouraging antitumor activity in the phase I study CC-93269-MM-001 treating patients with relapsed or refractory multiple myeloma. Identification of a recommended Phase III dose (RP3D) was a key objective, as such population pharmacokinetic (PopPK) and exposure-response analysis was critical. Intravenous (IV) alnuctamab was administered in fixed doses (0.15-10 mg) or in step-up doses to a maximum 10-mg target dose. Subcutaneous (SC) step-up doses of 3 and 6 mg were followed by a target dose range of 10-60 mg. Concentration data from IV and SC alnuctamab administration was pooled and was well described by a two-compartment PopPK model with first-order absorption and elimination. Covariate analysis determined that the inclusion of baseline soluble BCMA (sBCMA) on clearance significantly improved model fitting. Individual exposure parameters were estimated from the final model to characterize exposure-response relationships. Switching from IV to SC administration improved the safety profile of alnuctamab by limiting the frequency of grade ≥2 CRS events. A significant exposure-CRS relationship was observed after the first SC dose, but not subsequent dose administrations. Exposure-safety analysis did not find a statistically significant relationship between increasing exposure and the probability of key safety events of interest. Logistic regression analysis for patients administered SC alnuctamab identified that increased exposure significantly increased the probability of response, although the additional benefit was minimal at exposures above 30 mg target dose. Considering the totality of exposure-response data, the clinical pharmacology assessment supported a SC RP3D of 3/6/30 mg.

Bioanalytical Methods for Characterization of CAR-T Cellular Kinetics: Comparison of PCR Assays and Matrices.

Recently, multiple chimeric antigen receptor T-cell (CAR-T)-based therapies have been approved for treating hematological malignancies, targeting CD19 and B-cell maturation antigen. Unlike protein or antibody therapies, CAR-T therapies are "living cell" therapies whose pharmacokinetics are characterized by expansion, distribution, contraction, and persistence. Therefore, this unique modality requires a different approach for quantitation compared with conventional ligand binding assays implemented for most biologics. Cellular (flow cytometry) or molecular assays (polymerase chain reaction (PCR)) can be deployed with each having unique advantages and disadvantages. In this article, we describe the molecular assays utilized: quantitative PCR (qPCR), which was the initial platform used to estimate transgene copy numbers and more recently droplet digital PCR (ddPCR) which quantitates the absolute copy numbers of CAR transgene. The comparability of the two methods in patient samples and of each method across different matrices (isolated CD3+ T-cells or whole blood) was also performed. The results show a good correlation between qPCR and ddPCR for the amplification of same gene in clinical samples from a CAR-T therapy trial. In addition, our studies show that the qPCR-based amplification of transgene levels was well-correlated, independent of DNA sources (either CD3+ T-cells or whole blood). Our results also highlight that ddPCR can be a better platform for monitoring samples at the early phase of CAR-T dosing prior to expansion and during long-term monitoring as they can detect samples with very low copy numbers with high sensitivity, in addition to easier implementation and sample logistics.

Allergen-specific T cells and clinical features of food allergy: Lessons from CoFAR immunotherapy cohorts.

BACKGROUND: Allergen-specific IL-4+ and IL-13+ CD4+ cells (type 2 cells) are essential for helping B cells to class-switch to IgE and establishing an allergic milieu in the gastrointestinal tract. The role of T cells in established food allergy is less clear. OBJECTIVE: We examined the food allergen-specific T-cell response in participants of 2 food allergen immunotherapy trials to assess the relationship of the T-cell response to clinical phenotypes, including response to immunotherapy. METHODS: Blood was obtained from 84 participants with peanut allergy and 142 participants with egg allergy who underwent double-blind placebo-controlled food challenges. Peanut- and egg-responsive T cells were identified by CD154 upregulation after stimulation with the respective extract. Intracellular cytokines and chemokine receptors were also detected. The response to peanut epicutaneous immunotherapy (Peanut Epicutaneous Phase II Immunotherapy Clinical Trial [CoFAR6]; 49 participants receiving epicutaneous immunotherapy) and egg oral immunotherapy or a baked egg diet (Baked Egg or Egg Oral Immunotherapy for Children With Egg Allergy [CoFAR7]; 92 participants) was monitored over time. RESULTS: Peanut-specific type 2 and CCR6+ T cells were negatively correlated with each other and differently associated with immune parameters, including specific IgE level and basophil activation test result. At baseline, type 2 cells, but not CCR6+ cells, were predictive of clinical parameters, including a successfully consumed dose of peanut and baked egg tolerance. Exposure to peanut or egg immunotherapy was associated with a decrease in type 2 cell frequency. At baseline, high egg-specific type 2 cell frequency was the immune feature most predictive of oral immunotherapy failure. CONCLUSION: Food-specific type 2 T cells at baseline are informative of threshold of reactivity and response to immunotherapy.

Open-label, add-on trial of cetirizine for neuromyelitis optica.

OBJECTIVE: This pilot study preliminarily examined the efficacy and tolerability of cetirizine as an add-on to standard therapy for neuromyelitis optica (NMO). METHODS: Eligible participants met the Wingerchuk 2006 diagnostic criteria or had a single typical episode along with positive NMO immunoglobulin G. After baseline clinical and laboratory assessments, participants began treatment with cetirizine 10 mg orally daily, in addition to their usual disease-modifying therapy for NMO, and continued for 1 year. The primary end point was the annualized relapse rate (ARR) while on the same disease-modifying therapy before starting cetirizine compared with after taking cetirizine. Additional end points included disability (Expanded Disability Status Scale [EDSS]), relapse severity, tolerability, especially with respect to drowsiness measured by the Epworth Sleepiness Scale (ESS), and laboratory parameters. RESULTS: The ARR before cetirizine was 0.4 ± 0.80 and after cetirizine was 0.1 ± 0.24 (p = 0.047). There was no statistically significant difference in the EDSS (mean 3.9 ± 2.18 before the start of the study and 3.2 ± 2.31 at the conclusion of the study, p = 0.500). The ESS remained fairly consistent throughout the study (mean 6.5 ± 5.33 at baseline and 6.9 ± 4.50 at month 12, p = 0.740). Laboratory studies were unrevealing. CONCLUSIONS: In this pilot study, cetirizine was well tolerated, and the prespecified primary efficacy end point was satisfied. However, the open-label design and the small sample size of this pilot study preclude definitive conclusions. Further research is needed. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that in patients with NMO, the addition of cetirizine to standard therapy is safe, well tolerated, and reduces relapses.

Egg-specific IgE and basophil activation but not egg-specific T-cell counts correlate with phenotypes of clinical egg allergy.

BACKGROUND: Egg allergy is phenotypically heterogeneous. A subset of patients with egg allergy can tolerate egg in an extensively heated form. Inclusion of baked egg (BE) into the diet accelerates resolution of egg allergy. Conversely, BE reactivity is associated with persistent disease. The immune basis of this clinical heterogeneity is unknown. OBJECTIVES: We sought to study egg-specific antibody, basophil, and T-cell responses in children with reactivity or tolerance to BE. METHODS: All participants underwent double-blind, placebo-controlled challenges to BE, and those who tolerated BE were challenged with unheated egg white protein to confirm clinical egg reactivity. Laboratory studies included serum antibody measurements, basophil activation tests, and CD154-based detection of egg-responsive T cells by using flow cytometry. RESULTS: Of the 129 children studied, BE-reactive participants had significantly greater levels of egg-, ovalbumin-, and ovomucoid-specific IgE; lower ratios of egg-specific IgG4/IgE; and increased basophil activation in response to egg. Among all participants, CD154-based profiling revealed egg-responsive T cells producing IL-4 and IL-13 but little IL-10 or IFN-γ, as well as the presence of egg-responsive Foxp3+CD25+CD127low regulatory T cells. Egg-responsive T cells expressed CCR4, CCR6, and CXCR5, indicating capacity for homing to the skin, mucosa, and B-cell follicles. However, neither the frequency nor phenotype of egg-responsive T cells was different in those with tolerance or reactivity to BE. CONCLUSIONS: Egg-specific antibody and basophil responses, but not T-cell responses, are greater in those with reactivity versus tolerance to BE. Egg-specific antibody and T-cell responses were highly heterogeneous in this cohort. The clinical implications of this immune heterogeneity will need to be studied longitudinally.

Increased Tolerance to Less Extensively Heat-Denatured (Baked) Milk Products in Milk-Allergic Children.

BACKGROUND: Most milk-allergic children tolerate baked milk. OBJECTIVE: To investigate the effect of more frequent versus less frequent introduction of higher doses of more allergenic (less heat-denatured) forms of milk (MAFM) on progression to tolerance. METHODS: Milk-allergic children were challenged with increasing doses of MAFM; baked foods were incorporated into the diet; challenges were repeated at 6- or 12-month intervals over 36 months. RESULTS: A total of 136 children (70% males) were enrolled in the active group (median age, 7 years). At baseline, 41 (30%) reacted to muffin, 31 (23%) to pizza, 11 (8%) to rice pudding, 43 (32%) to non-baked milk; and 10 (7%) tolerated non-baked milk. Children who tolerated baked milk but reacted to non-baked liquid milk were randomized to MAFM challenges every 6 months (n = 41) or 12 months (n = 44). At month 36, 61% children in the 6-month and 73% in the 12-month escalation groups tolerated MAFM. Overall, 41 (48%) children who ingested baked-milk diet became tolerant to non-baked milk; no difference was seen between 6- and 12- month escalations. Among children who reacted to muffin at baseline and continued avoidance, 20% developed tolerance to baked milk and 0% tolerated non-baked milk. None of the 34 children who qualified for inclusion but chose not to take part in the active study became tolerant to any form of milk by history. CONCLUSIONS: Majority of children tolerated baked milk at baseline. Baked-milk diets were associated with progressive immunomodulation. Most children who incorporated baked milk into their diet progressed to tolerating MAFM, but there was no advantage to more frequent attempts to escalate to MAFM, per intention-to-treat analysis.

Impact of granulocyte contamination on PBMC integrity of shipped blood samples: Implications for multi-center studies monitoring regulatory T cells.

In centralized immune monitoring for a multi-center allergen immunotherapy trial, we observed frequent loss of CD4+ T cell integrity following staining of cultured PBMCs with our regulatory T cell flow cytometry panel. Samples were marked by a loss of total cellular events, altered scatter properties, and reduced CD3+CD4+ events. This occurred only in samples that were stained with Foxp3 and were therefore treated with Foxp3 fixation-permeabilization buffer. We identified granulocyte contamination in samples associated with a loss of integrity, and went on to test the impact of granulocyte depletion on day-old blood samples. Granulocyte depletion prevented loss of cell integrity and CD3+CD4+ events, and reduced variability in detection of Foxp3+ cells. Addition of purified neutrophils back to PBMCs altered scatter properties and detection of CD4+ T cells. Implementation of a granulocyte depletion step in our standard operating protocols has reduced assay failure due to loss of sample integrity from 31% to 0%. Routine incorporation of a granulocyte depletion step during PBMC isolation is recommended prior to downstream immune monitoring in blood with next-day processing.

Mechanistic correlates of clinical responses to omalizumab in the setting of oral immunotherapy for milk allergy.

BACKGROUND: In our recent clinical trial, the addition of omalizumab to oral immunotherapy (OIT) for milk allergy improved safety, but no significant clinical benefit was detected. OBJECTIVE: We sought to investigate mechanisms by which omalizumab modulates immunity in the context of OIT and to identify baseline biomarkers that predict subgroups of patients most likely to benefit from omalizumab. METHODS: Blood was obtained at baseline and multiple time points during a placebo-controlled trial of OIT for milk allergy in which subjects were randomized to receive omalizumab or placebo. Immunologic outcomes included measurement of basophil CD63 expression and histamine release and casein-specific CD4+ regulatory T-cell proliferation. Biomarkers were analyzed in relationship to measurements of safety and efficacy. RESULTS: Milk-induced basophil CD63 expression was transiently reduced in whole blood samples from both omalizumab- and placebo-treated subjects. However, IgE-dependent histamine release increased in washed cell preparations from omalizumab- but not placebo-treated subjects. No increase in regulatory T-cell frequency was evident in either group. Subjects with lower rates of adverse reactions, regardless of arm, experienced better clinical outcomes. Pre-OIT basophil reactivity positively associated with occurrence of symptoms during OIT, whereas the baseline milk IgE/total IgE ratio correlated with the likelihood of achieving sustained unresponsiveness. A combination of baseline basophil and serologic biomarkers defined a subset of patients in which adjunctive therapy with omalizumab was associated with attainment of sustained unresponsiveness and a reduction in adverse reactions. CONCLUSIONS: Combining omalizumab therapy with milk OIT led to distinct alterations in basophil reactivity but not T-cell responses. Baseline biomarkers can identify subjects most likely to benefit from adjunctive therapy with omalizumab.

Basophil Degranulation Assay.

Basophil degranulation assay has gained importance over the last decade in both diagnosis of food allergy and evaluation of progression of immunotherapy. This assay involves the identification and quantification of the expression of CD63 molecule on basophil membrane. CD63 is a marker of multivesicular bodies that is exposed to cell membrane during the process of degranulation in which the contents of basophil granules are released. This chapter describes the methodology for performing this assay.

T-Cell Proliferation Assay: Determination of Immunodominant T-Cell Epitopes of Food Allergens.

Characterization of allergen-specific T cells is critical to understand their contribution to disease pathogenesis. The identification of immunodominant T-cell epitopes is crucial for development of T-cell-based vaccines. Peptide-specific T-cell proliferation studies are usually performed in a library of short synthetic peptides (15mer or 20mer) with 3 or 5 offset spanning the entire length of the allergen. T-cell peptide epitopes lack the primary and tertiary structure of the native protein to cross-link IgE, but retain the ability to stimulate T cells. The peptides sequences can also be obtained either by in silico approaches and in vitro binding assays. The efficacy of T-cell epitope-based peptide immunotherapy has been proven in certain allergies. The present methodology describes T-cell proliferation assays using whole blood sample from allergic subjects.

The false alarm hypothesis: Food allergy is associated with high dietary advanced glycation end-products and proglycating dietary sugars that mimic alarmins.

The incidence of food allergy has increased dramatically in the last few decades in westernized developed countries. We propose that the Western lifestyle and diet promote innate danger signals and immune responses through production of "alarmins." Alarmins are endogenous molecules secreted from cells undergoing nonprogrammed cell death that signal tissue and cell damage. High molecular group S (HMGB1) is a major alarmin that binds to the receptor for advanced glycation end-products (RAGE). Advanced glycation end-products (AGEs) are also present in foods. We propose the "false alarm" hypothesis, in which AGEs that are present in or formed from the food in our diet are predisposing to food allergy. The Western diet is high in AGEs, which are derived from cooked meat, oils, and cheese. AGEs are also formed in the presence of a high concentration of sugars. We propose that a diet high in AGEs and AGE-forming sugars results in misinterpretation of a threat from dietary allergens, promoting the development of food allergy. AGEs and other alarmins inadvertently prime innate signaling through multiple mechanisms, resulting in the development of allergic phenotypes. Current hypotheses and models of food allergy do not adequately explain the dramatic increase in food allergy in Western countries. Dietary AGEs and AGE-forming sugars might be the missing link, a hypothesis supported by a number of convincing epidemiologic and experimental observations, as discussed in this article.

Peanut T-cell epitope discovery: Ara h 1.

BACKGROUND: Identification of potential T-cell epitopes in the peanut major allergens is essential for development of peptide-based immunotherapy. Traditional methods of T-cell epitope discovery use overlapping short peptides spanning the full length of the protein in T-cell proliferation assays. Because large proteins, such as Ara h 1, require a large number of peptides, this limits screening to a small number of allergic subject-derived T-cell lines. OBJECTIVE: We sought to identify candidate peptides of Ara h 1 that display promiscuous binding to MHC class II and induce TH2 cytokine production by T cells. METHODS: In silico MHC class II binding prediction was performed with NetMHCIIpan 2.0 (peptide length, 15; 1-mer offset) and the most abundant class II alleles in the North American population and with an in vitro MHC class II peptide reporter assay performed in parallel, which used synthetic 15-mer peptides offset by 5 mer spanning the protein. High-resolution MHC class II typing and a T-cell proliferation assay using preselected peptides were performed with PBMCs from 98 subjects with peanut allergy and 14 healthy control subjects. IL-4, IL-13, IL-5, IFN-γ, and TNF-α levels were measured in culture supernatants. RESULTS: Thirty-six Ara h 1 peptides were identified by using in silico predictions and MHC class II binding assays. In combination with T-cell proliferation and cytokines secreted in T-cell assays, we have identified 4 vaccine candidate Ara h 1 peptides. CONCLUSIONS: Preselection of peptides by using in silico and in vitro approaches in combination with conventional methods appears to be an effective strategy for identifying peanut T-cell peptide vaccine candidates.

Dietary Elimination of Soybean Components Enhances Allergic Immune Response to Peanuts in BALB/c Mice.

BACKGROUND: Food allergy research is hampered by a lack of animal models that consistently mimic human food allergic responses. Laboratory mice are generally fed grain-based chow made with large amounts of soybeans rich in immunomodulatory isoflavones. We tested the role of dietary soy components in the induction of food allergic responses in the BALB/c mouse strain, which is known to be resistant to anaphylaxis when orally challenged by food allergens. METHODS: Mice were fed a soy-free diet for 2 generations. After weaning, mice were maintained on the same diet or fed a diet containing soy isoflavones, i.e. genistein and daidzein, followed by weekly oral sensitizations with crude peanut extract plus cholera toxin and finally challenged at week 7. The anaphylactic symptoms, body temperature, peanut-specific antibodies and mast cell degranulation were assessed. RESULTS: Soy-free diet mice showed significantly higher anaphylactic symptom scores and mast cell degranulation after challenge and higher peanut-specific antibody levels than mice fed regular chow. Introduction of a regular soy diet or an isoflavone diet to soy-free diet mice significantly suppressed the allergic reactions compared to the soy-free diet. CONCLUSION: Rodent diet is an important variable and needs to be taken into consideration when designing experiments involving animal models. Our results indicate that elimination of soy components from the diet enhances peanut sensitization in BALB/c mice. In addition to serving as a valuable tool to mimic human food allergy, the dietary influence on the immune response could have far-reaching consequences in research involving animal models.

Berberine and limonin suppress IgE production by human B cells and peripheral blood mononuclear cells from food-allergic patients.

BACKGROUND: Currently, there is no satisfactory treatment for IgE-mediated food allergy. Food Allergy Herbal Formula 2 (FAHF-2) and butanol-purified FAHF-2 (B-FAHF-2) have been shown to protect against peanut-induced anaphylaxis and inhibit IgE synthesis in a murine model. OBJECTIVE: To determine which herbs and compounds in FAHF-2 and B-FAHF-2 suppress IgE production. METHODS: The effect of FAHF-2 and B-FAHF-2 on IgE production was determined using a human B-cell line (U266). Individual compounds were isolated and identified using column chromatography, liquid chromatographic mass spectrometry, and nuclear magnetic resonance techniques. The potency of compounds on IgE suppression were investigated using U266 cells and verified using human peripheral blood mononuclear cells (n = 25) from peanut-allergic patients. Epsilon germline transcript expression was determined. Phosphorylated IκBα level was analyzed using the In-Cell Western assay. The mRNA expression of signal transducer and activator of transcription-3, T-box transcription factor TBX21, interferon-γ, forkhead box P3, GATA-binding protein 3, interleukin-10, and interleukin-5 also were analyzed using real-time polymerase chain reaction. RESULTS: FAHF-2 and B-FAHF-2 inhibited IgE production by U266 cells. B-FAHF-2 was 9 times more effective than FAHF-2. Two compounds that inhibited IgE production were isolated from Philodendron chinensis and identified as berberine and limonin. Berberine was more potent and inhibited IgE production by peripheral blood mononuclear cells by 80% at 0.62 µg/mL. Berberine significantly inhibited ε-germline transcript expression by peripheral blood mononuclear cells. Phosphorylated IκBα level was significantly suppressed and mRNA expressions of T-box transcription factor TBX21 and signal transducer and activator of transcription-3 were significantly increased by berberine. CONCLUSION: Berberine and limonin mediated IgE suppression. The mechanism by which berberine modulates ε-germline transcript expression might be through regulating the phosphorylated IκBα level and the expressions of signal transducer and activator of transcription-3 and T-box transcription factor TBX21. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT00602160.

Potential non-T cells source of interleukin-4 in food allergy.

BACKGROUND: Recently, a study from the Consortium of Food Allergy Research (CoFAR) showed that allergen-induced IL-4 expression in CD25(+) mononuclear cells was increased in allergic patients. However, they did not find the expected increase in GATA-3 expression, suggesting that allergen-induced IL-4 might not be of T-cell origin. We sought to determine whether other cell types were responsible for the increased IL-4 expression in the CD25(+) cell population. METHODS: Comparing six allergic patients and six healthy controls, we analyzed the CD25(+) isolated population from PBMC for the presence of potential IL-4-expressing non-T cells. We also compared spontaneous expression levels of surface markers (CD203c, CD63, CD25, and HLA-DR) on basophils from whole blood of 42 peanut-allergic patients and from 12 non-atopic controls. Expression of these markers was also evaluated following basophil activation in eight peanut-allergic patients selected from the previous cohort. RESULTS: In addition to CD4(+) T cells, a substantial proportion of non-T cells were found in the CD25(+) -isolated cell population: basophils, NK, and NK-T cells with a mean percentage ± s.e.m. of 5.24 ± 0.63%, 6.65 ± 1.01%, and 6.01 ± 1.04%, respectively. The majority of these cells exhibited positive intracytoplasmic staining for IL-4. Expression of CD63 and CD25 was significantly higher in allergic patients compared with controls (p < 0.05). Interestingly, we found a significantly higher proportion of activated basophils expressing HLA-DR, compared with non-activated basophils (p < 0.05). CONCLUSIONS: Our results support the suggested key role of non-T cells secreting IL-4 in food allergy, particularly basophils, which may also play a central role in antigen presentation.

Basophil reactivity, wheal size, and immunoglobulin levels distinguish degrees of cow's milk tolerance.

BACKGROUND: In our previous study about 75% of children with cow's milk allergy tolerated baked milk products, which improved their prognosis and quality of life. OBJECTIVE: We sought to identify biomarkers of varying degrees of clinical tolerance among a cohort of children with cow's milk allergy. METHODS: One hundred thirty-two subjects were initially classified as baked milk-reactive, baked milk-tolerant, or having "outgrown milk allergy" based on the results of oral food challenges. The baked milk-tolerant group was then divided into 3 groups based on the amount and degree of heat-denatured milk protein that they could tolerate. Serum was analyzed for allergen-specific IgE and IgG(4) levels, basophil reactivity was assessed in whole blood stimulated with serial 10-fold dilutions of milk protein, and skin prick tests (SPTs) were performed to commercial milk extract. Activated basophils were defined by using flow cytometry as CD63(bright)CD203c(+)CD123(+)HLA-DR(dim/-)CD41a(-)lineage(-). Data were analyzed by using the Jonckheere-Terpstra test. RESULTS: Significant differences across the 5 clinical groups were seen for median casein- and milk-specific IgE levels, casein-specific IgG(4) levels, and casein IgE/IgG(4) ratios; milk-specific to nonspecific basophil activation ratio, median basophil reactivity, and spontaneous basophil activation (CD203c expression after stimulation with RPMI); and milk SPT wheal diameters. Casein- and milk-specific IgE level, milk-specific basophil reactivity, and milk SPT wheal diameter are all significantly greater among patients with milk allergy who react to baked milk than among those who tolerate it. CONCLUSIONS: The majority of patients with milk allergy are able to tolerate some forms of baked milk in their diets. Different phenotypes of children with cow's milk allergy can be distinguished by casein- and milk-specific IgE levels, milk-specific basophil reactivity, and milk SPT mean wheal diameters. Spontaneous basophil activation is greater among patients with more severe clinical milk reactivity.

Isoflavones, genistein and daidzein, regulate mucosal immune response by suppressing dendritic cell function.

Lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, has been shown to have a strong adjuvant effect towards inhaled antigens contributing to airway inflammation. Isoflavones are anti-inflammatory molecules present in abundant quantities in soybeans. We investigated the effect of isoflavones on human dendritic cell (DC) activation via LPS stimulation and subsequent DC-mediated effector cell function both in vitro and in a mouse model of upper airway inflammation. Human monocyte-derived DCs (MDDC) were matured with LPS (or TNF-α) +/- isoflavones (genistein or daidzein). The surface expression levels of DC activation markers were analyzed by flow cytometry. Mature DCs +/- isoflavones were washed and cultured with freshly-isolated allogenic naïve CD4⁺ T cells for 5 days or with autologous natural killer (NK) cells for 2 hours. The percentages of proliferating IFN-γ⁺ CD4⁺ T cells and cytokine levels in culture supernatants were assessed. NK cell degranulation and DC cytotoxicity were measured by flow cytometry. Isoflavones significantly suppressed the activation-induced expression of DC maturation markers (CD83, CD80, CD86) and MHC class I but not MHC class II molecules in vitro. Isoflavone treatment inhibited the ability of LPS-DCs to induce IFN-γ in CD4⁺ T cells. NK cell degranulation and the percentage of dead DCs were significantly increased in isoflavone-treated DC-NK co-culture experiments. Dietary isoflavones suppressed the mucosal immune response to intra-nasal sensitization of mice to ovalbumin. Similar results were obtained when isoflavones were co-administered during sensitization. These results demonstrate that soybean isoflavones suppress immune sensitization by suppressing DC-maturation and its subsequent DC-mediated effector cell functions.

Regulation of the immune response by soybean isoflavones.

Soybeans are rich in immuno-modulatory isoflavones such as genistein, daidzein, and glycitein. These isoflavones are well-known antioxidants, chemopreventive and anti-inflammatory agents. Several epidemiological studies suggest that consumption of traditional soy food containing isoflavones is associated with reduced prevalence of chronic health disorders. Isoflavones are considered to be phytoestrogens because of their ability to bind to estrogen receptors. The literature is extensive on the chemistry, bio-availability, and bio-activity of isoflavones. However, their effects on immune response are yet to be fully understood, but are beginning to be appreciated. We review the role of isoflavones in regulation of the immune response and their potential clinical applications in immune-dysfunction. Special emphasis will be made regarding in vivo studies including humans and animal model systems.